Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Comparison of the properties of five pox virus strains isolated from monkeys

Summary Five strains of monkey pox viruses were compared with respect to their cultural characteristics in primary and continuous cell cultures and the lesions developed in embryonated eggs and in rabbit skin as well as to their hemagglutinating activity.Four strains (Copenhagen 65-31 65-32 and 7-61) appeared to be similar in their properties. The cytopathogenic effect (CPE) was identical to that induced by vaccinia virus. There was no detectable virus multiplication in an pig kidney cell line (PEK). All four strains produced small, white, compact, hemorrhagic pock-like lesions on the chorioallantoic membrane.The strain 64–7275, isolated from healthy monkeys kidneys, had all properties of variola virus. It multiplied in the PEK cell line with a CPE. The lesions on the CAM were more compact without hemorrhage. In rabbit skin no detectable reaction occurred after infection with this strain.     

Genes for C4b-binding protein α- and β-chains (C4BPA andC4BPB) are located on chromosome 1, band 1q32, in humans and on chromosome 13 in rats

C4b-binding protein is involved in the regulation of the complement system. It is a multimeric protein composed of seven identical -chains and a single copy of a unique -chain. The latter was identified only recently and its structure determined by cDNA cloning. Both subunits in C4b-binding protein belong to the same superfamily of proteins composed predominantly of tandemly arranged short consensus repeats (SCR) approximately 60 amino acid residues in length. The gene for the human -chain is known to be located in a gene cluster on chromosome 1, band 1q32, which is called the regulators of complement activation (RCA) gene cluster. We have used cDNA probes for both - and -chains of human C4b-binding protein to localize their genes with an in situ hybridization technique. We find the genes for both chains to be located on chromosome 1, band 1q32, in the human. This suggests that the -chain gene is also a member of the RCA gene cluster and that the - and -chain genes are located close to each other. The cDNA probes for the - and -chains also were used to screen mouse-rat somatic cell hybrids using Southern blotting to localize their genes in the rat. Both the - and -chain genes were shown to be located on chromosome 13 in the rat. These are the second and third genes to be located on rat chromosome 13, and the results suggest that the genes for the - and the -chains together with the gene for coagulation factor V represent a conserved chromosomal region in rat and man.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Regulation of interleukin-1β and tumor necrosis factor secretion by the human monocytic leukemia cell line,THP-1

Activated macrophages synthesize and release the potent polypeptides, interleukin-1 (IL-1) and tumor necrosis factor (TNF). In an effort to identify the cellular signals which control cytokine production by activated macrophages, we have developed anin vitro model employing the human THP-1 cell line. In the present study, THP-1 cells primed by 1.6 M phorbol 12-myristate-13-acetate (TPA) for 4 hr demonstrated a dose-and time-dependent release of IL-1 and TNF upon activation by 20 g/ml LPS. BSA/anti-BSA-coated latex beads were also a potent stimulus for IL-1 secretion. Moreover, the combination of a suboptimal concentration of LPS (200 ng/ml) plus interferon- (0.03–333 U/ml) greatly enhanced IL-1 production. Resting THP-1 monocytes not primed by TPA did not secrete IL-1 or TNF. These distinct patterns of cytokine production may be related to the developmental stages of macrophage activation.     

Investigation of the “valency” of antibodies by means of azoproteins

Summary The valency of antibodies was studied by the method of exhaustion of antisera against mono-and diazoproteins, and subsequent cross reactions both with the antibodies left over in the supernatant fluid of the serum and with the precipitating and nonprecipitating antibodies isolated from the precipitate.It was proved that the antibodies interact with the antigens as multivalent compounds.The valency determined with regard to the azoproteins is dependent upon the number of groups introduced.Thus, bivalent antibodies correspond to monoazoproteins and trivalent ones to diazoproteins.The valency of antibodies is, evidently, determined by the structural similarity of the heterologous and the immunizing antigens as well as by the less complete specific conformity between the individual structural peculiarities of the antigen and its antibody.From the Tashkent Pharmaceutical Institute (Director-Docent M. A. Azizov)Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov     

Inhibition of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) secretion but not IL-6 from activated human peripheral blood monocytes by a new synthetic demethylpodophyllotoxin derivative

A newly synthesized demethylpodophyllotoxin derivative, 4-O-butanoyl-4-demethylpodophyllotoxin (BDPT) or BN58705, has recently been shown to exert a potent cytotoxic activityin vitro against a variety of drug-resistant human tumor cell lines. The effect of this agent on effector cells of the immune system, however, has not been examined. The present study investigated the effect of BDPT on the response of activated human peripheral blood derived monocytes (PBM) to secrete cytokines. Activation of PBM overnight with LPS, IFN-, or PMA resulted in secretion into the supernatant of TNF-, IL-1, IL-6, and IL-8 as assessed by ELISA. The addition of BDPT to the stimulated cultures resulted in significant inhibition of TNF- and IL-1 secretion, whereas the secretion of IL-6 and IL-8 was not affected. The selective inhibition of TNF- and IL-1 secretion by BDPT-treated PBM was observed with all three stimuli tested. The inhibitory effect mediated by BDPT was concentration dependent and was optimal at 6–20 M. Time kinetic analysis indicated that the inhibition of secretion was rapid and detected as soon as 2 hr following stimulation of the PBM and lasted for as long as 24 hr. A comparison was made between BDPT and pentoxyfilline, a xanthine-derived phosphodisterase inhibitor that was reported to inhibit TNF- and IL-1 secretion by PBM. Both BDPT and PTX showed similar time kinetics and patterns of inhibition. However, the concentration used by BDPT to achieve optimal inhibition of secretion was 10- to 20-fold less than that needed by PTX. The selective inhibition of TNF- secretion by BDPT and PTX was corroborated by inhibition of TNF- mRNA but not IL-6 mRNA as assessed by RT-PCR analysis. These studies demonstrate that the antitumor cytotoxic compound BDPT is also an immunomodulatory agent that can inhibit selectively TNF- and IL-1 secretion by PBM. Further, the low toxicity and low concentrations of BDPT needed for optimal inhibition suggest that BDPT may have potential in its therapeutic application in diseases that are mediated by TNF- and IL-1 like septic shock, inflammatory responses, and infections.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Differential Regulation of IL-8 by IL-1β and TNFα in Hyaline Membrane Disease

Mechanisms that regulate cytokine-mediated inflammation in the lungs of preterm infants, including factors which regulate production of the chemokine IL-8, remain poorly defined. Sequential bronchoalveolar lavage samples were obtained from preterm newborns with hyaline membrane disease over a 28-day period. Bronchoalveolar lavage cell cytokine relationships were evaluated and the differential regulation of IL-8 by IL-1 and TNF was studied in a short-term culture system. In vivo, IL-8 and IL-l protein levels correlated closely with each other and with macrophage counts. In cell culture, exogenous anti-IL-1 antibody led to a 40% maximum inhibition (approximately) of IL-8 production by lipopolysaccharide stimulated lung inflammatory cells. Comparable amounts of exogenous anti-TNF antibodies achieved a 15% maximum inhibition (approximately) of IL-8 production. Anti-IL-1 and anti-TNF antibodies in combination did not inhibit IL-8 production beyond that achieved by anti-IL-l antibody alone. These results, in preterm newborns, support the concept of lung inflammation mediated in part by a macrophage, IL-1, and IL-8 cell cytokine pathway. The results also suggest that factors other than IL-1 and TNF regulate IL-8 expression in the lungs of preterm infants.     

Factors influencing serum neopterin and β2-microglobulin levels in a healthy diverse population

Sera and questionnaire data from a population-based random sample of healthy adults was used to evaluate factors influencing neopterin and 2-microglobulin (2m) values. Both neopterin and 2m levels increased with age and were higher among white than blacks (mean values for whites and blacks: neopterin, 5.06 vs 4.49 nmol/L; 2m, 1.36 vs 1.28 mg/L). Gender differences were noted for 2m but not neopterin values (2m males vs females: 1.37 vs 1.29 mg/L). Neopterin values were lower among current smokers than among nonsmokers (4.32 vs 5.16 nmol/L) and were higher among users of antihistamines (5.46 among users vs 4.65 nmol/L among nonusers). Neopterin and 2m were correlated in this healthy adult population (adjustedr=0.53,P=0.001), yet no other interrelationships with numerous biologic markers except between 2m and serum-soluble interleukin-2 receptor levels (adjustedr=.41,P=0.05) were observed. These findings provide important baseline information to consider before planning or evaluating studies utilizing neopterin or 2m levels.     

Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Candida albicans induces the release of inflammatory mediators from human peripheral blood monocytes

Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [¹⁴C]-AA labeled monocytes released 8.9 ±2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 161) (P=0.0002). Prior studies indicate that soluble-mannans and-glucans antagonize mannose and-glucan receptors, respectively. Preincubation of monocytes with-mannan (100g/ml) caused 45.8 ±5.7% inhibition of [¹⁴C]-AA release, whereas-glucan (100g/ml) yielded 43.7 ±6.0% inhibition (P<0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1 (IL-1), tumor necrosis factor- (TNF), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, a-mannan or-glucan failed to inhibit IL-1 release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by a-mannan and-glucan components of the fungus.     

Induction of β-family chemokines mRNA in human embryonic astrocytes by inflammatory cytokines

The cellular infiltration found during CNS inflammation consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during inflammatory responses. Astrocyte chemokine expression might contribute to site-specific leukocyte infiltration within the CNS. To investigate the factors that regulate astrocyte chemokine expression, we examined the ability of human fetal astrocytes to induce -family chemokine mRNA. Astrocyte-derived monocyte chemoattractant protein-1 (MCP-1), RANTES, macrophage inflammatory protein-1 (MIP-1), and MIP-1 mRNA were easily induced by lipopolysaccharide and/or the proinflammatory cytokines (IFN and/or TNF-), respectively. Addition of both IFN and TNF- together did not lead to an additive effect but resulted in the inhibition of MCP-1 and MIP-1 mRNA expression, indicating that interaction between chemokines and cytokines may play a key role in regulating the local immune response of resident and infiltrating cells at the site of lesion. Interestingly, ultraviolet light-inactivated measles virus, but not cytomegalovirus, strongly induced expression of MCP-1, RANTES, MIP-1, and MIP-1 mRNA in human embryonic astrocytes, especially MCP-1 and MIP-1. An association occurs between the -family chemokine expression in astrocytes and inflammatory factors/virus, suggesting a possible role for -family chemokines in the pathogenesis of CNS inflammatory disease.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Interaction of G-actin with thymosin β4 and its variants thymosin β9 and thymosin β 9 met

Summary Thymosin ₄ is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin ₉ and thymosin ₉ ^met are minor variants of thymosin ₄. The possible function of these peptides has been investigated by comparing the actin binding properties of these -thymosins. Thymosin ₉ and thymosin ₉ ^met were found to inhibit polymerization of ATP-actin with identical K _d s of 0.7–0.8 M (as compared to 2±0.3 M for thymosin ₄); like thymosin ₄, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin ₄ and thymosin ₉ ^met with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin ₄ to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin ₄ binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of 1.5 M^-1 s^-1 and 2s^-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin ₄ function in vivo is discussed.     

Virus-specific RNA formed in Newcastle disease virus-infected cells after suppression of protein synthesis by cycloheximide

Summary In Newcastle disease virus-infected cells the accumulation of virus-specific RNA is prevented if protein synthesis is inhibited by cycloheximide at an early stage of infection. At a later stage cycloheximide enhances the synthesis of smaller (18–35 S) virus-specific RNA while the synthesis of 49 S RNA is suppressed. This is accompanied by a corresponding change in the relative amounts of plus and minus RNA strands: the proportion of plus strand is sharply decreased. A possible significance of the phenomenon for the regulation of virus-specific RNA synthesis is discussed.