Greater Proliferative Response Research Articles

Immune Responses and Efficacy of Brucella Abortus Strain RB51 in Bison After Delivery in a Dry Dart Formulation or by Parenteral Inoculation.

Bison (Bison bison) heifer calves (n = 32) were randomly assigned to control or vaccination with 1010 colony-forming units of Brucella abortus strain RB51 (RB51) vaccine by single or boostered parenteral delivery, or by surgical implantation of a dry dart formulation (n = 8/trt). Serum and/or peripheral blood mononuclear cells (PBMC) were obtained at 0, 4, 8, 13, 16, 21, and 24 wks after initial vaccination and at 0, 4, 8, 12, 15, 22, and 27 wks after booster vaccination to characterize humoral and cellular immune responses to RB51. Bison in both RB51 vaccination treatments demonstrated greater (P < 0.0001) serum humoral responses when compared to non-vaccinates, with parenteral vaccinates demonstrating greater (P < 0.01) responses when compared to mean responses of bison inoculated with the dry dart. Only the booster vaccinated treatment demonstrated greater (P < 0.0001) humoral responses than control bison in samples collected after re-inoculation. At 4, 8, 12, 16, and 24 wks after initial vaccination, PBMC from parenteral RB51 vaccinates demonstrated greater proliferative responses to RB51 when compared to responses of control animals. In comparison, bison inoculated with the RB51 dry dart did not demonstrate greater (P > 0.05) proliferative responses when compared to responses of non-vaccinates. Bison were pasture bred and pregnant animals experimentally challenged in mid-gestation with 107 CFU of B. abortus strain 2,308. Bison in parenteral vaccination treatments had reduced (P < 0.05) abortions and infection in uterine and fetal samples as compared to non-vaccinated bison, with booster vaccinates tending to have the lowest colonization (CFU/gm) in tissues. In comparison, the dry dart formulation did reduce abortion (P < 0.05) but not infection (P > 0.05) in most tissues when compared to non-vaccinated bison. The results of this study reaffirm the efficacy of boostered parenteral vaccination of bison with RB51 in preventing brucellosis. Our data also suggests that the novel dry dart RB51 formulation does not induce sufficient efficacy in bison after a single inoculation.

Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity.

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice, but not in high affinity-primed mice. Mechanistically, low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge, low affinity-primed CD45RB(hi) cells became CD45RB(lo), demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge.

The Transcription Factor PU.1 Regulates γδ T Cell Homeostasis

BackgroundT cell development results in the generation of both mature αβ and γδ T cells. While αβ T cells predominate in secondary lymphoid organs, γδ T cells are more abundant in mucosal tissues. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi1) is essential for early stages of T cell development, but is down regulated during the DN T-cell stage.Methodology/Principal FindingsIn this study, we show that in mice specifically lacking PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1 lck−/−) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild-type mice. The increase in γδ T cell numbers in PU.1-deficient mice is consistent in γδ T cell subsets identified by TCR variable regions. PU.1-deficient γδ T cells demonstrate greater proliferation in vivo and in vitro.Conclusions/SignificanceThe increase of γδ T cell numbers in Lck-Cre deleter strains, where deletion occurs after PU.1 expression is diminished, as well as the observation that PU.1-deficient γδ T cells have greater proliferative responses than wild type cells, suggests that PU.1 effects are not developmental but rather at the level of homeostasis. Thus, our data shows that PU.1 has a negative influence on γδ T cell expansion.

Increased kon of TCR-pMHC interaction influences activation and development of CD4+ T cells (109.18)

Abstract While T cell activation has been correlated with half-life of the TCR-pMHC interaction, the importance of on-rate is only now being realized. In contrast to other studies, our lab has generated a system that separates the kon component from koff by comparing the response of two T cells, 3L2 and M2. 3L2 responds specifically to Hb(64-76) peptide presented on I-Ek. M2 differs from 3L2 by two point mutations in the CDR1α loop, resulting in a 4-fold higher affinity for Hb(64-76) due entirely to a faster kon. We generated an M2 TCR transgenic mouse to compare the affect of kon on T cell responses in vivo and in vitro to 3L2 T cells. M2 expressing T cells are positively selected and detected with a clonotypic antibody in the periphery though at a reduced number in comparison to 3L2. This could be due to decreased positive selection and/or increased negative selection of M2 T cells by alteration of the kinetics of M2 interactions with endogenous pMHC. M2 responds to 12 Hb(64-76) altered peptides (APLs) that are null peptides for 3L2 and has increased reactivity to APLs that are agonists for 3L2. M2 cells have a greater proliferative response at day 3 than 3L2 when exposed to Hb(64-76). While both M2 and 3L2 proliferate by day 3, a larger number of M2 cells than 3L2 cells have divided. This suggests that a fast kon increases early T cell responses. By selectively altering kon, we have revealed the importance of kon for TCR binding pMHC in T cell development and activation.

CCL20/CCR6 chemokine/receptor expression in bone tissue from osteoarthritis and rheumatoid arthritis patients: Different response of osteoblasts in the two groups

Subchondral bone remodeling in osteoarthritis (OA) and rheumatoid arthritis (RA) is mainly characterized by the formation of osteophytes/fibrosis and by the presence of infiltrating cells associated to bone resorption. In this study we analyzed CC (cysteine cysteine motif) chemokine ligand (CCL)20 and CC chemokine receptor (CCR)6 function in subchondral bone tissue and osteoblasts isolated from OA and RA patients. CCL20/CCR6 expression was evaluated by immunohistochemical techniques in bone tissue from OA and RA patients. CCL20-functional tests were performed on osteoblasts isolated from OA and RA patients to evaluate enzymatic response and cell proliferation. Moreover, we assessed Akt phosphorylation as the major signaling pathway for CCL20. In bone tissue biopsies we found that osteoblasts from both OA and RA patients expressed CCR6 while CCL20 was expressed only by RA osteoblasts. Both CCR6 and CCL20 were highly expressed in osteocytes and mononuclear cells from only RA patients. CCL20-stimulated OA osteoblasts showed a significant increase in beta-N-acetylhexosaminidase release compared to RA. Conversely, a significant increase in cellular proliferation was found only in CCL20-stimulated RA osteoblasts associated to Akt phosphorylation. These data were confirmed in bone tissue biopsies. This study demonstrates a different expression of CCL20-positive osteoblasts in OA versus RA disease that seem to be associated with the presence of infiltrating mononuclear cells. Moreover, CCL20 stimulation resulted in a greater proliferative response in RA osteoblasts compared to OA osteoblasts, mediated by Akt signaling, while OA osteoblasts showed increased enzymatic activity, thus suggesting a differential role of this chemokine in OA and RA.

HCV-Specific T-Cell Response in Relation to Viral Kinetics and Treatment Outcome (DITTO-HCV Project)

The second slope of viral decline induced by interferon treatment has been suggested to be influenced mainly by the hepatitis C virus (HCV)-specific T-cell response; however, this hypothesis needs to be validated by results derived from experimental studies. To address this issue, the HCV-specific T-cell response of 32 genotype-1-infected patients of the 270 patients enrolled in the dynamically individualized treatment of hepatitis C infection and correlates of viral/host dynamics phase III, open-label, randomized, multicenter trial was studied in relation to viral kinetics and treatment outcome. Greater proliferative responses by HCV-specific CD8 cells were found before treatment in patients with a fast viral decline and with a sustained viral response. However, no significant improvement of HCV-specific CD8 responses was observed in the first weeks of therapy in both rapid viral responder and non-rapid viral responder patients. A mild enhancement of proliferative T-cell responses and a partial restoration of the cytotoxic T-cell potential was expressed only late during treatment, likely favored by HCV clearance. Early restoration of an efficient T-cell response does not seem to be an essential requirement for a rapid viral decline in the first weeks of treatment. However, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline are more likely to present a rapid and sustained viral response. Therefore, future treatment protocols should consider the development of strategies aimed at improving HCV-specific T-cell responses.

Insulin-Like Growth Factor-I and Fibroblast Growth Factor, But Not Growth Hormone, Affect Growth Plate Chondrocyte Proliferation

Of the many factors that regulate linear growth, IGF-I has a central role in epiphyseal chondrocyte development. Whether IGF-I is solely of systemic or also of local origin is uncertain, as is how other growth factors interact with IGF-I at the growth plate. We studied the proliferative effects of IGF-I on juvenile bovine epiphyseal chondrocytes fractionated by density gradient centrifugation. Cell density correlated with size, glycogen content, and gene expression patterns. There was a gradient of response to IGF-I, with the greatest proliferative response in high-density cells corresponding to the reserve zone, as measured by [3H]thymidine uptake. Low-density (hypertrophic zone) cells proliferated only when exposed to IGF-I and basic fibroblast growth factor (FGF). The gradient of IGF-I response correlated with [125I]IGF-I binding as determined by Scatchard analysis: IGF-I receptor number was 10-fold greater in reserve zone cells than in hypertrophic cells. When exposed to basic FGF for 24 hours, IGF-I binding in hypertrophic cells increased 3-fold. In contrast, no specific binding of GH was demonstrated in juvenile bovine chondrocytes. GH produced neither signal transducer and activator of transcription phosphorylation, increased proliferation, nor increased IGF-I mRNA levels in any chondrocyte fraction. IGF-I mRNA levels were extremely low at 800-1100 copies/microg 18S RNA in bovine chondrocytes. These results suggest that the major regulator of chondrocyte proliferation is systemic IGF-I; FGFs may influence the actions of IGF-I at the growth plate by altering its receptor number in chondrocytes.

Antibody blockade of Thy‐1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti‐CD3 monoclonal antibody

Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.

Immunotherapy of murine prostate cancer using whole tumour cells killed ex vivo by cytosine deaminase/5‐fluorocytosine suicide‐gene therapy

To evaluate the efficacy of antitumour vaccines comprising irradiated allogeneic or autologous whole cells expressing cytosine deaminase (CD) which are first killed ex vivo by prodrug activation using 5-fluorocytosine (5-FC), as the immunogenicity of tumour cells used as irradiated vaccines depends both on antigen expression and on the mode of their death. The PA3 rat prostate cell line and MATLyLu, an androgen-insensitive subline, were grown and transfected with CD (designated PCD and MCD). In vitro drug-sensitivity was assessed in the cell lines using a viability assay, and the mode of cell death quantified by assessing apoptosis. Bax and bcl-2 expression were assessed by Western blot analysis. For in vivo experiments, male 8-10-week-old Lobund-Wistar rats were vaccinated (using vehicle in control groups) with 5 x 10(6) cells, all cells being irradiated before injection, to give groups with PA3, PCD, PCD killed with 5-FC, MatLyLu, MCD, and MCD killed with 5-FC. After 7 days all animals were given a subcutaneous tumour challenge of PA3 cells, and tumour volume measured subsequently. Immune responses were assessed in splenocytes. The efficiency of cell kill varied between the cell lines assessed, but cell death was by induced apoptosis. Single doses of vaccine were most effective in the allogeneic setting, causing significantly slower growth of syngeneic tumour challenge (P < 0.01), and 25% better survival at 50 days (P < 0.02) than irradiated untransfected cells. This was consistent with the greater proliferative response after allogeneic than autologous vaccination. The immunogenicity of irradiated tumour cells is enhanced when they are killed ex-vivo using suicide-gene therapy. This approach would be clinically applicable in terms of ease of vaccine production, safety, storage and avoidance of potential toxicities of in vivo gene transfer.

Effect of prior dietary exposure to cows' milk protein on antigen-specific and nonspecific cellular proliferation in mice

The impact of dietary components on the immune system is gaining increased attention in the effort to develop safe food products, some even with health-promoting potential, as well as to improve the basic understanding of the immunomodulatory potential of common food components. In such studies, which are mainly based on experiments in vitro, it is important to be able to differentiate nonspecific activation of immune cells induced by dietary components from ex vivo restimulation of antigen-specific cells that might be present in cell cultures owing to prior dietary exposure to the antigens in cell donors. Focusing on the immunostimulatory potential of cows' milk proteins and peptides, we studied the impact of prior dietary exposure to cows' milk on proliferation of murine immune cells upon ex vivo stimulation with bovine milk proteins. Nonspecific proliferation induced by beta-casein peptides was further assessed on cells from mice bred on a cows'-milk-free diet. Regarding the dietary effect, we found that prior oral intake of cows' milk proteins affected cell proliferation induced by culturing with cows' milk proteins in vitro, as spleen cells from mice fed a milk-containing diet showed a significantly greater proliferative response than did cells from mice bred on a cows'-milk-free diet. Studies of immune enhancing potentials of beta-casein peptides showed that some peptides stimulate proliferation of immune cells nonspecifically. In conclusion, these findings stress the importance of employing immune cells from mice unexposed to cows' milk for studies of the immunomodulating capacity of cows' milk proteins and peptides, in order to rule out the interference caused by antigen-specific immune responses. By using such cells, we here show that some beta-casein peptides possess the potential to induce proliferation in immune cells in a nonspecific manner.

Aging exacerbates neointimal formation, and increases proliferation and reduces susceptibility to apoptosis of vascular smooth muscle cells in mice

In response to injury, aging mediates exaggerated neointimal formation, the pathologic hallmark of obliterative vascular diseases. We assessed the development of neointima in a model of mechanical vascular injury in aging mice (18 months old) and young mice (2 months old). To investigate the mechanisms by which aging affects neointimal formation, we also carried out a set of in vitro studies to characterize the biologic properties of vascular smooth muscle cells (VSMCs) derived from aging and young mice. Aging and young mice were subjected to wire injury to the carotid artery. Four weeks later injured arteries were harvested, and neointimal formation was histologically assessed. The profiles of angiogenesis-related genes between aortic VSMCs derived from aging and young mice were compared with complementary DNA arrays. Expression of platelet-derived growth factor receptor-alpha (PDGFR-alpha) and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) by VSMCs were assessed. Susceptibility to apoptosis in aging and young VSMCs in response to nitric oxide and serum starvation was investigated. In addition, the level of apoptosis in neointimal VSMCs (by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay) was compared between aging and young animals. When compared with young mice, aging mice exhibited exaggerated neointimal formation (intima-media ratio, 1.17 +/- 0.57 vs 0.49 +/- 0.16; P < .0001). Aging VSMCs expressed higher levels of PDGFR-alpha (12.0% +/- 2.7% vs 3.2 +/- 0.67%; P = .034) and greater proliferative response (4-fold increase) to PDGF-BB, compared with young VSMCs. However, aging VSMCs were less susceptible to apoptosis when subjected to serum starvation (75% less) and exposure to nitric oxide (50% less). Furthermore, there was more apoptosis in the neointima of young arteries than in their aging counterparts (8.75% +/- 3.3% vs 2.8% +/- 1.9; P = .021). Age-dependent increases in PDGFR-alpha may alter VSMC proliferation, and when coupled with resistance to apoptosis could contribute to exaggerated neointima formation in aging animals. Of significance, our findings in the mouse will enable application of abundant molecular tools afforded by this species to further dissect the mechanisms of exaggerated neointimal formation associated with aging. Neointimal formation is the pathologic hallmark of obliterative vascular diseases, including primary atherosclerosis, post stent restenosis, graft occlusion after vascular bypass procedures, and transplant allograft vasculopathy. Aging is an independent risk factor for development of cardiovascular diseases, and aging exaggerates neointimal formation after vascular injury. Understanding the mechanisms responsible for this phenomenon may facilitate prevention or provide new therapies for vascular occlusive diseases, which are so prevalent in the aging population. Our ability to reproduce the model in the mouse will no doubt facilitate such understanding.

COMPARATIVE STUDY AND EFFECTS OF MACROPHAGE COLONY-STIMULATING FACTOR (M-CSF) ADMINISTRATION ON NK1.1+ CELLS IN MOUSE SPLEEN AND BONE MARROW

ABSTRACTWe conducted a comparative study of NK1.1+ cells in spleen and bone marrow and the effects of administration of M-CSF on them. Administration of M-CSF to mice increased the number of NK1.1+ cells in spleen but not in bone marrow. The NK1.1+ cells in spleen (Spl-NK1.1) and bone marrow (BM-NK1.1) were purified by magnetic cell sorter. Their cell surface markers and functions were then examined. The percentage of Mac-1 antigen-positive cells (and F4/80 antigen-positive cells) was higher among BM-NK1.1 than Spl-NK1.1. Moreover, the administration of M-CSF increased the number of Mac-1 and F4/80 antigen-positive cells in both Spl-Nk1.1 and BM-NK1.1. The functions (cytolytic activity and IFN-γ production) of Spl-NK1.1 and BM-NK1.1 were the same and were enhanced by the administration of M-CSF. But Spl-NK1.1 produced more IFN-γ than BM-NK1.1 when M-CSF was administered. BM-NK1.1 showed a greater proliferative response to IL-2 than Spl-NK1.1. Administration of M-CSF augmented this response. BM-NK1.1 proliferated in response to IL-4 and IL-15, but Spl-NK1.1 responded only slightly. However, administration of M-CSF stimulated Spl-NK1.1 to respond to these cytokines. Both Spl-NK1.1 and BM-NK1.1 showed only a weak response to M-CSF in vitro. But the expression of c-fms antigen (M-CSFR) increased after the M-CSF injections in vivo. These results suggested that there are phenotypical and functional differences between Spl-NK1.1 and BM-NK1.1. The administration of M-CSF led to an accumulation of NK1.1+ cells which were mobilized from bone marrow in spleen.

Effects of short-term high-dose and low-dose dermal exposure to Jet A, JP-8 and JP-8 + 100 jet fuels.

Occupational and environmental exposures to jet fuel recently have become a source of public and regulatory concern. This study investigates the cutaneous toxicity of three fuels used in both civilian and military aircraft. Pigs, an accepted animal model for human skin, were exposed to low-dose (25 microl or 7.96 microl cm(-2)) or high-dose (335 microl or 67 microl cm(-2)) Jet A, JP-8 and JP-8 + 100 under occluded (Hill Top) chamber or cotton fabric) and non-occluded conditions for 5 h, 24 h and 5 days. To mimic occupational exposure, fuel-soaked fabric (high dose) was used. Erythema, edema, transepidermal water loss (TEWL) and epidermal thickness were quantified. High-dose fabric occluded sites had slight erythema at 5 h with increased erythema at 5 days. No erythema was noted in any of the occluded (Hill Top) or non-occluded sites at any of the time points. Morphological assessments depicted slight intracellular epidermal edema at all time points. An increase in change in TEWL (DeltaTEWL) was observed at the 5-h and 24-h fabric and Hill Top occluded treatments and a decrease at the 5-day fabric and Hill Top occluded sites. In all 5-day JP-8 + 100 fabric sites, intracorneal microabscesses filled with inflammatory cells were observed. Epidermal thickening was significant (P < 0.05) in all three jet fuels at the high-dose fabric sites, with JP-8 + 100 being the thickest. The epidermal rete peg depth increased significantly (P < 0.05) at 24 h and 5 days with Jet A, JP-8, and JP-8 + 100 in the fabric sites. No significant differences were noted in the 5-day non-occluded fabric and Hill Top occluded and non-occluded sites. Jet fuel JP-8 + 100 tended to have the greatest proliferative response. In conclusion, the high-dose fabric-soaked exposure at 5 days to Jet A, JP-8 and JP-8 + 100 fuels caused the greatest increase in cutaneous erythema, edema, epidermal thickness and rete peg depth compared with high-dose non-occluded or low-dose exposure under Hill Top occluded and non-occluded conditions.

Bromo-deoxyuridine (BRDU) uptake in the lungs of rats inhaling amosite asbestos or vitreous fibres at equal airborne fibre concentrations.

Rats were exposed, by inhalation, to target airborne fibre concentrations of 1000 f/ml (PCOM fibres by WHO criteria) of a long amosite asbestos sample and a vitreous fibre sample; the target was closely attained for both fibre samples. The size distributions of the two fibre samples was closely similar. Rats were placed in the chambers for 7 hours and then, following a further 16 hours in room air, were injected with bromo-deoxyuridine (BRDU). The presence of BRDU-positive cells in terminal bronchioles/alveolar ducts was assessed in blocks taken from various parts of the left lung, from apex to base. There were significant differences in the proliferative responses between animals but there were also significant differences between the treatments. Lungs from rats exposed to vitreous fibres showed no greater response than the controls, but there was a markedly greater proliferative response in the lungs of rats inhaling long amosite. There was a decreasing gradient of proliferative response from the apex of the lung to the base with all treatments. This could be explained by different degrees of deposition in different areas of the lung. Similar amounts of fibre accumulated in the lungs of rats exposed to the two fibre types and it is unlikely that dissolution could be important over the timescale used here. We conclude that, when amosite asbestos deposits in the lungs of rats it stimulates a proliferative response and that deposition of an equal number of similar-sized vitreous fibres has no effect.

Lesion-Specific Activation of Cloned Human Tumor-Infiltrating Lymphocytes by Autologous Tumor Cells: Induction of Proliferation and Cytokine Production

To investigate the mechanisms by which tumor-infiltrating lymphocytes exert their antitumor effects, tumor-specific tumor-infiltrating lymphocyte clones, as well as autologous tumor cell lines from primary and secondary tumors of two patients during the course of melanoma progression were established. Enrichment for tumor-infiltrating lymphocytes expressing CD25, as well as low concentrations of interleukin-2 (30 IU/ml) in the culture medium, led to a preferential outgrowth of cells that express the high-affinity interleukin-2 receptor. All of these expressed CD2, CD3, CD11, and CD25. Coculture of tumor-infiltrating lymphocyte clones with irradiated, autologous tumor cells induced an up to 480% greater proliferative responses than recombinant interleukin-2 alone. Approximately 60% of the tumor-infiltrating lymphocyte clones showed cytotoxicity against the relevant tumor in a 4-h 51Cr-release assay. When tested in an 18-h 51Cr-release assay, the number of tumor-infiltrating lymphocyte clones exhibiting cytotoxicity against the relevant tumor increased to over 85%. In response to autologous tumor cells, nine of 15 clones secreted interferon-gamma, tumor necrosis factor-alpha, or both. Cytokine production was not restricted to either CD4+ or CD8+ T cells because both CD4+ and CD8+ tumor-infiltrating lymphocyte clones secreted cytokines. Tumor-infiltrating lymphocyte tumor interaction appears to be lesion specific because induction of proliferation and cytokine production, as well as susceptibility to cytolysis, was found not only restricted to the autologous system, but also to the specific lesion. The pattern of tumor-infiltrating lymphocyte tumor interaction specificity indicates a possible loss of antigens expressed on the tumor during disease progression.

The cellular proliferative response, humoral antibody response, and cross reactivity studies of Tetrahymena pyriformis with ichthyophthirius multifiliis in juvenile carp ( Cyprinus carpio L.)

The immune response of 10–12-week-old juvenile carp to T. pyriformis [CCAP 1630/w, 1939 (w)] was investigated. The humoral antibody response following one and two intraperitoneal injections of whole live T. pyriformis (protein concentration of 25 μg/g body weight), separated by an interval of 4 weeks, was monitored over 12–16 weeks. Peak antibody titres were detected 6 weeks following antigen administration. Antibody titres were elevated following the second injection, and relatively high levels were still maintained by week 16. Proliferative responses measured by autoradiography following intraperitioneal injection of methyl 3H thymidine (1–2 μCi/g body weight) were recorded in the pronephros, opisthonephros, and spleen at weekly intervals for 4 weeks following each injection of T. pyriformis. Immunised fish had higher counts than nonimmunised controls, with the greatest counts occurring at 2 and/or 3 weeks following one injection of antigen and 2 weeks following two injections. Activity was recorded prior to peak antibody production with the pronephros and opisthonephros having greater proliferative responses than the spleen. Cross antigenicity studies to examine protection of carp against I. multifiliis following administration of T. pyriformis, showed little evidence in support of protection using this strain of T. pyriformis. Further, antisera raised in either carp, rabbits, or rats failed to show any cross antigenicity between these two species of protozoa.

Lung lymphocytes in bleomycin-induced pulmonary disease

Previous studies have not clearly defined the role of cell-mediated immunity in bleomycin-induced lung injury. In this report the functional activity of T lymphocytes obtained from minced lung preparations, bronchoalveolar lavage, and blood of rabbits treated with bleomycin was examined in cell proliferation and cell-mediated cytotoxicity assays. Four days after instillation of bleomycin (10 units/kg) into the right lung, histologic examination revealed mononuclear cell interstitial infiltrates and alveolar exudates. Right lung bronchoalveolar lavage (BAL) cell counts were similar in both groups, but the percentage of lymphocytes and neutrophils was elevated in bleomycin-treated groups (25% vs. 7% and 35% vs. 0% respectively; p less than 0.05). Spontaneous proliferation of cultured BAL and blood lymphocytes was similar in bleomycin-treated rabbits and controls. After 24 h of incubation with interleukin-2 (IL-2), BAL lymphocytes from bleomycin-treated rabbits had nearly a 4-fold greater proliferative response than lymphocytes from untreated rabbits. Concanavalin-A-dependent cell-mediated cytotoxicity (CDCMC) assays were performed to evaluate cytolytic lymphocyte activity. Spontaneous CDCMC activity was not detected in BAL fluid or in blood lymphocytes from either treated or control animals. After 24 h of incubation with IL-2, significant CDCMC activity was detected in lung lymphocytes from bleomycin-treated animals, but not in lung lymphocytes from control animals. These results indicate that stimulated lymphocytes are present in the lungs of rabbits 4 days after exposure to bleomycin.

Genetic factors controlling inheritance of susceptibility to 1,2-dimethylhydrazine.

Reciprocal crosses were made between AKR/J, a 1,2-dimethylhydrazine (DMH)-resistant mouse strain, and SWR/J, a sensitive strain. The F1 hybrids were tested with DMH and methylazoxymethanol (MAM), two colon carcinogens. Either DMH (20 mg/kg body weight) or MAM (35 mg/kg body weight), a metabolic derivative of DMH, was injected weekly for 10 weeks. In each group of 35 mice, 10 were injected with tritiated thymidine (25 microCi) 1 week after the sixth injection of DMH and MAM for the evaluation of proliferative characteristics and the number of foci of dysplasia occurring in 325 microns of distal colonic mucosa. At 27 weeks after the first injection of the carcinogen, the colons of remaining mice were opened longitudinally and the number of tumors enumerated. Compared with DMH-treated mice, the number of foci of dysplasia per mouse, the percentage of tumor-bearing mice, the number of tumors per animal, and the number of tumors per tumor-bearing animal induced by MAM were severalfold higher. This would suggest the presence of a gene(s) repressing metabolism of DMH to MAM. Moreover, differences in response to the carcinogens were observed between the sexes. In contrast to males, females treated with both DMH and MAM had significantly greater numbers of tumors per animal, tumors per tumor-bearing mice, and a greater proliferative response with extension of S-phase cells to the upper third and luminal surface of crypts. Among males, those with the XAKR/YSWR heritage appeared more resistant than XSWR/YAKR males, particularly in their response to MAM. A twofold difference in the number of foci of dysplasia per mouse, tumors per animal, and the number of tumors per tumor-bearing animals was seen. Analyses of the response to DMH and MAM by F1 reciprocal hybrids of the AKR and SWR strains have shown a complex inheritance pattern governing susceptibility to DMH. Resistance to the carcinogen is provided by at least two specific repressor genes, one governing metabolism of carcinogen from DMH to MAM, and the other controlled by gender. Genetic factors contributed by the AKR female appear to convey additional resistance to male progeny, suggesting more than one gender-related gene.

Trenbolone Alters the Responsiveness of Skeletal Muscle Satellite Cells to Fibroblast Growth Factor and Insulin- Like Growth Factor I*

The potential role of satellite cells in mediating the effect of trenbolone [17 beta-hydroxyestra-4,9-11-trien-3-one (TBOH)] on skeletal muscle hypertrophy was examined. Young female Sprague-Dawley rats received TBOH injections daily for 2 weeks; growth, body composition, and the composition of selected muscles were assessed. Treated rats grew more rapidly and deposited less body lipid and more protein. The semimembranosus muscle from treated rats was larger and had approximately 60% more DNA per muscle than muscles from control rats. The addition of trenbolone directly to the medium of cultured satellite cells did not stimulate cell proliferation, nor did it augment the stimulatory response of these cells to fibroblast growth factor (FGF) or insulin-like growth factor I (IGF-I). In contrast, satellite cells cultured from TBOH-treated rats exhibited greater proliferative responses to FGF and IGF-I than satellite cells from control rats. In addition, serum from TBOH-treated rats stimulated greater cell proliferation in satellite cell cultures than serum from control rats. These experiments suggest that one possible mechanism responsible for the ability of TBOH to stimulate skeletal muscle hypertrophy may be through enhanced proliferation and differentiation of satellite cells as a result of the increased sensitivity of these cells to IGF-I and FGF.

Serum antibody and cellular responses in LEW and F344 rats after immunization with Mycoplasma pulmonis antigens.

Mycoplasma pulmonis causes a chronic respiratory disease in rats which is more severe in LEW than in F344 rats. This study compared the ability of each of these rat strains to produce specific immune responses to M. pulmonis antigens. By an enzyme-linked immunosorbent assay, LEW rats were found to produce approximately 10 times lower levels of specific immunoglobulin G (IgG) after immunization with M. pulmonis antigens than F344 rats, while no significant difference was found in the levels of IgM. The difference in IgG levels was due to much greater levels of specific IgG2b (about 50 times) in F344 rats; no differences were found in other subclasses. Nonimmune LEW rats were found to have as much total IgG2b in their sera as unimmunized F344 rats by a single radial immunodiffusion test; thus, the difference was not due to the inability of LEW rats to produce IgG2b. In contrast to the antibody response to M. pulmonis antigens, anti-keyhole limpet hemocyanin IgG responses in LEW and F344 rats were similar, but F344 rats produced significantly more (about 21 times) IgG2b than was found in M. pulmonis responses. Antisera from F344 rats recognized several additional M. pulmonis antigens than antisera from LEW rats; however, this could not explain the differences in the level of IgG2b in LEW and F344 rats. In vitro stimulation of splenic lymphocytes with M. pulmonis antigens from immunized F344 rats produced much greater proliferative responses than in LEW and nonimmune F344 cells. Thus, the susceptible rat strain LEW produced lower cellular and humoral immune responses to M. pulmonis antigens than the resistant rat strain F344 after immunization.