INVITED REVIEW: Evolution of meat animal growth research during the past 50 years: Adipose and muscle stem cells.

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.

Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice.

The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

Circadian distribution of serum cytokines in multiple sclerosis.

AIMS: The purpose of this study was to examine circadian distribution of selected cytokine levels (IL-2, IL-10, GM-CSF, TNF-alpha, and IFN-gamma) in serum of subjects with active Multiple Sclerosis (MS) and non-MS subjects. MATERIALS AND METHODS: Six females (36-56y) and five males (52-68y) with active MS volunteered and consented for the study conducted at Special Diagnostic Ward of this hospital. All subjects gave their medical history and were given complete physical examination. Low purine meals were served at 16:30, 07:30 and 13:00 h. Lights were "OFF' at 22:30 hr and "ON" at 06:30h. Blood collections were made at 3h intervals over a 24h period of time. Six healthy male subjects (53-76y) subjects' data were obtained from a study conducted 3 years previously using the same procedural protocol. Cytokine assays were assessed using commercial enzyme-linked immuno-absorbent procedure. Time series of average data and the range of change between the highest and lowest concentrations are presented for MS subjects along with data from non-MS subjects. RESULTS: IL-2, IL-10, and GM-CSF levels were significantly reduced in females with MS when compared with levels of healthy subjects while their IL-6 levels were increased. The IL-6, GM-CSF and TNF-alpha levels in males with MS were below detection limits. The TNF-alpha levels were essentially similar in MS females and healthy subjects. CONCLUSIONS: These preliminary studies, although with very small number of patients and healthy male controls appear to suggest that the circadian analysis of cytokines and other markers of immunity may have utility in understanding the pathogenesis of diseases like MS.

Psoas haematoma and femoral neuropathy associated with enoxaparin therapy.

Haemorrhage into the iliopsoas muscle causing femoral neuropathy is an infrequent complication of haemophilia or anticoagulant therapy. The association of an iliopsoas haematoma with enoxaparin therapy is very rare. We describe a case of femoral neuropathy secondary to psoas haematoma in a patient who was on enoxaparin therapy for suspected non-Q wave myocardial infarction. There is no clear consensus for the treatment of these haematomas, with both surgical and conservative options advocated. In this case, our patient recovered fully following conservative management.

Phase IB study of 25-hydroxyvitamin D(3) treatment to diminish suppressor cells in head and neck cancer patients.

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by an increased number of immune inhibitory CD34(+) progenitor cells in their peripheral blood and tumor. The CD34(+) cells suppress autologous T-cell functions. Our prior work had shown that the differentiation inducer 1alpha,25-dihydroxyvitamin D(3) could drive the differentiation of CD34(+) cells isolated from HNSCC patients into dendritic cells. A phase IB clinical trial was initiated with HNSCC patients to determine if 25-hydroxyvitamin D(3) treatment could diminish CD34(+) cell levels and improve immune function. Six patients per treatment group were orally administered 20 or 40 microg/day 25-hydroxyvitamin D(3) for six weeks. Peripheral blood was collected at 0, 1, 2, 4, 6, and 8 weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of these pilot studies showed that 25-hydroxyvitamin D(3) reduced the presence of immune suppressive CD34(+) cells and improved immune competence of HNSCC patients.

Assessment of melatonin's ability to regulate cytokine production by macrophage and microglia cell types.

Evidence in support of melatonin's role as an immunomodulator is incomplete and, in some cases, contradictory. The present studies determined whether melatonin modulates the activity of stimulated macrophages. In vitro lipopolysaccharide (LPS, 10-1000 ng/ml) treatment of alveolar, splenic and peritoneal macrophages isolated from mice and/or rats resulted in a dose-dependent increase in interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha) secretion. Treatment with melatonin (10(-10)-10(-6) M) prior to the addition of LPS, had no effect on IL-1beta or TNF-alpha release. Additionally, melatonin had no effect on stimulated BV2 microglial cell line cytokine secretion. To determine whether melatonin had an indirect effect on macrophage cytokine release via T cells, melatonin was added to unfractionated mouse spleen cells. Again, melatonin showed no priming effect on LPS-stimulated spleen cells. These results suggest that melatonin has no direct or indirect effect on mouse and rat macrophages. In vivo studies, where melatonin was continuously available in the drinking water, showed that melatonin did not have a priming effect on LPS-stimulated mouse peritoneal macrophages. These findings suggest that melatonin is not an important modulator of macrophage and microglia function.

Randomized controlled trial of two cigarette quit programmes in coronary care patients after acute myocardial infarction.

BACKGROUND: Tobacco cessation after acute myocardial infarction (AMI) substantially improves outcome but how effective individual programmes are needs to be established. To date, few studies have examined this factor. AIMS: To assess the outcome of two smoking cessation programmes after AMI. METHODS: One hundred and ninety-eight current smokers admitted to coronary care with an AMI participated in a randomized controlled study comparing two outpatient tobacco interventions, the Stanford Heart Attack Staying Free (SF) programme and a Usual Care (UC) programme. RESULTS: Log-rank analyses revealed that patients in the SF programme were retained longer (P < 0.001) and had higher cotinine validated abstinence rates (P < 0.001) compared with patients in the UC programme. Twelve months after intervention, 39% of the SF programme compared with 2% of the UC programme demonstrated cotinine validated tobacco cessation, representing a significant reduced relapse rate in the SF programme (chi2, P< 0.001). CONCLUSIONS: The SF smoking cessation programme initiated in hospital can significantly reduce smoking rates at 12 months after myocardial infarction. Although superior to the UC quit programme, Australian outcomes were lower than the American programme originators' published outcomes.

Increased levels of immune inhibitory CD34+ progenitor cells in the peripheral blood of patients with node positive head and neck squamous cell carcinomas and the ability of these CD34+ cells to differentiate into immune stimulatory dendritic cells.

OBJECTIVES: This study determined whether mobilization of immune inhibitory CD34+ cells by head and neck squamous cell carcinomas (HNSCC) is most prominent in patients who are node positive and whether these CD34+ cells could differentiate into immune stimulatory dendritic cells. STUDY DESIGN AND SETTING: Peripheral blood from patients with head and neck cancer was used to measure the frequency of CD34+ cells and their capacity to differentiate into immune stimulatory dendritic cells. RESULTS: This study demonstrated that increased CD34+ cell levels were most prominent in patients who were node positive and patients with recurrent disease. These CD34+ cells differentiated into dendritic cells that were able to present tetanus toxoid to autologous T-cells. CONCLUSIONS: Immune suppressive CD34+ cells that are prominent in patients with HNSCC who are node positive are able to develop into immune stimulatory dendritic cells. SIGNIFICANCE: Differentiation of tumor-mobilized CD34+ cells into dendritic cells may be an immunotherapeutic approach to stimulate antitumor reactivity.

Impact of aging on immune modulation by tumor.

Tumor development and aging can each alter immune competence. The present study aimed to determine the impact of Lewis lung carcinoma (LLC) presence on immune parameters of middle-aged (averaging 6.5 months) versus aged (averaging 21.3 months) mice. An age-associated decline in the CD4+ cell frequency was seen in freshly isolated spleen and lymph node cells, as well as in cultures stimulated with immobilized anti-CD3. This decline was not further exacerbated by tumor presence. What was prominently inhibited by tumor was the capacity of either splenic or lymph node CD4+ cells to become stimulated to express IFN-gamma. Spleen and lymph node cultures from aged tumor-bearing mice had the lowest frequency of CD4+IFN-gamma+ cells and the least amount of secreted IFN-gamma. CD8+ cells were not affected by aging, but tumor presence reduced the induction of CD8+IFN-gamma+ cells in lymph node cultures. We previously showed that LLC growth stimulates myelopoiesis, as seen by splenomegaly and the mobilization of immune inhibitory CD34+ progenitor cells. Tumor presence in middle-aged mice reduced spleen cell blastogenesis, which was mediated by CD34+ cells. Aged mice had reduced blastogenesis, and this was further reduced by presence of tumor. However, neither the age-associated immune dysfunction nor the tumor-induced immune suppression in aged mice was due to CD34+ progenitor cells. These studies show how tumor presence can further compromise the immune dysfunction that accompanies aging. In addition, they show that aging impacts on the mechanisms by which tumors inhibit T-cell capabilities, with myelopoiesis-associated CD34+ cells mediating the immune depression of middle-aged tumor-bearers and an independent mechanism being responsible for the immune depression in aged tumor-bearing mice.

Duration of neuronal stretch correlates with functional loss.

Postoperative cranial nerve weakness or paralysis is not uncommon in many otolaryngologic surgical procedures. Our study used a rat model to test the hypothesis that the length of time that a nerve is under tension may be an important variable in the amount of postoperative paresis. Forty Sprague-Dawley rats were divided into 4 groups that underwent either a sham operation or a traction injury for 1, 2, or 5 minutes. The traction injury was performed with a vessel loop placed around the sciatic nerve with 50 g of tension. Traction injury for 1 or 2 minutes did not result in any statistical differences in the motor capabilities of the lower limb. However, those animals with a stretch injury for 5 minutes had a significant loss of function (P < 0.01) when compared with all other groups. Histologic examination of nerves harvested on postoperative day 7 showed no evidence of mechanical injury. This study demonstrates that even minimal tension, if maintained for a significant amount of time, may result in postoperative weakness.

Transformation nonresponsive cells owe their resistance to lack of p65/nuclear factor-kappaB activation.

Clonal variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to tumor promoter-induced neoplastic transformation exhibit differential activator protein-1 (AP-1) response. Transactivation of AP-1 appears to be necessary but not sufficient to promote transformation in JB6 cells. Inhibition of AP-1 is invariably accompanied by inhibition of nuclear factor-kappaB (NF-kappaB) when transformation is suppressed, suggesting that NF-kappaB may also play a role in neoplastic transformation. We report here that transactivation of NF-kappaB is inducible by tumor promoters in P+ but not in P- JB6 cells. Inhibition of NF-kappaB using a nondegradable mutant of IkappaBalpha suppressed inducible anchorage-independent transformation of P+ JB6 cells, suggesting that NF-kappaB activation is required for tumor promotion. Induced degradation of IkappaBalpha occurred in both P+ and P- JB6 cells, indicating that failure to activate NF-kappaB in P- JB6 cells cannot be attributed to failure to degrade IkappaBalpha. Slightly higher levels of nuclear p65 were seen in P+ than in P- JB6 cells. The p65-specific DNA binding activity was also higher in P+ cells upon induction by tumor necrosis factor-alpha, suggesting that differential NF-kappaB activation may be attributable to changes in p65 activity. Transactivation of p65 protein was substantially higher in P+ than in P- JB6 cells, as determined by assay of Gal4-p65 fusion constructs. Thus activated, p65 may be a limiting factor for NF-kappaB activation and transformation responses. Stable expression of p65 in P- JB6 cells conferred not only inducible NF-kappaB and AP-1 activation but also transformation response to tumor promoters. Therefore, p65/NF-kappaB appears to be not only necessary for but also sufficient to confer tumor promotion response. Although stable expression of p65 in P- cells produced p65 increases in whole cell extracts, only the transfectants exhibiting increased nuclear p65 showed transformation response. Thus, elevation of nuclear p65 appears to be a necessary step for a transformation response. The P-/p65 transfectants showing acquired transformation response also showed elevated p65-specific transactivation response, thus recapitulating the NF-kappaB phenotypes seen in P+ cells. Expression of a transactivation-deficient mutant of Jun or dominant-negative extracellular signal-regulated kinase suppressed both AP-1 activation and p65-specific transactivation in JB6 cells, suggesting that AP-1 activity is needed for p65 transactivation and consequently for NF-kappaB activation. Thus, the transformation nonresponsive P- JB6 cells owe their resistance to lack of NF-kappaB activation and p65 transactivation that appears in turn to be attributable to insufficient AP-1 activation.

Human squamous cell carcinomas of the head and neck chemoattract immune suppressive CD34(+) progenitor cells.

CD34(+) progenitor cells have previously been shown to be mobilized in patients with squamous cell carcinoma of the head and neck (HNSCC). The present study showed that these CD34(+) cells inhibit the capacity of intratumoral lymphoid cells to become activated in response to stimulation through the TCR/CD3 complex. The mechanisms that could lead to the accumulation of CD34(+) cells within the tumor tissue were assessed. This was accomplished through in vitro studies that determined if HNSCC produce soluble factors that chemoattract CD34(+) cells. The migration of cord blood CD34(+) cells, which were used as a readily available source of progenitor cells, was stimulated by products derived from HNSCC explants and primary HNSCC cultures. This stimulated migration was due to chemotaxis because it was dependent on an increasing gradient of HNSCC-derived products. CD34(+) cells that were isolated from the peripheral blood of HNSCC patients were similarly chemoattracted to the HNSCC-derived products. The majority of the chemotactic activity produced by HNSCC could be attributed to vascular endothelial cell growth factor (VEGF). These studies indicate that HNSCC can chemoattract immune inhibitory CD34(+) progenitor cells through their production of VEGF.

Induced expression of dominant-negative c-jun downregulates NFkappaB and AP-1 target genes and suppresses tumor phenotype in human keratinocytes.

Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor kappaB (NFkappaB) transcription factors. The present study addresses the question of whether elevated NFkappaB in addition to elevated AP-1-dependent gene expression is necessary for maintaining the tumor cell phenotype. When a tetracycline-regulatable dominant-negative c-jun (TAM67, having a truncated transactivation domain) was expressed in tumorigenic human keratinocytes, AP-1- and NFkappaB- but not p53-dependent reporter activity was inhibited by 40-60%. Tumor phenotype, as measured by anchorage-independent growth, was inhibited by 90%. Neither AP-1/NFkappaB activation nor expression of tumor phenotype was inhibited in TAM67-harboring keratinocytes under noninducing conditions. Electrophoretic mobility shift analysis showed that induction of TAM67 expression slightly increased AP-1- but reduced NFkappaB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFkappaB p65, suggesting that inhibition of NFkappaB by TAM67 is mediated by direct protein-protein interactions, possibly producing decreased binding to DNA or inactivating p65. To analyze the putative effector genes that may be targeted by TAM67, expression of genes responsive to AP-1 or NFkappaB was measured by reverse transcriptase-polymerase chain reaction in TAM67 transfectants with or without TAM67 induction. Induction of TAM67 inhibited or reduced the expression of collagenase I, stromelysin I (AP-1 responsive), and interleukins 1 and 6 (NFkappaB responsive). These results indicate that genes controlled by NFkappaB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFkappaB activation through direct interaction with NFkappaB p65. Moreover, the findings provide proof for the principle of using inducible TAM67 as a gene therapy to suppress tumor phenotype in human carcinoma cells.

CD34+ immune suppressive cells in the peripheral blood of patients with head and neck cancer.

Patients with head and neck squamous cell carcinoma (HNSCC) have profound defects in their immune defenses. Using immunofluorescent staining and flow cytometric analysis, we found that most patients with HNSCC have increased levels of CD34+ cells within their peripheral blood. These circulating CD34+ cells contribute to the depressed functional competence of the peripheral blood T-lymphocytes. This was demonstrated by the increased level of proliferative responsiveness to interleukin-2 by the patients' peripheral blood T-cells after depletion of CD34+ cells. These results show the importance of CD34+ cells in contributing to the depression of T-lymphocyte function in patients with HNSCC and suggest that strategies designed to reduce the levels of circulating CD34+ cells may enhance the immune reactivity of the patients' circulating T-lymphocytes against the HNSCC.

Activator protein 1 (AP-1)- and nuclear factor kappaB (NF-kappaB)-dependent transcriptional events in carcinogenesis.

Generation of reactive oxygen species (ROS) during metabolic conversion of molecular oxygen imposes a constant threat to aerobic organisms. Other than the cytotoxic effects, many ROS and oxidants are also potent tumor promoters linking oxidative stress to carcinogenesis. Clonal variants of mouse epidermal JB6 cells originally identified for their differential susceptibility to tumor promoters also show differential reduction-oxidation (redox) responses providing a unique model to study oxidative events in tumor promotion. AP-1 and NF-kappaB, inducible by tumor promoters or oxidative stimuli, show differential protein levels or activation in response to tumor promoters in JB6 cells. We further demonstrated that AP-1 and NF-kappaB are both required for maintaining the transformed phenotypes where inhibition of either activity suppresses transformation response in JB6 cells as well as human keratinocytes and transgenic mouse. NF-kappaB proteins or extracellular signal-regulated kinase (ERK) but not AP-1 proteins are shown to be sufficient for conversion from transformation-resistant to transformation-susceptible phenotype. Insofar as oxidative events regulate AP-1 and NF-kappaB transactivation, these oxidative events can be important molecular targets for cancer prevention.

Interrelationship between protein phosphatase-2A and cytoskeletal architecture during the endothelial cell response to soluble products produced by human head and neck cancer.

Tumor neovascularization is necessary for the progressive development of all solid tumors, including head and neck squamous cell carcinomas (HNSCCs). The angiogenic process includes increased endothelial cell motility. Our prior studies have shown the importance of protein phosphatase-2A (PP-2A) in restricting endothelial cell motility. Because motility is regulated by the polymerization/depolymerization of the cellular cytoskeleton, the present study defined the interrelationship between PP-2A and the cytoskeleton during endothelial cell responses to HNSCC-derived angiogenic factors. PP-2A was shown to colocalize with microtubules of unstimulated endothelial cells. However, exposure to HNSCC-derived products resulted in a more diffuse distribution of PP-2A staining and a loss of filamentous tubulin. The feasibility of pharmacologically preventing this cytoskeletal disorganization as a means of blocking tumor-induced angiogenesis was tested. This was accomplished by use of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and all-trans -retinoic acid to indirectly stimulate PP-2A activity through their capacity to elevated intracellular levels of the second messenger ceramide. Pretreatment of endothelial cells with either 1,25(OH)(2)D(3) or retinoic acid prevented the cytoskeletal disorganization that otherwise occurs in endothelial cells on exposure to HNSCC-derived products. These studies support the feasibility of using elevation of PP-2A to prevent the morphogenic component of the angiogenic process that is stimulated by HNSCC-derived factors.

In vivo indomethacin treatment causes microglial activation in adult mice.

The current study was undertaken to study the role of prostaglandins in regulating microglial activation. Mice were treated with indomethacin (2 microg/ml) in their drinking water to selectively inhibit cyclooxygenase activity. After 4-8 days, the effect of inhibiting prostaglandin synthesis on microglial activity was evaluated. This was accomplished by analyzing microglial expression of Mac-1 (C3 complement receptor) as an indicator of activation. Mac-1 expression was assessed by immunohistochemistry of fixed brain cryosections, and by flow cytometric analysis of immunostained single cell suspensions. Both methods demonstrated that compared to age-matched, untreated controls, brains of indomethacin-treated mice had increased levels of Mac-1 expression, suggesting an increase in the state of microglial activation. These results demonstrate the importance of prostaglandins in down regulating microglial activity, and that inhibition of prostaglandin synthesis with indomethacin may act to increase the reactivity of the brain's immune system.