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.     

Transient expression of myosin heavy chain MHCIα in rabbit muscle during fast-to-slow transition

The expression of an -cardiac-like myosin heavy chain, MHCI, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI isoform was found to be identical to the -cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI mRNA preceded that of the MHCI mRNA. At more advanced stages of the transformation, MHCI mRNA decayed while MHCI mRNA persisted at high levels. The expression of MHCI, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI protein as seen at themRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCI + MHCI (28%), MHCI+MHCI (18%), and MHCIIa + MHCI (11%). According to these combinations, the upregulation of MHCI may be assigned as an intermediate step in the transformation of existing fibres during theMHCIIa MHCI transition. A small fraction of fibres contained, in addition to the MHCI + MHCI and MHCIIa + MHCI combinations, developmental myosin, suggesting that MHCI was also expressed in regenerating fibres originating from satellite cell-derived myotubes.     

Evidence of the co-activation of α-motoneurones and static γ-motoneurones of the sartorius medialis muscle during locomotion in the thalamic cat

Summary The activity in and efferent axon populations and in group I and group II afferent fibre populations innervating a flexor muscle, the sartorius medialis, was observed during spontaneous locomotor movements in the thalamic cat. Multi-unit discharges of each kind of fibre were obtained by electronic sorting of the action potentials from the overall activity of a thin, intact branch of the sartorius medialis nerve. The following results were obtained: (1) The -motoneurones have a phasic behaviour characterized by a single discharge period during the hip flexion (swing phase of the step-cycle). (2) The -motoneurones are co-activated with the homonymous -motoneurones. (3) Between rhythmic and discharges, i.e. during the hip extension (stance phase of the step cycle), both - and -motoneurones were normally silent. However, in 5 out of 17 experiments, a few units of the population fired at very low frequency. (4) Two observations indicate that the -motoneurones that are co-activated with the -motoneurones by central locomotor commands are predominantly of the static type. In actual locomotion, the rhythmic fusimotor discharges over-compensate the depressor effect on the firing rate of the group II afferents of the unloading of muscle spindles by the active shortening of the parent muscle. In fictive locomotion, when the transmission of the excitation is blocked by selective curarization in alpha skeleto-motor junctions alone, the rhythmic fusimotor discharges elicit in-phase modulations not only of the group I but also of the group II fibres. The group II afferent population consists almost entirely of fibres arising from spindle secondary endings which are located primarily on intrafusal muscle fibres whose contraction is exclusively controlled by static fusimotor motoneurones. In the two experimental circumstances, the analysis of the group I fibre discharge does not allow to decide whether dynamic motoneurones are firing or silent during rhythmic discharge. (5) The group I and group II afferent discharges during the step-cycle showed two frequency peaks, one static-fusimotor dependent while the contracting muscle shortened during the hip flexion (swing) phase, the other length-change dependent while the relaxed muscle was rapidly stretched during the first part of the hip extension (stance) phase. Then, during the second part of hip extension when the muscle was slowly stretched in the absence of fusimotor drive, the firing rate of the spindle afférents decreased to a low level. The spindle sensory endings during the extension phase showed low dynamic and static responsiveness like deefferented spindles. (6) The results obtained in sartorius medialis (flexor) muscle are discussed in comparison with the results previously obtained in gastrocnemii (extensor) muscles (Bessou et al. 1986). The consequences of the predominant activation of the static or dynamic fusimotor system in functionally different muscles are considered with respect to the proprioceptive or motor role of musclespindles during muscle contraction.     

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     

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.     

Electromechanical behaviour of human muscles in vertical jumps

Summary The relationships of muscle structure to the potentiation of myoelectrical activity and to the use of prestretching in five lower limb muscles were studied in different vertical jumping conditions. The subjects for the study were six male students, divided according to the muscle fiber distribution in m. vastus lateralis into fast and slow groups. The subjects performed vertical jumps (1) from a static squatting position (SJ), (2) with a preliminary counter movement (CMJ) and (3) after dropping (DJ) from five different heights. Myoelectrical (EMG) activity was recorded from mm. gluteus maximus, vastus lateralis, vastus medialis, rectus femoris and gastrocnemius in each jumping condition and integrated (IEMG) for the eccentric and concentric phases of contact. EMG activity showed potentiation during the eccentric phase of movement when compared to the concentric phase. The fast and slow groups did not differ significantly in this respect, whereas in DJ conditions the relative (% from SJ) height of rise of the center of gravity was greater in the slow than in the fast group. The result indicated that the utilization of elastic energy during jumping was possible better in subjects having a high percentage of slow twitch muscle fibres in their vastus lateralis muscles.     

Effects of delayed myelination by oligodendrocytes and Schwann cells on the macromolecular structure of axonal membrane in rat spinal cord

Summary The macromolecular structure of axonal membrane from dorsal funiculi of control and irradiated spinal cord of 45-day-old rats was examined with freeze-lracture electron microscopy. In control spinal cords, virtually all myelination is mediated by oligodendrocytes, and the internodal axonal membrane of these fibres displays highly asymmetrical partitioning of intramembranous particles (IMPs). The internodal P-face particle density is 2350 IMPs per m², whereas the E-face IMP density is 150 per m². In control dorsal spinal roots, myelination is mediated by Schwann cells, and the ultrastructure of the internodal axolemma of the myelinated fibres is similar to that displayed by myelinated fibres of dorsal funiculi. On the internodal P-face of Schwann cell-myelinated fibres the IMP density is 2350 per m², whereas on the E-face the density is 175 per m². Irradiation of the lumbosacral spinal cord at 3 days of age results in a glial cell-deficient region within the spinal cord such that myelination in irradiated dorsal funiculi is delayed and subsequent myelination is mediated by both oligodendrocytes and Schwann cells. By 45 days of age, dorsal funiculi of irradiated spinal cords are well populated with fibres myelinated by oligodendrocytes and Schwann cells. However, fibres myelinated by oligodendrocytes display very thin myelin sheaths whereas Schwann cell-myelinated fibres exhibit myelin sheaths with normal thicknesses. Internodal membrane of fibres myelinated by Schwann cells and oligodendrocytes exhibit similar macromolecular structure, with 2400 IMPs per m² on P-faces and 150 IMPs per m² on E-faces. Occasional large (>1.5 m diameter) axons without glial-Schwann cell ensheathment are observed. These axons display a high density of P-face particles (2000 per m²) and a moderate density (350 per m²) of E-face IMPs on their fracture faces. These results demonstrate that CNS fibres exhibit similar axonal membrane ultrastructure irrespective of whether they are myelinated by Schwann cells or oligodendrocytes, or whether myelination is delayed. Moreover, when myelination does not occur, the axolemmal E-face IMP density, which may be related to the density of voltage-sensitive sodium channels, is not reduced.     

Electron microscopic observations on the possible retinohypothalamic projection in the rat

Summary Degenerating nerve fibres and boutons were searched with the aid of the electron microscope in the arcuate nucleus of rats 2–7 days after bilateral destruction of the retina.In the arcuate nucleus of the control animals as well as in the operated animals, 4 types of boutons were distinguished on the basis of vesicular contents and glial ensheathment. In the operated animals changes interpreted as degenerating were found in small myelinated axons and boutons of type II (boutons containing both synaptic and granular vesicles). The changes were similar to those described in the literature as the dark type of degeneration in experimentally interrupted axons and boutons. Similar changes were not found in the unoperated animals. The conclusion is reached, that a small number of fibres of the optic tract reach the arcuate nucleus to terminate here.Financial support for this work was given by the Calouste Gulbenkian Foundation and by the 3rd Development Plan of the Portuguese Ministry of Education.     

The tubular vacuolation process in amphibian skeletal muscle

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes vacuolation in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface (detubulation), particularly if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for the first time two types of vacuole, open and closed, whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed close to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. Open vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. Closed vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca2+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by open vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole closure took place well after formation of open vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of open vacuole formation. These findings identify electrophysiological detubulation of striated muscle with closure of initially open vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.This revised version was published online in September 2005 with corrections to the Cover Date.     

Two mechanically distinct types of fast twitch muscle fibres of the frog and their temperature sensitivity,as detected by sinusoidal analysis

Summary The mechanical responses to sinusoidal oscillations were recorded from tetanically contracting fast twitch fibres from the anterior tibialis muscle of the frog, Rana japonica. Two distinct fibre types were recognized. One type of fibres (tentatively referred to as worker) were similar to skinned rabbit skeletal muscle fibres in that three exponential processes (each represents a single exponential viscoelastic decay) were resolved. The second fastest process (process b) had a negative polarity and caused the fibres to produce net work during oscillations in a range of frequencies. In the other type of fibres (tentatively referred to as idler), this negative process was much smaller, and no net work was produced at any frequency. No intermediate fibre has been found so far. In the worker type of fibres, the tension response to oscillation had a modest amount of harmonic components at frequencies just above the range for work production. In the idler type, the corresponding harmonic components were much greater. In spite of these large apparent differences, the two types of fibres showed similar temperature sensitivity, raising a possibility that the basic contractile mechanisms and their rate constants are common to both types of fibres. In the idler type, the rate constant for the fastest process (process c) is possibly distributed over a wide range, thus masking the work-producing process (process b).     

Denervated chicken breast muscle displays discoordinate regulation and differential patterns of expression of αf and β tropomyosin genes

Summary The expression of the fast (f) and tropomyosin (TM) genes has been analysed with muscle-specific and common cDNA probes after unilateral nerve section of the pectoralis major muscle (PM) in 4-week-old chickens. The following were observed in denervated muscles. (1) The TM mRNA, which was repressed during development, reaccumulates in a biphasic curve with the increase in the TM protein lagging behind the changes in its mRNA. Accordingly, no TM is seen in products translated in vitro from total and polyA⁺ RNA obtained 1 week after denervation. No such translation block is seen with RNA obtained from control or muscles denervated for 6 weeks. (2) No changes in the fTM mRNA and corresponding protein are observed. (3) RNA processing of the two genes is not changed. (4) In the contralateral muscles, transitory increases in f and TM mRNAs are observed while the corresponding proteins remain unchanged. Our data suggest that muscle fibres display early and long-term responses to the loss of neural input which might result from a combination of changes produced by regenerative processes and reprogramming of existing fibres. Moreover, in contrast to normal development, no reciprocal changes of f and TM expression are seen in denervated muscles.     

Cationic proteins of human granulocytes Effects on human platelet aggregation and serotonin release

Immune-aggregate and thrombin-mediated [³H]serotonin release from human platelets are shown to be enhanced when platelets are preincubated with the antibacterial chymotrypsin-like cationic protein isolated from human granulocytes. The enhancement is dose dependent and inhibited by heating of the cationic protein. Release with chymotrypsin-like cationic protein alone was not observed, although the protein was shown to micro-aggregate platelets irreversibly by an ADP-dependent reaction. Platelet macro-aggregation induced by immune-aggregate was also enhanced by chymotrypsin-like cationic protein whereas platelet macro-aggregation induced by thrombin was inhibited competitively. Platelet micro-aggregation induced by chymotrypsin-like cationic protein was inhibited when preincubated for more than 5 min with a 2-fold molar excess of-1-antitrypsin. Chymotrypsin-like cationic protein interaction with several platelet reactions suggests a close relationship between neutrophils and platelets in the inflammatory process.     

Action of a serum protein on muscular contraction

Summary The mechanism of action of a serum protein isolated from human serum was assessed in several experimental preparations including glycerol-treated muscle fibers, rat heart papillary muscle and isolatedin vitro perfused rat heart. The action of the serum protein was studied also on canine and human heart papillary muscles which were made to respond to electrical stimulation with ultrasonication modified epinephrine. In addition the action of the protein on adenosine 5 triphosphate generated precipitation of purified human actomyosin was investigated.The serum protein enhanced and intensified the generation of ATP induced tension in glycerol-extracted muscle fibers. It intensified the developed tension (DT) and increased the rate of development of tension (dT/dt) without influencing the time peak tension (TPT) of capillary muscles from rat, canine and human hearts in response to electrical stimulation. The serum protein increased the force of contraction of the isolatedin vitro perfused rat heart, and accelerated the adenosine 5 triphosphate generated precipitation of purified human heart actomyosin.     

Molecular heterogeneity of histochemical fibre types: a comparison of fast fibres

Summary The relationship between histochemical fibre type and contractile protein expression was analysed in three rabbit skeletal muscles, the erector spinae, the plantaris and the diaphragm. A procedure for staining fibre bundles was developed using the same histochemical methods as those for typing fibres in cross-section. This allowed pretyped fibres to be selected and their molecular composition to be analysed by gel electrophoresis.The balance of expression of the two predominant fast troponin species, TnT_1f and TnT_2f, and and tropomyosin subunits were studied in type IIA and IIB fast fibres. Type IIA fibres exhibited a restricted pattern of thin filament expression, exhibiting TnT_1f and both tropomyosin subunits in all three muscles. The expression in type IIB fibres, however, ranges from predominantly TnT_2f and the tropomyosin subunit in the erector spinae to TnT_1f with both and subunits in the diaphragm.These results indicate that there is not a simple one-to-one relationship between the fast muscle fibre subtypes and the expression of different thin filament protein isoforms.     

Efficacy of the two-microelectrode voltage clamp technique in crayfish muscle

Crayfish muscle fibres of different dimensions were voltage clamped and white noise current was injected into the fibres at various distances from the voltage clamp current electrode. The clamp current was measured and power spectral densities were calculated. This method revealed the efficacy of the voltage clamp in these fibres. In large fibres (l=1.8–2.0 mm; =100–180m) a space clamp was achieved only for a band width f=40Hz. At a distance of 100m from the clamp electrodes f was 250–500Hz. In fibres of medium size (l=1.0–1.3mm; =60–120m) f was about 80Hz and about 800 Hz at a distance of 100m. In experiments with very small muscle fibres (l=400–600m; =30–50m) f was more than 500Hz. The improvement of the space clamp for the smaller muscle fibres resulted mainly from the reduced total membrane capacity,c _m, of these fibres. The limitations of the space clamp could be derived from the impedance properties of the fibres. The band width of the space clamp correlated with the band width for which the square of the absolute impedance, |Z _p|², of the muscle fibre could be described by a simple RC-model. This correlation was demonstrated in a model circuit.Power density spectra of membrane current fluctuations were measured also. To optimize the resolution of these measurements the contribution of instrumental noise was minimized. The effects of instrumental noise are discussed.This investigation was supported by the Deutsche Forschungs-gemeinschaft     

Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein

The cellular responses to alpha and beta interferons (IFN- and -) are mediated through the IFN-/ (type I) receptor, while the response to IFN- is mediated through the IFN- (type II) receptor. The receptors for IFN-/ and IFN- are encoded by genes on human chromosomes 21 and 6q, respectively. The presence of chromosome 21q confers both ligand binding and responsiveness to human IFN-/, whereas chromosome 6q confers binding of Hu-IFN-, but not cellular responsiveness on somatic cell hybrids. Chromosome 6q (i.e., the Hu-IFN- receptor gene) and chromosome 21q are both necessary for the cellular response of somatic cell hybrids (from fibroblasts) to Hu-IFN-. It is conceivable that the factor mediating activity through the IFN- receptor is, in fact, the IFN- receptor, or that the two genes are distinct but part of an interferon response region. Here we more precisely localize on human chromosome 21 the genes for the IFN- receptor and for the factor(s) mediating the action of IFN- through the chromosome 6-encoded receptor. Hamster-human somatic cell hybrids containing various fragments of human chromosome 21 were used. The presence of the human IFN-/ receptor was determined by binding³²P-labeled human IFN- to cells, covalently cross-linking the [³²P]IFN--receptor complex, and analyzing it by SDS-polyacrylamide gel electrophoresis. The presence of the IFN- receptor-related factor mediating cellular responsiveness was determined by HLA induction in hybrid cells containing the IFN- receptor (chromosome 6q), a transfected copy of the human HLA-B7 gene, and various portions of chromosome 21. In all hybrids examined, the two genes cosegregate. Specifically, both genes are localized to the region of chromosome 21 containing the markers D21S58, D21S65, and GART and appear to be proximal to D21S58. The implications for IFN action are discussed.     

A monovalent ion-selective cation current activated by noradrenaline in smooth muscle cells of rabbit ear artery

We have studied chloride influx and efflux in a highly purified preparation of type n cells freshly isolated from adult guinea-pig lung using ³⁶Cl^–. Chloride uptake was time-dependent, saturable (K_m<10 mM) and was inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS; K_i80 M). In the absence of external chloride (substituted by gluconate), ³⁶Cl^– uptake exhibited an overshoot above equilibrium. The rate of ³⁶Cl^– entry was strongly inhibited by addition of external nitrate; sulphate was a weaker inhibitor. ³⁶Cl^– efflux was stimulated by external bromide > bicarbonate chloride citrate; and was inhibited by proprionate > acetate > oxalate. Although the chloride channel blocker 4-nitro-2-(3-phenylpropylamino)benzoate (0.14 mM) caused an inhibition, ³⁶Cl^– influx did not appear to be electrogenic. These data are compatible with the existence of a substantial electroneutral anionexchange pathway for chloride transport in freshly isolated adult type II pneumocytes.     

Effect of stimulating a sympathetic nerve on the ionic constitution of frog skeletal muscle perfusate

Summary The author investigated the nature of the substances which influenced the skeletal muscle in stimulation of the sympathetic nerve. It was established that by stimulating the sympathetic nervous system, active substances appear in the perfusate of the frog's skeletal muscle. Evaporation of the perfusate, with subsequent calcination of the dry residue and its disolution in distilled water, does not remove the positive effect exerted on the frog's heart. Of the ions tested (Na, Mg, and Ca), the picture analogous to the action of the sympathetic perfusate is obtained only in the case of Ca.From the Pharmacological Laboratory (Head-Professor N.P. Sinitsyn) S.M. Kirov Gor'kii Medical InstitutePresented by Active member of the AMN SSSR V. V. Zakusov     

Organization of DNA topoisomerase II isotypes during the cell cycle of human lymphocytes and HeLa cells

We have monitored the organization of DNA topoisomerase II (Topo II) in relation to chromatin disaggregation during mitogen stimulation of lymphocytes and to the mitotic chromosome condensation cycle by immunofluorescence microscopy with isozyme-specific antibodies. Labelling for both Topo II and Topo II was diffusely nucleoplasmic and non-nucleolar in resting lymphocytes and the pattern changed little during stimulation. Topo II labelling intensity increased in parallel with the extent of cell stimulation, but a fraction of fully stimulated cells was labelled very brightly. Topo II labelling intensity was also greater in stimulated cells, but all partially and fully stimulated cells were labelled at the same, higher, intensity. In addition, anti-Topo II detected a few small spots within nucleoli of stimulated cells that coincided with regions containing fibrillarin. In lymphocytes and HeLa, chromosome association of Topo II began in prophase and lasted throughout mitosis. In contrast, Topo II stayed nucleoplasmic in prophase, was diffusely cytoplasmic during mitosis, and was first detected post-mitotically in nuclel with decondensing chromosomes and a reformed nuclear envelope. The results are consistent with a role for Topo II, but not for Topo II, in mitotic chromosome condensation, and indicate that the isotypes may play independent roles in the reorganization of chromatin structure during lymphocyte mitogenic activation.accepted for publication by T. D. Allen     

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.