The binding and processing of monoclonal human IgG1 by cells of a human macrophage-like cell line (U937)

The goal of these experiments was to assess the relationship between the binding and processing of IgG by Fc-receptor-bearing cells. Cells of the U937 human macrophage-like cell line were incubated with 125I- labeled monomers, dimers, oligomers (composed of 2-4 IgG1 subunits), and HP (heavy polymers composed of 5 or more subunits per polymer) of monoclonal human IgG1 in vitro. Binding was assessed by spinning cells through a layer of phthalate oils. Internalization of IgG1 was assessed by quantitating residual binding to cells after surface-bound IgG was removed by a brief treatment with a solution containing 0.25 M acetic acid and 0.5 M sodium chloride. Catabolism was assessed by measuring the release of radioactive fragments of IgG1, which were not precipitated by 10% trichloroacetic acid. Unstimulated U937 bound about 10,000 molecules per cell of IgG1 monomer, with an equilibrium binding constant (Ka) of 5 X 10(8) M-1. After stimulation with a conditioned medium in vitro, binding per cell was increased 3-7--fold, and the Ka was decreased 2-4--fold. Both unstimulated and stimulated cells internalized and catabolized labeled IgG1 HP, but stimulated cells internalized and digested much more IgG1 HP per cell than unstimulated cells. Both monomers and dimers of IgG1 were internalized and degraded very slowly by stimulated cells, even though both preparations readily bound to cells. In contrast, oligomers and (to an even greater extent) IgG1 HP were internalized and degraded much more rapidly. Internalization of IgG1 HP was markedly inhibited by incubation at 4 degrees C, but not by incubation with a variety of metabolic inhibitors. Catabolism was inhibited by chloroquine and monensin (inhibitors of lysosomal acidification) and by cytochalasin (an inhibitor of microfilament polymerization). Binding to the surface of cells was not markedly inhibited by any agent tested. The capacity of cells to bind labeled IgG1 was markedly reduced by prior incubation in the presence of unlabeled IgG1. This reduction was in part due to the steric blockade of receptors caused by the avid, but reversible, binding of IgG1. In addition, IgG1 oligomers or HP (but not IgG1 monomers or dimers) also caused an irreversible reduction in the number of Fc receptors by a process analogous to receptor down-regulation, as observed in other receptor--ligand systems.     

Possibility of selection against mtDNA mutations in tumors

Several studies of tumors have revealed substantial numbers of clonally expanded somatic mutations in mitochondrial DNA (mtDNA), not observed in adjacent intact tissues. These findings were interpreted as indicating the involvement of mtDNA mutations in tumorigenesis. Such comparisons, however, ignore an important confounding factor: the monoclonal origin of tumors as opposed to the highly polyclonal nature of normal tissues. Analysis of recently published data on the incidence of somatic mutations in nontumor monoclonal cells suggests that, contrary to the prevailing view, the process of tumorigenesis may be accompanied by active selection against detrimental mtDNA mutations.     

Secondhand smoke exposure and respiratory symptoms among casino, club, and office workers in Victoria, Australia

OBJECTIVE: To examine the association between smoke-free policies, exposure to secondhand smoke (SHS) at work, and self-reported respiratory and sensory symptoms of workers. METHOD: Ninety-one nonsmoking workers recruited from three workplaces with varying smoking policies completed a telephone-administered questionnaire and provided saliva samples (before and after usual work shift) for cotinine analysis. RESULTS: Mean before-after shift saliva cotinine per hour worked was significantly higher among club (0.42 ng/mL/hr worked) than casino workers (0.18 ng/mL/hr worked) (P < 0.001), club than office workers (0.03 ng/mL/hr worked) (P < 0.001), and casino than office workers (P < 0.001). Casino and club workers reported similar levels of respiratory morbidity and were more likely to have sore eyes (odds ratio [OR] = 5.5, P < 0.01) and a sore throat (OR = 4.3, P < 0.05) compared with office employees. CONCLUSION: Air-conditioning interventions reduce, but fail to eliminate, exposure of hospitality workers to SHS. Such exposure is associated with measurable increases in the risk of respiratory symptoms.     

Indications for the involvement of a CYP3A-like iso-enzyme in the metabolism of chlorobornane (Toxaphene) congeners in seals from inhibition studies with liver microsomes

The different isoforms of the cytochrome P450 (CYP) system can metabolise a suite of classes of lipophilic, anthropogenic compounds. The bioaccumulative potential as well as the toxicity of xenobiotics may be significantly altered in the process. To compare the metabolic ability of different wildlife species, it is important to identify the different iso-enzymes of CYP, which are responsible for the metabolism of different classes of compounds. This can be achieved with in vitro incubation assays. In the present study, preparations of hepatic microsomes of a harbour seal (Phoca vitulina) and a grey seal (Halichoerus grypus) demonstrated that the chlorobornane (CHB) congeners CHB-32 and -62 were metabolised enzymatically to their hydroxylated derivatives. These derivatives were partially characterised by their NCI mass-spectra. Inhibition studies were carried out to identify the specific CYP isoform(s) responsible for the metabolism of CHB-32 and -62. Ketoconazole has been shown to inhibit CYP3A enzymes in human and rat studies. In this study, ketoconazole caused concentration-dependent inhibition of metabolism of CHB-32 and -62, reaching 80% at the 1.0 microM treatment level. Ellipticine (1.0 microM), which has been shown to inhibit CYP1A1/2, also inhibited CHB-32 and -62 metabolism in the microsomes of grey seal, but to a much lower degree of less than 10 and 24%, respectively. In the same experiment the metabolism of 4,4'-dichlorobiphenyl was already inhibited 70% by ellipticine treatment at the same concentration. This non-ortho substituted PCB congener can easily attain a planar molecular configuration, and therefore served as a model CYP1A substrate. Inhibition of chlorobornane metabolism was not observed after the addition of goat anti-rat CYP2B antibodies or Aldrin, which is a model CYP2B substrate in rat. Cautious interpretation is advised for results obtained with so-called selective competitive inhibitors. Regardless, these studies indicated for the first time the possible involvement a CYP3A isoform in the mediation of chlorobornane metabolism in seals. The immunochemical cross-reactivity of mouse, rabbit or sheep anti-rat antibodies in the hepatic microsomes of harbour seal confirmed the presence of CYP1A1/2, CYP1A1, CYP2B1/2, CYP3A and CYP4A isoenzymes. Enantioselective metabolism by the microsomes of harbour seal was observed for both CHB-32 and -62. Stereochemical preferences of biotransformation enzymes can have an influence on the environmental distribution of both enantiomers of optically active compounds.     

RENAL FUNCTION IN CHRONICALLY CATHETERIZED CONSCIOUS DIABETIC RATS USING CONSTANT AND SERVO-CONTROLLED INFUSION

1. Infusion experiments were performed on chronically catheterized conscious rats to assess kidney function before and after the induction of diabetes mellitus with streptozotocin. 2. Two infusion regimens were used, a conventional constant-infusion protocol and a novel computer-driven, servo-controlled fluid replacement technique. The latter enables body fluid status to be maintained throughout a study occasion by servo-controlled replacement of spontaneous urinary fluid losses. 3. The chronically catheterized conscious rat infused using a servo-controlled system appears to be the optimum model for a study of diabetic renal function. The conscious preparation circumvents problems associated with anaesthesia and acute surgery. The servo-controlled infusion protocol maintains the altered fluid status of the diabetic condition. Both hyperfiltration and polyuria, characteristics of human diabetes often absent in anaesthetized and/or constantly infused diabetic rats, were seen in all conscious servo-controlled diabetic animals. 4. The new regimen enables a more accurate assessment of renal function in experimental diabetes than with previous protocols. It should prove useful in future studies, particularly those assessing the role of anti-diabetic drugs on the kidney.     

Unexplained diarrhoea and failure to thrive in 2 siblings with unusual facies and abnormal scalp hair shafts: a new syndrome

A family is described in which 2 siblings born to healthy parents presented with abnormal facies, persistent diarrhoea, and early death. Exhaustive pathological and biochemical investigations failed to find a cause. The scalp hair of both babies had an abnormal amino-acid composition, and presented an appearance that was unique on scanning electron microscopical examination; this fact and the clinical picture probably represents a new syndrome.