Isolation of rainbow trout neutrophils with an anti-granulocyte monoclonal antibody.

A magnetic cell sorting system has been optimised for the purification of rainbow trout neutrophils using a monoclonal antibody (E3D9) raised against Atlantic salmon neutrophils. The purified neutrophils have good viability (85%) and purity (approximately 92%), and were functional in respiratory burst and migration assays. The isolated neutrophils responded rapidly to PMA stimulation, producing levels of superoxide anion (4.85 nmols superoxide min-1/10(6) cells) approximately twice as high as macrophages from the same species. In the migration assay, there was a four-fold increase in migrating cells using the purified neutrophils compared with unfractionated blood leucocytes, and a relatively high neutrophil migratory activity was seen in the absence of serum.

Interleukin 2 receptor expression and interleukin 2 localisation in human solid tumor cells in situ and in vitro: evidence for a direct role in the regulation of tumour cell proliferation.

Frozen sections of 52 human solid tumours (38 malignant and 14 benign) of varied histogenesis were immunohistochemically stained with well characterised monoclonal antibodies (MAbs) to human interleukin 2 (IL-2) and the alpha and beta chains of its receptor (R). In all malignant specimens, the tumour cells expressed the IL-2R beta subunit (p75) but not the IL-2R alpha subunit (CD25). In 36 of 38 malignant tumours examined, there was conspicuous staining for IL-2 in the tumour cell nuclei/nucleoli and perinuclear cytoplasm. In the human solid tumour cell lines G361 (melanoma), A549 (lung), MCF-7 (breast) and WiDR (colorectal), both subunits of the IL-2R appeared to be expressed, although the alpha subunit only weakly. Exogenous addition of human recombinant (r) interleukin 2 altered cell numbers in 3 of the 4 cell lines (WiDR was refractory). When grown in the absence of exogenously added rIL-2, IL-2 staining was observed in all cell lines. The pattern of distribution was similar to that exhibited by the tumour cells in situ (i.e., a nuclear/nucleolar localisation). In G361 melanoma cells, this IL-2 staining was present in proliferating cells but disappeared as the cultures approached confluence. Addition of an IL-2R beta subunit blocking antibody to growing G361 cultures (grown in the absence of rIL-2) resulted in a significant reduction in cell numbers. We propose, therefore, that the presence of immunoreactive IL-2 and IL-2R expression is characteristic of human malignant cells and that IL-2 may play a role in the autocrine stimulation of proliferation of malignant cells, such as G361 melanoma cells.

Modulation of the cytokine and acute-phase response to major surgery by recombinant interleukin-2.

Major surgery suppresses host immune reactivity through alterations in monocyte and T cell-derived cytokine, eicosanoid and acute-phase protein release. Recombinant interleukin (IL) 2 augments T lymphocyte and monocyte activity in vitro. Eighteen patients, with localized colorectal cancer, were randomized to receive either recombinant IL-2 or placebo for 3 days by subcutaneous injection before surgery. Serum levels of IL-1 beta, IL-6, tumour necrosis factor alpha, soluble IL-2 receptor, C-reactive protein (CRP) and albumin were measured, and T lymphocyte surface expression of HLA-DR and CD25 and neutrophil phagocytosis were determined, before and for 21 days after surgery. Significant augmentation of IL-6, CRP and soluble IL-2 receptor production, enhanced expression of activation markers and increased neutrophil activity were found. Recombinant IL-2 may have a role in ameliorating the immunosuppression found after major surgery.

Calcitonin gene related peptide stimulates differentiation of neonatal rat myogenic cultures.

The effect of the nerve derived peptide, calcitonin gene related peptide (CGRP) on myoblast fusion/differentiation was studied in rat myogenic cell cultures, using creatine kinase activity as an index of fusion. Addition of CGRP at 10(-7) M to myoblast cultures resulted in an enhancement of kinase activity after 11 days of treatment. This response was not accompanied by increased protein content of cultures implying an effect primarily on fusion rather than on myotube growth/size. Neither was it a direct effect on enzyme activity alone since no increase in creatine kinase activity occurred when CGRP was added to mature myotube cultures. These data suggest a role for CGRP in the myoblast fusion process itself and raise questions as to its importance in the growth and development of muscle in vivo.

Tissue specific responses to clenbuterol; temporal changes in protein metabolism of striated muscle and visceral tissues from rats.

The beta-adrenoceptor agonist, clenbuterol, was administered orally to male weanling rats for a period of up to 10 days. The composition and fractional rate of protein synthesis (Ks) in skeletal and cardiac muscle, gut and liver were determined. There were few changes in the visceral tissues, but there was marked protein accretion in the muscles. The results suggested that in skeletal muscles there is an increase in both Ks and the amount of protein synthesised per unit RNA. In cardiac muscle, the results indicated that there was only a very transient increase in Ks and that changes in translational capacity (RNA/prot) may account in part for the increase in protein content. It is concluded that the mechanistic basis for the increased protein gain may be different between skeletal and cardiac muscles.

The effect of the beta-adrenergic agonist clenbuterol on growth and protein metabolism in rat muscle cell cultures.

Cultures were established from neonatal rat muscle cells, satellite cells, and L6 myoblasts and changes in protein metabolism were determined as development proceeded. For all three cell types, culture protein content increased with increasing myotube content. The beta-adrenergic agonist clenbuterol (added to a final concentration of 10(-7) M) significantly stimulated fusion (as indicated by creatine kinase activity) in neonatal muscle cultures and also increased culture protein content. This was associated with a stimulation in both the fractional (ks, percentage/day, +13%, P less than .05) and absolute (As, micrograms/day, +19%, P less than .05) rates of protein synthesis within 24 h after drug administration. At 48 h, As was increased by 42% above that of controls (P less than .01). In contrast, in satellite cell cultures, clenbuterol had no consistent effects on either protein accretion, creatine kinase activity, or protein synthesis (ks and As). Similarly, the drug had no stimulatory effect on protein synthesis and protein accretion in L6 myoblast or L6 myotube cultures (and no effect in neonatally derived fibroblast cultures). It is concluded that the fusion response to clenbuterol and, therefore, changes in protein metabolism and protein accretion are greatly dependent on the origin and genetic integrity of muscle cells.

Protein synthesis in trout liver is stimulated by both feeding and fasting.

The response of protein synthesis in the liver of the rainbow trout to feeding and fasting was investigated in 3 experiments. In the first experiment, the fractional rate of protein synthesis (k s ), %/day) appeared to cycle with daily feeding events being increased by 46%, 123%, and 72% at 1.5h, 3h, and 6h, after a meal. In Experiment 2, liver protein synthesis fell progressively with fasting to a basal level at 4d which was only 20% of the value at 3h after feeding. Liver weight (hepatosomatic index, HSI, % body weight), total RNA and total protein also fell gradually. Between 4d and 6d, both the RNA/protein ratio and the rate of protein synthesis were significantly increased (11% and 74%). At this time, however, there was also a large loss of liver protein suggesting a concomitant increase in protein breakdown. In the last experiment, when trout were pre-fed a low ration (0.6%/d for 2 wks, LR group), the HSI and liver total RNA and protein were largely unaffected by the 6d fast (i.e., relative to the body weight). In this group, however, protein synthesis at 3h was significantly higher than in fish pre-fed a high ration (1.5%/d, HR group). In addition, at 6d after feeding, protein synthesis had increased back to fed levels in the LR group only. It is concluded that protein synthesis in the liver of the trout is influenced both by feeding events and by ration size and also by the degree to which the trout is fasted.

Effects of the cyclo-oxygenase inhibitor, fenbufen, on clenbuterol-induced hypertrophy of cardiac and skeletal muscle of rats.

1. When rats were fed with clenbuterol for 7 days skeletal muscle mass increased by 21% in the tonic soleus and phasic plantaris muscles and a 16% hypertrophy of the heart was also induced. Fenbufen, fed to rats for the same period, blocked the hypertrophy of the heart but not that of the skeletal muscles. 2. When feeding of fenbufen commenced 3 days before the administration of clenbuterol, plasma prosta-glandin F2 alpha (PGF2 alpha) was reduced by 79%; there was again no effect of fenbufen on clenbuterol-induced increases in the RNA or protein content of plantaris, nor in the increased area of fast or slow twitch fibres in the soleus. In the heart the clenbuterol-induced increases in the RNA (+21%) and protein content (+20%) were totally inhibited. 3. The effects of clenbuterol on heart muscle appear to be mediated by a cyclo-oxygenase metabolite of arachidonic acid whilst the effects on skeletal muscle are not.

Changes in protein turnover in hypertrophying plantaris muscles of rats: effect of fenbufen--an inhibitor of prostaglandin synthesis.

Plantaris muscle of the right hind limb of rats was subjected to hypertrophic stimulus by section of the tendons of the right gastrocnemius muscle. The RNA and protein content and the fractional rate of protein synthesis were elevated both 3 and 7 days after operation compared both with the unoperated left limb and with sham-operated control rats. The rate of protein degradation, calculated from the difference between the fractional rates of protein synthesis and protein gain of the muscles, was elevated in the plantaris 3-7 days after tenotomy. Dietary administration of the drug fenbufen reduced the RNA content and the ratio of RNA:protein in muscles from control animals. In one group of tenotomised rats administration of fenbufen commenced 3 days before tenotomy and resulted in a reduction in the ratio RNA:protein of the muscles of the left limb 3 days after the operation. Four days later, i.e. 7 days after tenotomy, both the ratio RNA:protein and the fractional rate of protein synthesis were significantly reduced in the fenbufen treated rats. In spite of these effects, fenbufen did not impair the ability of the plantaris to hypertrophy since the drug also reduced the rate of protein degradation.

Protein synthesis in skeletal muscle measured at different times during a 24 hour period.

Six groups of 5 male rats (starting body weight 109 g) were allowed free access to a conventional rat diet. At 4 hourly intervals, starting at 10.00 h muscle protein synthesis was measured. By relating the weights of the gastrocnemius and soleus muscles to the initial body weights of the animals (i.e., at 09.30, day 1), a linear increase in muscle weight throughout the day was demonstrated. The fractional rate of muscle protein synthesis varied from 16.8% per day to 20.3% per day in gastrocnemius muscle and from 17.9% per day and 22.1% per day in the soleus. It was calculated that the maximum error incurred in estimating daily muscle protein synthesis by extrapolation of the value at any one time was 6% in gastrocnemius and 9% in soleus. It is concluded that calculations of the average rate of muscle protein degradation based on the difference between the rates of synthesis and deposition are generally valid in rats allowed free access to an adequate diet.