The effect of fetal calf serum on intracellular calcium in GH3 cells

We have investigated the mechanism of action of fetal calf serum (FCS) on GH3 pituitary tumour cells by measuring intracellular free calcium levels. On the addition of FCS (1%) there was a transient increase in intracellular Ca2+ levels which was attenuated in conditions of reduced extracellular calcium concentrations. The Ca2+ response was abolished by the prior addition of lanthanum chloride (1mM). In contrast, the elevation of cytosolic calcium levels by TRH (100nM), an agonist which causes the mobilisation of calcium from the endoplasmic reticulum, was attenuated but not abolished by lanthanum chloride (1mM). We suggest that FCS (1%) causes the release of calcium from the plasma membrane and the influx of calcium from the extracellular milieu, but does not mobilise calcium from the endoplasmic reticulum.     

Effect of fetal calf serum on the cadmium clastogenicity

Inconsistent results among reports on cadmium genotoxicity revealed that certain confounding factors might significantly influence the outcomes of assessment. In Chinese hamster ovary (CHO-W8) cells, chromosome aberration induced by six different cadmium compounds was found positively associated with intracellular cadmium concentration. A parallel association was also observed among different CHO strains treated with same cadmium compound, the cadmium acetate. Both the cadmium-induced chromosome aberration and cadmium uptake were influenced by the presence of fetal calf serum (FCS). The presence of 10% FCS during the 2 h treatment period greatly retarded the cellular cadmium uptake, and concurrently reduced the chromosome aberration induction. Other factors such as specific cadmium anion involved and the duration of cadmium treatment period in the investigation also influenced the assessment results of cadmium-induced chromosome aberration. In the protocol with a 2 h pulse treatment, cadmium acetate, chloride and sulfate induced more chromosome aberration than cadmium nitrate, carbonate and oxide. When cadmium was present in the culture of the entire treatment period for 18 h, the results went the opposite way. Cadmium nitrate, carbonate and oxide induced significant chromosome aberration, while other three cadmium compounds gave negative results. Cadmium compounds did not induce significant SCE at the same dose level that yielded significant chromosome aberration induction, either in the protocol with the short pulse or long treatment period.     

In vitro tumoricidal activity of peritoneal macrophages and splenic lymphocytes of rats under diet with or without fish

Rats were fed with or without a diet supplemented with fish meal over a 4-week period. The moderate fish fatty acids intake had no influence on the tumoricidal activity of peritoneal and splenic lymphocytes.     

In vivo and in vitro effect of trehalose dimycolate on tumoricidal activity of peritoneal macrophages from rats

Trehalose dimycolate (TDM), a well-defined component of Mycobacterium tuberculosis cell wall has been tested in vivo and in vitro for its effect on the tumoricidal activity of Rat peritoneal macrophages. Macrophages could be rendered cytolytic against syngeneic tumor cells by an intraperitoneal injection of an aqueous suspension of TDM. However, as we failed to render them tumoricidal in vitro, we consider that the activation process is not due to a direct effect on macrophages.     

Differences in the tumoricidal activation of rat and mouse macrophages by endotoxins

Conventional and specific pathogen-free rat resident peritoneal macrophages were lytic to tumor cells in the presence of endotoxins even when not elicited or not stimulated in vivo or in vitro. In contrast, conventional mouse resident peritoneal macrophages were not cytolytic in the presence of endotoxins. The induction by endotoxins of rat macrophage-mediated cytolysis was only obtained after the binding of tumor cells by macrophages. Rat resident peritoneal macrophages bound faster and stronger to tumor cells than mouse resident peritoneal macrophages. These differences in binding could explain the species differences in the tumoricidal response to endotoxins.     

T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.     

Separation of soluble amoebicidal and tumoricidal activity of activated macrophages.

Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.     

Structural differences among guinea pig Fc gamma 1/gamma 2 receptors on macrophages, polymorphonuclear cells, and lymphocytes.

Using the cDNA, D-3, coding for Fc gamma 1/gamma 2 receptor of guinea pig macrophages that binds IgG1 and IgG2 (Fc gamma 1/gamma 2R), we examined the cell distribution of this receptor by RNA blot analysis. The Fc gamma 1/gamma 2R mRNA was expressed in polymorphonuclear cells and B cells as well as in macrophages, but not at the detectable level in T cells. The cDNA amplified from RNA of polymorphonuclear cells in the polymerase chain reaction was the same as D-3. The cDNA of B cells was found to have about 140 bp cDNA segment inserted to the cytoplasmic tail of D-3. We found that the cDNA amplified from T cell RNA differed in signal peptide and extracellular domain sequence from cDNAs of other cell types. This cDNA does not seem to be amplified from the mRNAs of contaminating other cell types.     

The ability of fetal calf serum, new-born calf serum and normal steer serum to promote the in vitro development of bovine morulae

The objective of this study was to compare fetal calf serum, new-born calf serum and normal steer serum as medium supplements in the development of bovine morulae in vitro . Bovine morulae were cultured in Hams F-10 tissue culture medium (HF-10) supplemented with 5% or 10% (v/v) fetal calf serum (FCS), new-born calf serum (NBCS) or normal steer serum (NSS). Embryos were recovered at slaughter from mixed bred donor cows of mixed breeding following estrus synchronization with prostaglandin and superovulation with follicle stimulating hormone. A total of 88 morulae were recovered, washed in HF-10 + 1% Bovine Serum Albumin and randomly assigned to treatments. Embryos were cultured in microdrops of medium under paraffin oil at 37 degrees C in a 5% CO(2) humidified atmosphere. Observations for stage of development were made every 24 hours. In vitro development was analyzed by assigning to each embryo a value of 0-5 based on the most advanced stage reached (0= no development, 5= development to a hatched blastocyst). Analysis of variance of these data revealed a significant treatment effect (P<.001) while no level effect or treatment x level interaction was apparent. Comparison of treatment means by Duncans new mulitple range test showed that NSS was superior to NBCS (P<.05) which was in turn superior to FCS (P<.05) as supplements of HF-10 in promoting the in vitro development of bovine morulae.     

The synergistic tumoricidal activity of anticancer drugs and oxidative burst-triggered macrophages

Summary The anticancer drugs adriamycin (ADR) and actinomycin D (AMD) were tested for their effect on the oxidative burst (OB) of mouse peritoneal macrophages (MPM) and on the killing of tumor cells by OB-stimulated MPM. The oxidative burst of MPM determined by hydrogen peroxide (H₂O₂) production was severely impaired by ADR (10 g/ml) and AMD (40 g/ml) after a 1 h treatment and by lower concentrations of the drugs following a 24 h treatment. The toxicity of the drugs against MPM was comparable to their effect on EL4 cells. Pretreatment of EL4 and TLX-9 tumor cells with sublethal amounts of ADR for 4 h rendered the cells sensitive to the cytotoxic effect of OB-stimulated MPM which were otherwise unable to kill these cells. It seems that anticancer drugs and OB-stimulated macrophages can cooperate in the destruction of tumor cells in vitro.     

A novel muramyl peptide derivative stimulates tumoricidal activity of macrophages and antibody production by B cells

A novel analog of MDP, the 3'-iodo-4'-azido-L-phenylalanine methyl ester derivative of N-acetyl-L-alanyl-D-isoglutamine, has been prepared. This compound is capable of activating macrophages to the tumoricidal state and increasing the specific immune response of B cells. It thus appears to exhibit similar biological activities to MDP. Moreover, this compound is of potential interest for receptor photolabelling studies.     

Prostaglandin E2 does not inhibit tumoricidal activity of mouse macrophages against adherent tumor cells

We determined whether endogenously produced PGE2 can down-regulate the tumoricidal properties of macrophages by a negative feedback mechanism. Peritoneal exudate macrophages or resident peritoneal macrophages of mice were incubated in medium (control) or in medium containing IFN-gamma and LPS. Activated macrophages were highly tumoricidal against syngeneic melanoma cells and secreted high levels of PGE2. Treatment with indomethacin or diclofenac sodium (voltaren) completely inhibited the production and secretion of PGE2 but not the tumoricidal activity of activated macrophages measured either immediately after activation or 1 to 3 days thereafter. Finally, the addition of exogenous PGE2 did not alter the ability of peritoneal exudate macrophages to respond to IFN-gamma or of LPS to produce high levels of tumor cell lysis. Collectively, these results show that PGE2 produced by activated macrophages is not a down-regulator of their tumoricidal activity against adherent tumor cells.     

2',5'-Oligoadenylate trimer core and the cordycepin analog augment the tumoricidal activity of human natural killer cells

Interferon (IFN) augments the lytic activity of natural killer (NK) cells, inhibits the transformation of human peripheral blood lymphocytes (PBL) by Epstein Barr virus (EBV), and induces a 2',5'-oligoadenylate (2',5'-An) synthetase. Exogenous 2',5'-An by itself can inhibit the transformation of human PBL by EBV. The present studies report that 2',5'-An and its cordycepin analog also augmented the tumoricidal activity of human NK cells. Incubation of nylon wool-passed PBL for 1 to 2 hr with the 5'-dephosphorylated core trimer of 2',5'-An boosted natural killing of tumor target cells modestly, but consistently. The cordycepin analog (3'-deoxyadenylate) also augmented NK activity. The optimal concentration both of 2',5'-A3 core and of 2',5'-3'dA3 core was 50 microM, and the optimal time for this effect was 2 hr of treatment. Kinetic analysis revealed that 2',5'-A3 core increased the lytic rate of NK cells by about one-third. This increase was due to an even greater increase (about 50%) in the lytic activity of individual NK cells, coupled with a slight decrease in the number of actual NK effector cells. In contrast, 3',5'-A3 core did not increase NK activity even at 300 microM, at which point it was toxic. In addition, to rule out a pro-drug effect as the basis for the boosting of NK activity by 2',5'-A3 core and by 2',5'-3'dA3 core, the effect of adenosine and cordycepin monomers on NK activity was tested. Neither adenosine nor cordycepin, tested at 150 microM (three times the optimal concentration of the trimer cores), boosted NK activity. The addition of 2'-deoxycoformycin (2 microM) had no effect on the actions of adenosine and cordycepin monomers. The data presented here demonstrate that 2',5'-A3 core and its analog 2',5'-3'dA3 core have another IFN-like action, augmentation of NK activity, in addition to inhibiting EBV-induced transformation.     

Protective effect of D-glucose,L-leucine and fetal calf serum against oxidative stress in neonatal pancreatic islets

B-cell destruction during the onset of diabetes mellitus is associated with oxidative stress. In this work, we investigated the mechanisms of defense against oxidative stress present in neonatal islets and their modulation by D-glucose, L-leucine and fetal calf serum (FCS). Culturing neonatal rat islets in the presence of low D-glucose concentrations (2.8-5.6 mmol/l) and 1 mmol/l H(2)O(2) increased the D-glucose uptake by islets sixfold compared to control levels. This effect was dose-dependently inhibited by D-glucose or FCS and by high concentrations of L-leucine. These supplements allowed islets to increase cytoplasmic catalase (CAT) activity only in response to H(2)O(2), with no decrease in NO formation. Although L-leucine increased CAT activity and restored D-glucose uptake, it did not prevent damage to the islets. These data indicate that the most important H(2)O(2) scavenger system in the islets is CAT and that this system can be modulated by metabolic substrates.     

The effect of bovine serum albumin and fetal calf serum on sperm quality,DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen

The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72 h at 5 °C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37 °C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40 × 10⁶ sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5 mg/ml), FCS (10%) or no additive (control) at 37 °C, cooled to 5 °C and stored for up to 72 h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48 h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72 h storage period, compared to the control (P < 0.01). The supplementation of BSA and FCS had a protective effect on motility (P < 0.05), plasma membrane integrity (P < 0.01) and acrosomal integrity (P < 0.01) at 72 h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72 h during storage period, compared to the control (P < 0.001). Although supplementation of BSA and FCS caused significant (P < 0.01) decreases in malondialdehyde (MDA) level at 48 h and 72 h, they significantly (P < 0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72 h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties.