Effects of activated complement components on enzyme secretion by macrophages

Purified cleavage products of the guinea-pig complement component C3, namely C3b and C3a, interact with guinea-pig and mouse macrophages in culture to induce a dose- and time dependent release of lysosmal enzymes into the medium. In the case of C3b the selectivity of the release of hydrolases, which occurs without cell killing, is shown by morphological observations and the failure of lactate dehydrogenase to appear in the medium. However, lysosomal enzyme release in the presence of C3a is accompanied by loss of cellular lactate dehydrogenase. Preincubation of C3b with anti-C3 Fab inhibits its attachment to macrophages, after which there is hardly detectable enzyme release into the medium. We have found that stimulated macrophages release enzyme(s) which can cleave C3, generating more C3b either directly or via the alternative pathway; the C3b so formed would induce further enzyme release. This amplification system may provide an explanation for the ability of macrophages to generate mediators of inflammation and cause tissue damage and degradation at sites of chronic inflammation while retaining their ability for long periods of time.     

Effects of altitude and latitude on ambient UVB radiation

PURPOSE: Spitting as an ictal automatism has been rarely reported. We aimed to establish its potential lateralizing and localizing significance. METHODS: Review of patients undergoing surgery for intractable epilepsy at two comprehensive epilepsy centers. RESULTS: Five patients were found who had spitting as a stereotyped automatism of their complex partial seizures. All had evidence of right temporal ictal onset and underwent resective surgery. Two had tumors; one, a cavernous angioma; one, hippocampal gliosis, and one, hippocampal sclerosis. We found no instances of ictal spitting in patients with left hemisphere onset. CONCLUSIONS: Spitting as an automatism in complex partial seizures, although uncommon, may be a localizing sign to the nondominant temporal lobe.     

Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers

Epidemiology shows a relationship between solar exposure and all types of skin cancer. Understanding the mechanisms of skin cancer requires knowledge of the photomolecular events that occur within the relevant epidermal cell types in vivo. Studies to date have focused on UVR-induced DNA lesions in keratinocytes, the majority epidermal cell population which gives rise to most skin cancers. Malignant melanoma, arising from melanocytes (5%-10% of epidermal cells), accounts for most skin cancer deaths. We report on new techniques to detect DNA photolesions in human epidermal melanocytes in situ. Previously nonexposed buttock skin of volunteers of skin types I/II was exposed to clinically relevant doses of narrow bandwidth UVB (300 nm) and UVA (320 nm, 340 nm, 360 nm) radiation. Biopsies were taken immediately afterwards and processed for routine histology. Microscope sections were prepared and double-stained with fluorescent-tagged monoclonal antibodies for thymine dimers and melanocytes. UVR dose-response curves for dimer levels within melanocyte nuclei were determined by image analysis and compared with dimer levels in adjacent basal cell keratinocytes. Our data show that UVB and UVA readily induce thymine dimers in melanocytes at levels that are comparable with those found in adjacent keratinocytes. This new technique will enable melanocyte specific studies, such as DNA repair kinetics, to be done in vivo.     

Identification of complement receptor type 1-related proteins on primate erythrocytes

The purpose of this study was to characterize the structure and function of the immune adherence receptor (CR1, CD35, C3b/C4b receptor) of primates. Western blotting, immunoprecipitation, ELISA, and affinity chromatography with homologous C3b and C4b were utilized. The major cross-reactive E membrane protein of ten species of primates tested was lower in m.w. than was human CR1 and fell into two size groups of 55 to 75 and 130 to 165 kDa. There was 10- to 100-fold more CR1 per primate E than human E. Five species also expressed lesser quantities of a protein similar in m.w. (approximately 200 kDa) to human CR1. In contrast to E, the major cross-reactive protein on PBMC was similar in size to human CR1. Four species also expressed lesser amounts of a lower m.w. protein on their PBMC of the same M(r) as that found on their E. Affinity chromatography demonstrated that the approximately 200-kDa form, if present, was recovered with a similar efficiency to that of human CR1. Three patterns of binding, however, were identified among the lower m.w. proteins: 1) C3b > or = C4b; 2) C4b > C3b; and C3b only or predominantly. The fact that these E proteins cross-react with Ab to human CR1, bind homologous C3b and, in most cases, C4b, and for some species represent the only such protein expressed on their E identifies them as immune adherence receptors. The 70-kDa CR1 of the chimpanzee E seems to arise by alternative splicing of the mRNA encoding the 200-kDa protein. These data raise interesting questions relative to the evolution of CR1 in primates and provide a basis for analysis of structure-function relationships among these size forms of CR1.     

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.     

Rapid activation of the complement system by cuprophane depends on complement component C4

Hemodialysis with cuprophane dialyzer membranes promotes rapid activation of the complement system, which is thought to be mediated by the alternative pathway. Complete hereditary deficiency of complement C4, a classical pathway component, in two hemodialysis patients provided the opportunity to investigate a possible role of the classical pathway. In two hemodialysis patients with both C4 isotypes, C4A and C4B, and in one patient with C4B deficiency complement activation occurred immediately after the onset of hemodialysis, with peak levels of C3a and terminal complement complex (TCC) after ten to fifteen minutes. In patients with complete C4 deficiency, C3a and TCC remained unchanged for fifteen minutes and increased thereafter, reaching the highest level after thirty minutes. The leukocyte nadir was also delayed from fifteen to thirty minutes. In vitro incubation of normal, C4A- or C4B-deficient serum with cuprophane caused complement activation after fifteen minutes. In contrast, no activation was observed in sera of four C4-deficient patients. The addition of normal serum or purified human C4 restored the capacity for rapid complement activation. In one patient with severe immunoglobulin deficiency, C3a and TCC levels increased only moderately after 25 minutes of cuprophane dialysis. This patient's serum also exhibited delayed complement activation in vitro, which was normalized after pretreatment of cuprophane with immunoglobulins. Preincubation of normal serum with MgEGTA, a blocker of the classical pathway, inhibited rapid complement activation through cuprophane. As basal levels of C4a are markedly increased in hemodialysis patients (3450 +/- 850 ng/ml) compared to healthy controls (224 +/- 81 ng/ml), no further elevation of C4a was detectable during cuprophane hemodialysis. Incubation of normal serum with cuprophane, however, caused a slight increase in C4a after five minutes. These results indicate that the initial deposition of complement C3b on the cuprophane membrane, necessary for activation of the amplification loop of the alternative pathway, is mediated by the classical pathway C3-convertase C4b2a. We propose an extended concept of complement activation through cuprophane, which is based on four steps: (a) binding of anti-polysaccharide antibodies, (b) classical pathway activation, (c) alternative pathway activation and (d) terminal pathway activation.     

Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes

The inhabitants living in the neighbourhood of a deserted mercury-contaminated industrial site are subjected to an age-group differentiated mercury exposure assessment based on a scenario-linked calculation. Analytical input data for the calculation procedure are provided for from soil, air and plants in a large number. The most sensitive group are small children being mainly exposed by soil ingestion which makes up nearly 80% of the ADI, followed by inhalation of mercury contaminated indoor air. On the other hand, inhalation of indoor air has a predominant impact on youth and adults.     

Optimum amino acid complement for protein synthesis by rat mammary cells in tissue culture

The amino acid requirement of rat mammary cells for milk protein synthesis was investigated in dispersed cell culture. A three-dimensional central composite design utilizing three variables (X1 = lysine; X2= methionine, valine, and arginine; X3 = isoleucine, tryptophan, threonine, phenylalanine, and histidine) at five concentrations each, was duplicated twice with mammary cells from lactating Sprague-Dawley rats. The optimum combination of amino acids for maximum milk protein synthesis from multiple regression models was X1 15.0-, X2 4.5-, and X3 1.5-fold their quantities in Eagle's minimal essential medium with leucine, tyrosine, cystine, and glutamine at the base 1-fold in the medium.     

Effects of interferons on tumour necrosis factor alpha production from human keratinocytes

Interferons (IFNs) have been reported to have pleiotrophic effects including the ability to induce the production of other cytokines in several cell types. Tumour necrosis factor alpha (TNF-alpha) is pro-inflammatory cytokine a known to be produced by a variety of cells including human keratinocytes. In the present study, we sought to determine the effects of IFNs on TNF-alpha production from human keratinocytes. IFN-gamma (50-100 ng/ml) induced TNF-alpha production dose dependently, but no induction of TNF-alpha was observed with IFN-alpha or IFN-beta. Since in the epidermis cytokines often work with in a cascade fashion and keratinocytes are a source of primary cytokine, IL-1 alpha, whether combined treatment with IFN-gamma and IL-1 alpha had a synergistic effect on TNF-alpha production was examined. Combined treatment with IFN-gamma (100 ng/ml) and IL-1 alpha (10 ng/ml) induced 2-3-fold higher level of TNF-alpha than IL-1 alpha alone. These results suggest that IFN-gamma is a positive regulator for the production of TNF-alpha from human keratinocytes and likely to increase skin inflammation.     

Effects of areca nut, inflorescence piper betle extracts and arecoline on cytotoxicity, total and unscheduled DNA synthesis in cultured gingival keratinocytes

Betel quid (BQ) chewing has a strong correlation with oral leukoplakia, submucous fibrosis and oral cancer. For elucidation of its pathogenesis, we investigated the effects of areca nut (AN) and inflorescence piper betle (IPB) extracts and arecoline on the growth, total DNA synthesis (TDS) and unscheduled DNA synthesis (UDS) of cultured human gingival keratinocytes (GK). Arecoline and AN extract suppressed the growth of GK over 5 days of incubation in a dose-dependent fashion. At concentrations of 100, 200 and 400 microg/ml, AN extract suppressed the growth of GK by 31%, 46% and 90%, respectively. The IPB extracts exerted less inhibitory effect on the growth of GK. IPB extract (200-400 microg/ml) decreased cell numbers by 20-40% over 5 days of incubation. Moreover, at a concentration of 0.1, 0.2 and 0.4 mM, arecoline suppressed cell growth by 44%, 77% and 96%, respectively. However, only AN extract induced TDS and UDS in cultured GK within 6 h of exposure. Induction of UDS by AN extract was concomitant with the presence of apparent intracellular vacuolization. Arecoline was also toxic to GK, but did not induce intracellular vacuolization. At a concentration range of 200-1600 microg/ml, AN extract induced TDS by 2.1- to 6.5-fold. Furthermore, at a concentration of 400-1600 microg/ml, AN extract elevated the UDS by 2.4- to 5.5-fold more than that of untreated control. On the contrary, IPB extract (200-1600 microg/ml) and arecoline (0.2-1.6 mM) inhibited the TDS and UDS of GK to a different extent. Simultaneous exposure of confluent GK to AN extract, IPB extract and arecoline for 1 to 5 days led to different degrees of cytotoxicity that was dose- and time-dependent. These results indicate that AN, IPB and arecoline take part in the pathogenesis of BQ chewing-related oral mucosal lesions, possibly through both genotoxic and non-genotoxic mechanisms.     

Effects of thyrotropin-releasing hormone on rat motoneurons are mediated by G proteins

Numerous transmitter receptors are linked via GTP-binding proteins (G proteins) to membrane phosphoinositide metabolism by phospholipase C (PLC) and generation of second messengers such as activated protein kinase C (PKC), inositol trisphosphate (IP3) and/or elevations in intracellular calcium. In many cases, these same receptors also inhibit a resting ('leak') potassium current (IK(L)), thereby depolarizing neurons. It is unclear if activation of this PLC pathway mediates inhibition of IK(L) by neurotransmitter receptors. Therefore, we tested the contribution of this pathway to the TRH-induced inhibition of IK(L) in rat hypoglossal motoneurons (HMs) using conventional intracellular recording in brainstem slices. When HMs were recorded with electrodes containing 3 M KCl or 30 mM GTP (in KCl), TRH induced a depolarization that recovered quickly (within 8-10 min) and could be repeated with only modest tachyphylaxis (< 20%). However, with electrodes containing the non-hydrolyzable G protein activator, GTP gamma S (10 mM), the TRH-induced depolarization was long lasting (up to 1 h); with electrodes containing the G protein inhibitor, GDP beta S (20 mM) the tachyphylaxis with repeated TRH application was exaggerated (approximately 60%). Activation of PKC by phorbol dibutyrate (10 microM in perfusate) neither mimicked nor occluded the effects of TRH. There were no effects on membrane potential, input resistance (RN) or the response to TRH in HMs during long recordings with electrodes containing high concentrations of IP3 (60 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of homologous complement via the alternative complement pathway by quail cells transformed by Rous sarcoma virus

After incubation of the cells with fresh quail serum, deposition of the third component of complement (C3) was demonstrated on the cell surface of various quail cell lines transformed by Rous sarcoma virus (RSV) as well as on that of primary quail embryo (QE) cells transformed by RSV. The C3 deposition occurred irrespective of virus production. On the other hand, the C-3 deposition was not observed on two quail cell lines transformed by a chemical carcinogen, QE cells infected with avian leukosis virus or normal QE cells. Moreover, QE cells infected with a temperature-sensitive mutant of RSV activated the complement at 37 degrees C but not at 41 degrees C. Since the progeny virus was generated even at 41 degrees C, viral molecules on the cell surface may not play an essential role for the activation. The activation of complement was blocked by EDTA but not by EGTA-Mg++. Therefore, the complement activation on the transformed cells appears to be mediated via the alternative complement pathway (ACP). Similar results were obtained with the complement consumption test; the residual cytolytic activity of fresh quail serum via ACP was markedly reduced by pre-incubation of the serum with transformed cells.     

Effects of phosphorothioate oligodeoxyribonucleotide and oligoribonucleotides on human complement and coagulation

We have synthesized and studied the effects of phosphorothioate (PS) oligodeoxyribonucleotide (DNA) and oligoribonucleotides (RNA, 2'-O-methyl-RNA and 2'-5'-RNA) on complement activation and prolongation of activated partial thromboplastin time (aPTT) in vitro. These results suggest that a PS-DNA prolongs aPTT, and inhibits complement lysis more than do the PS-RNA analogs.     

Effects of a streptococcal lipoteichoic acid on complement activation in vitro

This study describes activation of serum complement by lipoteichoic acid (LTA) from Streptococcus mutans OMZ 176, while in solution. Serum from 16 healthy students was taken. Test samples were incubated with increasing doses (1-5,000 micrograms/ml) of LTA or lipopolysaccharide (LPS) from Escherichia coli 0111:B4 for 1 h at 37 degrees C; then assayed for degradation of C3, C4 or factor B by crossed immunoelectrophoresis. Each preparation caused a significant (p < 0.05) dose-dependent conversion of C3. The response curves obtained were not statistically different. LPS was a stronger activator of the alternative pathway than LTA, as judged from analysis of C3 degradation in the presence of Mg2+/EGTA, and from their effects on factor B cleavage. LTA caused, however, pronounced alterations in the shape of C4 precipitation in the gels. Functional (hemolytic) assays showed that, when tested at 200 micrograms/ml, LTA and LPS triggered significant (p < 0.05) consumptions of both classical and alternative pathway proteins. LPS was a significantly (p < 0.05) stronger activator than LTA. Apparently, the C3 degradation found for this LTA involved the alternative pathway to a small extent; thus some other mechanism of fluid-phase C3 cleavage seemed also to be operative.     

Effects of in vivo hypoxia on acetylcholine synthesis by rat brain synaptosomes

Synaptosomes from normoxic and hypoxic rats were incubated aerobically in the presence and absence of veratridine. In the absence of veratridine, no significant difference was observed between the two types of preparation regarding either ATP/ADP ratio or 14CO2 or [14C]acetylcholine synthesis from D-[U-14C]glucose. However, in the presence of veratridine, significant reductions in the output of 14CO2 and [14C]acetylcholine by synaptosomes from hypoxic rats were apparent. It was concluded that irreversible metabolic lesions occur at the synapse as a result of hypoxia, which are apparent only when the metabolism of the preparation is accelerated to a level comparable with the maximal rate occurring in vivo. The presence of such lesions is further evidenced by the significant reductions in ATP/ADP ratio, 14CO2 output, and [14C]acetylcholine synthesis that occur in synaptosomes from hypoxic rats made anoxic in vitro and permitted to recover. Such decreases are not seen when synaptosomes from normoxic rats are similarly treated.     

Adsorption of complement, cytokines, and proteins by different dialysis membrane materials: evaluation by confocal laser scanning fluorescence microscopy

The membranes tested in the present study were cellulose triacetate (CTA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN). The adsorption by each membrane of albumin, IgG, C3a, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), human neutrophil elastase (HNE), and tumor necrosis factor alpha (TNFalpha) was examined and semiquantitatively graded by confocal laser scanning fluorescence microscopy (CLSFM). After clinical use the dialyzers were treated with antibodies for these proteins and cytokines. Then the samples were incubated with fluorescein isothiocyanate-labeled anti-IgG antibody and observed by CLSFM. The changes in the blood levels of C3a and cytokines were also studied. In the CTA membrane, the adsorption of these substances, except for albumin and HNE, was less than in the synthetic membranes. The PAN membrane revealed the most abundant adsorption, especially for IL-1beta, IL-6, and TNFalpha. Although a marked elevation of C3a in the blood was observed in the CTA membrane, considerable adsorption was evident in the PMMA and the PAN membranes. Because the changes in the blood levels could be affected by membrane adsorption, both the blood levels and the adsorption of the biocompatibility parameters should be evaluated when membrane biocompatibility is discussed.