Reactive nitrogen intermediates implicated in the inhibition of Encephalitozoon cuniculi (phylum microspora) replication in murine peritoneal macrophages

Encephalitozoon cuniculi (phylum microspora) is a protozoan parasite that can replicate within parasitophorous vacuoles in macrophages. Thioglycollate-elicited BALBjc peritoneal macrophages treated with murine recombinant interferon-γ (rIFN-γ; 10u/ml) in combination with lipopolysaccharide (LPS; 10ng/ml) for 24 h killed E. cuniculi as determined by significant reductions in the number of parasites and percent of infected macrophages 48 h later compared with cultures treated with medium only. Treatment of the elicited macrophages with murine rIFN-γ (10u/ml or 100u/ml) only, resulted in microbistatic activity. Significantly higher levels of nitrite (NO₂) were detected in supernatants from macrophage cultures treated with rIFN-γ (10 u/ml or 100 u/ml) which induced microbistatic macrophage activity as well as from macrophage cultures treated with LPS + rIFN- when compared with levels of nitrite detected in supernatants of infected macrophages treated with medium only. Addition of the L-ariginine analogue, N³ monomethyl-L-arginine (NMMA) at concentrations of 50, 100 or 250 uM significantly inhibited nitrite synthesis and prevented microsporidia killing. Addition of exogenous L-arginine at concentrations of 5mM or 10 mM reversed the NMMA-induced inhibition of parasite killing. These results indicate that reactive nitrogen intermediates contribute to the killing of E. cuniculi by LPS + rIFN-γ-activated murine peritoneal macrophages in vitro.     

Antiplasmin Activity of Rat Serum Slow α-Globulins

The antiplasmin activity of serum slow α₁- and α₂-globulins has been studied in rats injected with streptokinase/human serum and human or rat plasmin. Highly significant falls in serum slow α₁ -globulin were noted soon after administration of streptokinase and human or rat plasmin; this suggested a reaction between plasmin and slow α₁-globulin with rapid removal of the resultant complexes. Rats with immune complex nephritis (ICN) given streptokinase showed a highly significant increase in slow α₂-globulin, but no change in slow α₂-globulin was noted following injection of human plasmin. Using fibrin agar plates additional evidence was obtained that rat slow α₁-globulin inhibited digestion of fibrin by plasmin. We conclude that of the two globulins in the rat only slow α₁-globulin shows antiplasmin activity and we believe that this represents the functional analogue of α₁-macroglobulin in the human.     

A Study of 'Haemosiderin'in the Marrow of the Femur of Normal Young Adult Rabbits Compared with that in Rabbits 4 Months after an Intravenous Injection of 239Pu(NO3)4

A study of the distribution of cells containing iron, as demonstrated by Perls'reagent, in the marrow of the femur of normal young adult rabbits in comparison with that in rabbits of the same age group 4 months after an intravenous injection of ²³⁹Pu(NO₃)₄ is described.
The number of iron containing cells, which are probably macrophages, was found to be variable in the length of the bone and also from one normal rabbit to another. The amount of pigment in any one cell was also variable.
A significant increase in the number of iron containing cells and in the amount of pigment the cells contained was noted in the marrow of the femur of rabbits 4 months after a single intravenous injection of ²³⁹Pu(NO₃)₄, compared with the normal controls. ²³⁹Pu aggregates close to the mineralized surface were not characteristically associated with haemosiderin.
This work was carried out at the Medical Research Council Unit for Research on Bone-Seeking Isotopes, Oxford, while the author was holding a World Health Organization Fellowship.
I am indebted to Dr. Janet Vaughan and Mrs. Betty Bleaney for much helpful advice and discussion. I also wish to thank Dr. David Taylor, Department of Biophysics, The Royal Cancer Hospital, for injecting the rabbits with ²³⁹Pu(NO₃)₄. I appreciate the technical help of Mr. John Halfacre.     

Nitrogen dioxide pneumonitis in ice hockey players

Karlson-Stiber C, Höjer J, Sjöholm Å, Bluhm G, Salmonson H (Swedish Poison Information Centre, the Department of Internal Medicine, Söder Hospital, and the Department of Environmental Health and Infectious Disease Control, Karolinska Hospital, Stockholm; and the Department of Internal Medicine, Löwenströmska Hospital, Upplands Väsby; Sweden). Nitrogen dioxide poisoning with toxic pneumonitis in ice hockey players (Case report). J Intern Med 1996; 239: 451–6.
Exposure to the toxic gases carbon monoxide and nitrogen dioxide (NO₂) in indoor ice arenas occasionally occurs and may result in severe symptoms. The gases are produced by ice resurfacing machines operating on hydrocarbons, and in certain conditions toxic levels accumulate. The damage to lung tissues caused by NO₂ may not be evident until after a latency time of ½–2 days. The role of corticosteroids in the treatment is controversial, but there are clinical experiences as well as experimental data supporting their use.
We report two cases of toxic pneumonitis, with delayed onset, due to NO₂ exposure during an ice hockey game in an indoor arena. Signs and symptoms were cough, dyspnoea, haemoptysis, hypoxaemia and reduced peak expiratory flow. Chest radiographs showed parenchymatous infiltrative lesions and alveolar consolidation. Both patients were treated with high doses of corticosteroids by inhalation and orally or intravenously. Their condition rapidly improved and pulmonary function was restored.     

Absence of Intrinsic Factor from Human Portal Plasma during 57CoB12 Absorption in Man

S ummary The ⁵⁷CoB₁₂ activity in blood obtained from the portal vein during ⁵⁷CoB₁₂ absorption did not have the biological or the immunological features in vitro of intrinsic factor from normal human gastric juice.
This indicates that ⁵⁷CoB₁₂ probably is not absorbed from the intestine with intrinsic factor.     

IL-10 deficit correlates with chronic, hypersplenomegaly syndrome in male CBA/J mice infected with Schistosoma mansoni

Twenty weeks after moderate level infections with Schistosoma mansoni, approximately 20% of male CBA/J mice develop hypersplenomegaly syndrome (HSS) while the rest present with moderate splenomegaly syndrome (MSS). HSS and MSS mice differ pathophysiologically (degree of splenomegaly, anaemia, ascites, periportal fibrosis, portal hypertension) and immunologically with regard to antibodies (idiotypic expression, isotype levels) to schistosome soluble egg antigens (SEA), and spleen cell phenotypic profiles. This study compared in vitro proliferative responses and IL-2, IFNγ, IL-4, and IL-10 production by spleen cells from uninfected mice and mice with acute (8 wk), MSS or HSS schistosomiasis mansoni, upon exposure to anti-CD3_ε or SEA. Spleen cells from uninfected mice produce IL-2 to anti-CD3_ε, but exposure of cells from all three groups of infected mice to anti-CD3_ε or SEA led to only very low levels of supernatant IL-2. Anti-CD3_ε- or SEA-stimulated production of IFNγ or IL-4, and anti-CD3_ε-stimulated production of IL-10, displayed similar patterns: highest cytokine production by cells from mice with acute infections and lower levels of production that did not differ between the two chronic groups. In contrast, while SEA-stimulated IL-10 production was again highest with cells from mice with acute infections, spleen cells from mice with MSS produced significantly more IL-10 than did those from mice with HSS. This association of low levels of antigen-induced IL-10 with severe pathology is consistent with the theory that IL-10 plays a role in the immunoregulation that occurs in chronic schistosomiasis .     

In vivo and in vitro effects of the pineal gland and melatonin on [Ca2++ Mg2+]-dependent ATPase in cardiac sarcolemma

Abstract: The possible diurnal variation in cardiac [Ca²⁺+ Mg²⁺]-dependent ATPase (Ca²⁺ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemma] membrane [Ca²⁺+ Mg²⁺]-dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time-dependent changes in [Ca²⁺+ Mg²⁺]-dependent ATPase activity did not occur in either pinealectomized or light-exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca²⁺+ Mg²⁺]-dependent ATPase activity. In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca²⁺ pump. The half-maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca²⁺+ Mg²⁺]-dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.     

Arginine-dependent generation of reactive nitrogen intermediates is instrumental in the in vitro killing of protoscoleces of Echinococcus multilocularis by activated macrophages

The interaction between protoscoleces of Echinococcus multilocularis and activated murine macrophages was examined in this study. Marked protoscolicidal activity was displayed by peritoneal macrophages (PM) activated with interferon-γ (IFN-γ), or IFN-y plus lipopolysaccharide. Pretreatment of the parasites with heat-inactivated specific murine infection serum, but not with normal serum rendered them more susceptible to PM killing. N^G-monomethyl-Larginine, a competitive inhibitor of L-arginine completely inhibited the killing activity of activated PM. while reconstitution of arginine-free medium with L-arginine restored the killing properties of the activated PM. The results show that activated PM have the ability to kill E. multilocularis protoscoleces in vitro and suggest that reactive nitrogen intermediates (RNl) play an important role in the mechanism. An oxygen-mediated mechanism did not appear to play a role because scavengers of reactive oxygen species did not reduce the killing activity. The arginine-dependent killing mechanism was enhanced by superoxide dismutase (SOD), probably because SOD might prolong the effect of nitric oxide. Secretion of RNI by activated macrophages may be capable of a significant role in preventing of the dissemination of E. multilocularis infection in vivo.     

Effect of Terminal (Dry) Heat Treatment on Non-Enveloped Viruses in Coagulation Factor Concentrates

Terminal dry heat treatment effectively inactivated hepatitis A virus (HAV) and canine parvovirus added to high-purity factor VIII. After 24 h at 80°C, HAV infectivity was reduced by ≥4.3 log₁₀ TCID₅₀, as measured in a newly developed infectivity assay. The same reduction in virus titer was achieved after 2 h and before 6 h at 90°C. Inactivation of hepatitis A virus was also seen in the freeze-drying step prior to heat treatment with an approximately 2.0 log₁₀ reduction in titer. Similar results were obtained with a high-purity factor IX concentrate. Canine parvovirus was also inactivated at both temperatures, with residual infectivity being undetected after 48 h at 80°C or 10 h at 90°C. Canine parvovirus was not affected by lyophilisation. Canine parvovirus measurements by PCR did not reflect the levels of infectivity measured by the tissue-culture-based method. The addition of the terminal dry heat treatment to solvent/detergent could effectively eliminate the potential contamination of solvent/detergent-treated coagulation factor concentrates by non-lipid-enveloped viruses. However, careful evaluation for any increased induction of non-antigens for factor VIII, as a consequence of such treatment, is needed before use in patients can be recommended.     

Red Cell Hydrolases

Abstract. Human erythrocyte plasma membranes solubilized in 0.1% Triton X-100, with tritium-labeled 5-acetamido-3,5-dideoxy- l -arabino-2-heptulosonic acid fetuin as substrate, were found to contain considerable neuraminidase activity. The enzyme was purified 470-fold, with 17% recovery, by centrifugation and gel chromatography on Sephadex G-100 and Sephadex G-150. The highest activity of the purified enzyme was at pH 4.2, but the neuraminidase was active over a broad pH range. No cofactors were necessary for neuraminidase activity; the enzyme was inhibited by 0.05 m Ca acetate, 0.1 m CuSO₄, 0.01 m FeCl₃, 0.01 m FeCl₂, 0.01 m HgCl₂, and 0.005 m p -chloromercuribenzene sulfonate. The purified enzyme had maximal activity at 30–40 °C, and a linear relationship was demonstrated for activity versus time and for activity versus amount of enzyme.
Using the Eadie-Hofstee plot and a fetuin molecular weight of 48,000, a V_max of 4.5 × 10⁴ cpm ċ h⁻¹ ċ mg⁻¹, and a Km of 9 × 10⁻⁶ m were calculated. On whole-cell electrophoresis, it was demonstrated that purified human erythrocyte plasma membrane neuraminidase, like Clostridium perfringens neuraminidase, could cause release of terminal sialic acid residues from the external surfaces of intact human erythrocytes.     

Non-specific binding of IgG1 to Heligmosomoides polygyrus: adult worm homogenate superantigen is a target for immunoglobulin-induced inhibition

Previous evidence had indicated that selected antigens contained in H. polygyrus adult worm homogenate (AWH) could bind non-specifically to mouse IgG1. To determine whether H. polygyrus superantigen was one of these binding molecules, an inhibition assay was carried out using monoclonal antibodies (MoAb) to block the in vitro superantigen response. The results indicated that non-specific IgG1 binding could inhibit the cellular response to the superantigen. This assumption was tested using affinity chromatography to extract those antigens which bound non-specifically to mouse IgG1. Both the protein fraction which bound to the column and the unfractionated AWH demonstrated superantigen activity, as described previously. In contrast, the unbound fraction contained no superantigen activity. None of the tested fractions exhibited non-specific mitogen activity. These results indicate that the superantigen produced by H. polygyrus binds to host IgG1 of any specificity and this binding can inhibit further host recognition of this molecule. Additionally, it was demonstrated that an apparently similar superantigen is also contained in L₄ homogenate, and is strongly represented in the excretory/secretory (E/S) proteins produced by both adult and L₄ parasitic stages. Therefore, it is probable that H. polygyrus superantigen influences the host during both the L₄ and adult stages of the life-cycle.     

Hormonal Regulation of Pre-natal Haemoglobin Synthesis by Erythropoietin

S ummary . Developmental changes in the response of mouse, rat and rabbit foetal liver cells and of rat foetal spleen cells to erythropoietin have been investigated in vitro .
Liver cells from 10¹/₂-14¹/₂-day mouse foetuses and 12–13-day rat foetuses show little haemoglobin synthesis in vitro but marked response to erythropoietin. This is followed, in the mouse, by a brief period in which the rate of synthesis on explantation is high and cannot be increased by erythropoietin. In the rat from 14 to 16 days the rate of synthesis on explantation increases progressively while the response to erythropoietin persists. Liver cells from later foetuses of both animals show variable haemoglobin synthesis and are not erythropoietin sensitive. 14–18-day foetal rabbit liver cells and 18–19-day foetal rat spleen cells are also sensitive to erythropoietin. It is suggested that development of sensitive stem cell populations precedes erythropoietin production in vivo so that critical population numbers are reached which enable feedback controls to be established.
Isotopically labelled haenioglobins produced in vitro by rat and mouse foetal liver cells were electrophoretically characterized. Erythropoietin caused increased production of a haemoglobin type already present.     

Antileishmanial and immunomodulatory activity of Xylopia discreta

This study aimed at determining the in vitro antileishmanial activity of the essential oil and eight extracts obtained from Xylopia discreta . J774 and U937 macrophages were exposed to the different substances to establish the median lethal concentration (LC₅₀). The median effective concentration (EC₅₀) was obtained by determining the reduction of Leishmania panamensis - infected cells. A selectivity index (SI) (LC₅₀/EC₅₀) ≥ 20 defined a specific activity for one Xylopia discreta leaf extracts and for the essential oil, being these the two that showed the highest activity (SI = 64·8 and 110, respectively in J774 cells). To assess the substances' immunomodulatory activity, pro- and anti-inflammatory soluble mediators produced after treating infected macrophages were quantified by flow cytometry. The leaf methanol extract and the essential oil induced a differential production of monocyte chemoattractant protein-1, a chemokine associated with a Leishmania -resistant phenotype (Th1).     

In pregnant mice, the infection of Toxoplasma gondii causes the decrease of CD4+CD25+ -regulatory T cells

Acute maternal infection with Toxoplasma gondii during pregnancy is associated with adverse pregnancy outcomes. Although previous reports have indicated that T. gondii may result in abortion without direct transmission of the parasite to the foetus, the molecular mechanism remains unclear. CD4⁺CD25⁺-regulatory T cells are known to be involved in maternal tolerance toward the foetus-bearing alloantigens. With a model of pregnant mice infected with T. gondii , we found that Foxp3 mRNA expression levels in both splenocytes and placenta were reduced markedly during the process of infection. Furthermore, the numbers of splenic CD4⁺CD25⁺-regulatory T cells and placental Foxp3⁺ cells decreased synchronously in the infected mice, and the reduction of splenic CD4⁺CD25⁺-regulatory T cells were associated with apoptosis induced by the infection. Additionally, injection of pregnant mice with excretory–secretory antigens (ESA) of T. gondii also resulted in foetal loss, which could be partly prevented by adoptive transfer of CD4⁺CD25⁺-regulatory T cells from normal pregnant mice. These data suggest that foetal loss caused by T. gondii can be independent of vertical infection and that the decrease of CD4⁺CD25⁺-regulatory T cells during infection may represent a previously unrecognized mechanism for the pathogenesis of abortion caused by this parasite.     

On the Mechanism of Inhibition of KATP Channels by Glibenclamide in Rat Ventricular Myocytes

Glibenclamide Block of K_ATP Channels. Introduction: The mechanism by which glibenclamide inhibits K_ATP channel activity has been examined in membrane patches from isolated rat ventricular cells. Methods and Results: Inside-out patches were exposed to zero, or low, [ATP] to activate K_ATP channels. Glibenclamide did not affect single channel conductance, but reversibly reduced channel open probability from either side of the membrane. Internal (cytoplasmic) glibenclamide inhibited with half-maximal inhibitory [glibenclamide] = 6 μM, Hill coefficient = 0.35. Complete channel inhibition was not observed, even at 300 μM [glibenclamide]. The response to step increases of internal [glibenclamide] could be resolved into two phases of channel inhibition (t_{1/2, fast}, < 1 sec, t_{1/2, slow}= 10.5 ± 0.9 sec, n = 8). Step decrease of [glibenclamide] caused a single resolvable phase of reactivation (t_1/2= 20.4 ± 0.7 sec, n = 16). Channel inhibition by internal glibenclamide could be relieved by ADP, but only in the presence of Mg²⁺.
Conclusion: Glibenclamide can inhibit K_ATP channels from either side of the membrane, with block from one side being competitive with block from the other. Internal MgADP antagonizes the blocking action of glibenclamide. Glibenclamide inhibition of cardiac K_ATP channels differs quantitatively and qualitatively from the inhibition of pancreatic K_ATP channels.     

Uptake of Tritiated Folates by Human Bone Marrow Cells in Vitro

S ummary . Using incubation periods up to 4 hr, it was demonstrated that uptake of tritiated pteroylglutamic acid ([³H]PteGlu) by human bone marrow cells in vitro was in the range of six-fold greater than uptake by reticulocytes. Uptake was temperature-dependent, increasing during 4 hr at 37°C but not at 4°C; a similar temperature dependence was found for the uptake of [³H]methyltetrahydrofolate ([³H-CH₃]H₄PteGlu). The pH optimum for [³H]PteGlu uptake was in the range of 7.4. Percentage uptake decreased as concentration of [³H]PteGlu increased. Preincubation with unlabelled PteGlu reduced uptake of subsequently added [³H]PteGlu by twice as much as did preincubation with methotrexate, suggesting that methotrexate may only partly share the uptake mechanism for [³H]PteGlu. [³H]PteGlu uptake was not affected by preincubation with diphenylhydantoin or a sulphydryl inhibitor. Uptake of [³H-CH₃]H₄PteGlu by human bone marrow cells appeared to be approximately twice the uptake of [³H]PteGlu. The findings support the concept of two mechanisms for folate uptake by human reticulocytes and bone marrow cells: an energy-dependent, active carrier mechanism probably of primary physiologic significance, and a seemingly passive diffusion-like mechanism, probably primarily of pharmacologic significance.     

Efficacy of artemisinin and mefloquine combinations against Plasmodium falciparum. In vitro simulation of in vivo pharmacokinetics

The activity of artemisinin in combination with mefloquine was tested in vitro against a chloroquine‐sensitive (F32) strain of Plasmodium falciparum . A method of repetitive dosing and extending the culture observation period to 28–30 days was used to mimic the in vivo pharmacokinetic situation. Plasmodium falciparum was exposed to artemisinin from 10⁻⁸ to 10⁻⁵  m , mefloquine from 3×10⁻⁹ to 10⁻⁵  m and their combinations. The exposure time for artemisinin was 3 hours twice daily and for mefloquine 24 hours. The drug‐dosing duration was 3 days.
Neither artemisinin nor mefloquine alone provided radical clearance of P. falciparum , even when maximum concentrations (10⁻⁵  m ) were applied. The antiparasitic activity of artemisinin and mefloquine were significantly higher when dosed alone. Effective concentrations for different degrees of inhibition (EC 50, 90 and 99) of both artemisinin and mefloquine respectively were significantly lower when used in combination. At concentrations normally reached in vivo , this effect was clearly synergistic ( P =0.016)
Our in vitro model of intermittent dosing of artemisinin and mefloquine combinations for 3 days provides significant evidence of positive interaction between the two compounds. Lower combination concentrations around the MIC‐values for the individual compounds showed synergistic effect, and high concentrations showed additive effect. This indicates that such drug combinations may provide radical clearance at concentrations lower than those required for single‐drug treatment.     

Hereditary Spherocytosis: The Metabolism of Erythrocytes in the Peripheral Blood and in the Splenic Pulp

S ummary . Na⁺ transport was studied in intact red cells and membrane preparations ('ghosts') of patients with hereditary spherocytosis (h. s.). Ouabain-insensitive efflux was greater than normal in ghost preparations as well as in intact cells, findings which arc consistent with a membrane defect in h.s. red cells. Ouabain-sensitive ('pump') sodium efflux was normal in h.s. membrane preparations, but was increased in intact cells and the latter probably reflects changes secondary to the increased Na⁺ influx in these cells.
In h.s., red cells are trapped and destroyed in the spleen and we studied splenic pulp red cells in a group of h.s. patients at splenectomy. These cells showed greater osmotic fragility, higher Na⁺ and lower K⁺ concentrations than did peripheral red cells. Membrane permeability to Na⁺ was not greater than in peripheral red cells but the ouabain-sensitive ('pump') Na⁺ efflux was reduced. Despite this, ATP concentration and ATPase activity were found to be normal. Low levels of 2,3-diphosphoglyceric acid were noted in splenic red cells but ATP turnover was normal. Since failure of the Na⁺ pump occurs in h.s. splenic red cells despite adequate substrate (ATP) and an intact ATPase system, it is suggested that the abnormality results from a change in the red cell membrane 4 which reduces the function of the pump. These characteristics of splenic red cells are similar to those found in normal red cells following in vitro incubation at 37°C. This supports the concept that cell death in h.s. results from a deterioration in membrane function secondary to substrate deprivation in the stagnant circulation of the splenic pulp.     

Na+/Ca2+ Exchanger Expression and Function in a Rabbit Model of Myocardial Infarction

Introduction: In general, sarcolemmal Na⁺/Ca²⁺ exchanger (NCX) protein and activity is increased in hearts with ventricular dysfunction. However, in a subset of studies, reduced activity of NCX has been reported. Left ventricular dysfunction (LVD) was induced in the rabbit eight weeks after an apical myocardial infarction.
Methods: Using single microelectrode voltage clamp to assess the NCX activity in isolated ventricular cells, a decrease in NCX activity by ∼30% was observed. Immunoblot analysis indicated increased NCX protein levels by ∼20% in the LVD group. The cause of this paradox is unknown. Overexpression of the protein sorcin increased the activity of NCX without affecting NCX protein levels.
Results: Sorcin protein (dimer) levels were significantly lower in the LVD group (0.67 ± 0.05 n = 15, P < 0.05) compared to sham (1.0 ± 0.16, n = 15). Sorcin monomer levels were not significantly different (sham: 1.0 ± 0.26, LVD: 0.83 ± 0.13). Mathematical modeling of NCX suggests that a reduction of NCX activity during diastole to that in LVD could be achieved by holding the diastolic membrane potential at −60 mV instead of −80 mV. Holding E_m at −60 mV decreased NCX-mediated Ca²⁺ efflux rates to values comparable to those seen in LVD and increased SR Ca²⁺ content and peak systolic [Ca²⁺] in sham and LVD cardiomyocytes.
Conclusions: In conclusion, reduced sorcin expression may be linked to the lower NCX activity in the rabbit model of LVD. Reduced NCX activity during diastole increases SR Ca²⁺ content and Ca²⁺ transient amplitude.     

Ethanol Suppresses Mycobacteria tuberculosis-Induced mRNA for Nitric Oxide Synthase in Alveolar Macrophages, In Vivo

Acute ingestion of alcohol [ethanol (ETOH)] adversely affects the immunocompetence of both naive individuals as well as chronic alcohol abusers. An increased incidence and severity of tuberculosis is found in chronic alcohol abusers. Nitric oxide (NO) produced by alveolar macrophages (AMs) may play a role in the in vitro killing of Mycobacterium avium and Mycobacterium tuberculosis (MTB). Moreover, tumor necrosis factor-α (TNF-α) is believed to be a primary cytokine mediator of NO production by AMs. Recent studies from our laboratory demonstrated that ETOH suppressed endotoxin-induced increases in both TNF-α and NO in AMs, in vivo. We tested the postulate that acute ingestion of ETOH can interfere with myco-bacteria-induced upregulation of the NO system in AMs, in vivo. We show that heat-killed M. avium complex (MAC) and human virulent MTB instilled into rat lungs rapidly increased mRNA for inducible NO synthase II (iNOS) of AMs in fluid obtained by bronchoalveolar lavage (BAL fluid). This was associated with production of reactive nitrogen intermediates [(RNIs); NO_2- and NO_3-] in BAL fluid, lung homogenate, and AMs in the absence of a significant increase in BAL fluid TNF-α. A single dose of ETOH (5.5 g/kg, ip) administered 30 min before intratracheal administration of MAC or MTB attenuated both MAC and MTB-induced increases in RNI in BAL fluid, lung, and AMs, and the increase in mRNA for iNOS. Thus, mycobacteria upregulate iNOS mRNA and enhance RNI production by AMs without any increase in the production of TNF-α. Moreover, ETOH attenuates mycobacteria-induced upregulation of mRNA for iNOS and RNI production in the absence of ETOH-mediated suppression of TNF. Speculatively, ETOH-mediated inhibition of the AM NO system may offer an explanation for the increased severity of mycobacterial infections in alcoholics.