Synthetic bactericidal peptide based on CAP37: a 37-kDa human neutrophil granule-associated cationic antimicrobial protein chemotactic for monocytes

CAP37 (cationic antimicrobial protein of molecular mass 37 kDa) is a multifunctional protein isolated from the granules of human neutrophils. It is antibiotic and chemotactic and binds lipopolysaccharide. A synthetic peptide, amino acid sequence NQGRHFCGGALIHARFVMTAASCFQ, based on residues 20-44 of CAP37 protein mimics its antibiotic and lipopolysaccharide binding action. Peptide 20-44, at the concentrations tested, has antibacterial activity against Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. The bactericidal action of the peptide was pH dependent, with maximum activity at pH 5.0 and pH 5.5 and decreased activity at pH 7.0. Various truncations, substitutions, and other modifications in the sequence deteriorate its activity. Free sulfhydryl groups and/or disulfide bridge formation are required for optimum antibiotic activity, since substitution of serines for, or alkylation of, cysteine residues 26 and 42 eliminates bactericidal activity. Evidently amino acids 20-44 represent an important, perhaps principal, antibacterial domain of CAP37. This peptide should provide new insight into the mechanism of antimicrobial activity of CAP37 and may serve as a model for new, useful, synthetic antibiotics.     

The molecular and functional characterization of E2F-5 transcription factor

1. Endotoxaemia is associated with the expression of the inducible isoform of cyclo-oxygenase, cyclo-oxygenase-2 (COX-2), and an overproduction of arachidonic acid (AA) metabolites. The role of the AA metabolites generated by COX-2 in the circulatory failure and multiple organ dysfunction caused by endotoxin is unclear. Dexamethasone prevents the expression of COX-2 and exerts beneficial effects in animal models of shock. 2. Here we compare the effects of two inhibitors of COX-2 activity, namely NS-398 (5 mg kg(-1), i.p., n=7) and SC-58635 (3 mg kg(-1), i.p., n=9) with those of dexamethasone (3 mg kg(-1), i.p., n=9) on the circulatory failure and organ dysfunction caused by lipopolysaccharide (LPS, E. coli, 6 mg kg(-1), i.v., n=11) in the rat. 3. Endotoxaemia for 6 h caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of 6-keto-PGF1alpha (indicator of the induction of COX-2) and nitrite/nitrate (indicator of the induction of iNOS). 4. Pretreatment of rats with dexamethasone attenuated the hypotension, the renal dysfunction, the hepatocellular and pancreatic injury and the induction of COX-2 and iNOS caused by LPS. In contrast, inhibition of COX-2 activity with SC-58635 or NS-398 neither attenuated the circulatory failure nor the multiple organ failure caused by endotoxin. 5. Thus, the prevention of the circulatory failure and the multiple organ injury/dysfunction caused by dexamethasone in the rat is not due to inhibition of the activity of COX-2. Our results suggest that an enhanced formation of eicosanoids by COX-2 does not contribute to the development of organ injury and/or dysfunction in rats with endotoxaemia.     

Sperm immobilizing activity of a synthetic bioactive peptide 20-44 of 37-kDa cationic antimicrobial protein (CAP37) of human neutrophils

We have previously reported that human sperm coincubated with human peripheral blood neutrophils in the presence of complement (C)-fixing antisperm antibody (ASA)-positive sera are rapidly internalized and degraded within the neutrophil phagolysosome. However, the mechanism by which motile sperm are processed within the phagolysosome is unknown. Various spermicidal/antimicrobial proteins contained in azurophilic granules that can be secreted into the phagolysosome may play a role in sperm disposal. In this study, we examined the expression of a 37-kDa cationic antimicrobial protein (CAP37) during sperm phagocytosis and the effect of its synthetic bioactive fragment, Peptide 20-44 (P20-44), on sperm motility, acrosomal integrity, and mitochondrial functionality. CAP37 expression by neutrophils undergoing ASA- and C-dependent sperm phagocytosis was increased as measured by flow cytometry. Exposure of motile sperm to a cationic P20-44, the bioactive antimicrobial fragment of CAP37, resulted in the loss of sperm motility without disruption of the acrosomal membrane. The sperm immobilizing activity (SIA) of P20-44 was modulated by the length of incubation, the concentration of the peptide, and the pH of the assay medium. SIA induced by P20-44 was partially reversible and was unaffected by the presence of anionic heparin or seminal plasma. Similar to the antimicrobial activity of P20-44, the SIA was also dependent on the presence of a disulfide bond between cysteine residues at positions 26 and 42 and was inhibited by Lipid A. However, the mechanism of action of P20-44 on sperm is not totally dependent on the molecule's cationicity, because five other cationic antimicrobial peptides had no detectable effect on sperm viability. Thus, the mechanism of action of P20-44 on human sperm is different from its cationic antibactericidal effect. These findings established that motile human sperm are sensitive to CAP37 or its synthetic bioactive peptide and suggested that this protein could play a role in neutrophil-mediated immune destruction of sperm in the female genital tract. P20-44 of CAP37 may be useful in investigating the regulation of human sperm motility and to construct "hybrid peptides" with enhanced potency as a component of vaginal contraceptive that could doubly be effective by killing infectious agents and inhibiting sperm transport.     

Suppression of murine endotoxin response by E5531, a novel synthetic lipid A antagonist

As a consequence of blood-borne bacterial sepsis, endotoxin or lipopolysaccharide (LPS) from the cell walls of gram-negative bacteria can trigger an acute inflammatory response, leading to a series of pathological events and often resulting in death. To block this inflammatory response to endotoxin, a novel lipid A analogue, E5531, was designed and synthesized as an LPS antagonist, and its biological properties were examined in vitro and in vivo. In murine peritoneal macrophages, E5531 inhibited the release of tumor necrosis factor alpha (TNF-alpha) by Escherichia coli LPS with a 50% inhibitory concentration (IC50) of 2.2 nM, while E5531 elicited no significant increases in TNF-alpha on its own. In support of a mechanism consistent with antagonism of binding to a cell surface receptor for LPS, E5531 inhibited equilibrium binding of radioiodinated LPS ([125I]2-(r-azidosalicylamido)-1, 3'-dithiopropionate-LPS) to mouse macrophages with an IC50 of 0.50 microM. E5531 inhibited LPS-induced increases in TNF-alpha in vivo when it was coinjected with LPS into C57BL/6 mice primed with Mycobacterium bovis bacillus Calmette-Guérin (BCG). In this model, the efficacy of E5531 was inversely correlated to the LPS challenge dose, consistent with a competitive antagonist-like mechanism of action. Blockade of the inflammatory response by E5531 could further be demonstrated in other in vivo models: E5531 protected BCG-primed mice from LPS-induced lethality in a dose-dependent manner and suppressed LPS-induced hepatic injury in Propionibacterium acnes-primed or galactosamine-sensitized mice. These results argue that the novel synthetic lipid A analogue E5531 can antagonize the action of LPS in in vitro and suppress the pathological effects of LPS in vivo in mice.     

A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides

During the last decade, episodes of sepsis have increased and Escherichia coli has remained the most frequent clinical isolate. Lipopolysaccharides (LPS; endotoxin) are the major toxic and antigenic components of gram-negative bacteria and qualify as targets for therapeutic interventions. Molecules that neutralize the toxic effects of LPS are actively investigated. In this paper, we describe a murine monoclonal antibody (MAb; WN1 222-5), broadly cross-reactive and cross-protective for smooth (S)-form and rough (R)-form LPS. As shown in enzyme-linked immunosorbent assay and the passive hemolysis assay, WN1 222-5 binds to the five known E. coli core chemotypes, to Salmonella core, and to S-form LPS having these core structures. In immunoblots, it is shown to react with both the nonsubstituted core LPS and with LPS carrying O-side chains, indicating the exposure of the epitope in both S-form and R-form LPS. This MAb of the immunoglobulin G2a class is not lipid A reactive but binds to E. coli J5, an RcP+ mutant which carries an inner core structure common to many members of the family Enterobacteriaceae. Phosphate groups present in the inner core contribute to the epitope but are not essential for the binding of WN1 222-5 to complete core LPS. Cross-reactivity for clinical bacterial isolates is broad. WN1 222-5 binds to all E. coli clinical isolates tested so far (79 blood isolates, 80 urinary isolates, and 21 fecal isolates) and to some Citrobacter, Enterobacter, and Klebsiella isolates. This pattern of reactivity indicates that its binding epitope is widespread among members of the Enterobacteriaceae. WN1 222-5 exhibits biologically relevant activities. In vitro, it inhibits the Limulus amoebocyte lysate assay activity of S-form and R-form LPS in a dose-dependent manner and it neutralizes the LPS-induced release of clinically relevant monokines (interleukin 6 and tumor necrosis factor). In vivo, WN1 222-5 blocks endotoxin-induced pyrogenicity in rabbits and lethality in galactosamine-sensitized mice. The discovery of WN1 222-5 settles the long-lasting controversy over the existence of anti-core LPS MAbs with both cross-reactive and cross-protective activity, opening new possibilities for the immunotherapy of sepsis caused by gram-negative bacteria.     

Pharmacological activity and safety profile of P10358, a novel, orally active acetylcholinesterase inhibitor for Alzheimer's disease

1-[(3-Fluoro-4-pyridinyl)amino]-3-methyl-1(H)-indol-5-yl methyl carbamate (P10358) is a potent, reversible acetylcholinesterase inhibitor that produces central cholinergic stimulation after oral and parental administration in rats and mice. P10358 is a 2.5 times more potent acetylcholinesterase inhibitor than THA in vitro (IC50 = 0.10 +/- 0.02 microM vs. IC50 = 0.25 +/- 0.03 microM). It also inhibits butyrylcholinesterase activity as potently as THA (IC50 = 0.08 +/- 0.05 microM vs. IC50 = 0.07 +/- 0.01 microM). Ex vivo, P10358 (0.2 - 20 mg/kg, p.o.) produced dose-dependent inhibition of brain acetylcholinesterase activity. At 10 and 20 mg/ kg, it produced profound and long-lasting hypothermia in mice. P10358 enhanced performance in rats in a step-down passive avoidance task (0.62 and 1.25 mg/kg) and in a social recognition paradigm (0.32, 0.64 and 1.25 mg/kg) in mice. It reversed scopolamine-induced deficits in the Morris Water maze in rats (1.25 and 2.5 mg/kg) and a higher dose elevated striatal homovanillic acid levels. These behavioral and biochemical effects are consistent with central cholinergic stimulation. Hemodynamic studies in the rat demonstrated a 16-fold separation between behaviorally active doses (1.25 mg/kg) and those that elevated arterial pressure (20 mg/kg). Lethality in rats occurred at an oral dose of 80 mg/kg, but not at lower doses. Chemically, P10358 is an N-aminoindole and may not have the hepatotoxic liability associated with aminoacridine structure of tacrine. P10358 had weak affinity (>10 microM) at a variety of aminergic and peptidergic receptors and uptake carriers. These properties suggest that P10358 may be a safe and promising symptomatic treatment for Alzheimer's disease.     

Pyrrolidine dithiocarbamate improves the septic shock syndromes in spontaneously hypertensive rats

1. The aim of the present study was to evaluate the effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor-kappa B (NF-kappa B), on septic shock induced by Escherichia coli lipopolysaccharide (LPS) in spontaneously hypertensive rats (SHR). 2. After injection of LPS in SHR, a marked decrease in blood pressure was observed at 3 h and vascular hyporeactivity to noradrenaline (NA) was observed after 1 h. A marked increase in plasma levels of tumour necrosis factor-alpha (TNF-alpha) and nitrite (an indicator of nitric oxide) was also observed in SHR. 3. The delayed hypotension and hyporeactivity to NA induced by LPS were significantly reserved by pretreatment of rats with PDTC (10 mg/kg). The increase in plasma levels of TNF-alpha and nitrite in LPS-treated groups was also significantly suppressed by PDTC pretreatment. In addition, the survival time of SHR treated with LPS was significantly prolonged by PDTC pretreatment. 4. The present ex vivo study demonstrates that the NA-induced contraction is attenuated and the L-arginine-induced relaxation is enhanced in aortic rings obtained from LPS-treated SHR. Both the reduction of the NA-induced contraction and the increase of L-arginine-induced relaxation were reversed by pretreatment with PDTC. However, the relaxation elicited by acetylcholine (ACh) was not affected in LPS-treated SHR when compared with sham-operated SHR. In addition, the ACh-induced relaxation in LPS-treated SHR was not affected by PDTC pretreatment. 5. In normotensive Wistar-Kyoto (WKY) rats, LPS had mild effects on blood pressure, vascular hyporeactivity and plasma levels of TNF-alpha and nitrite. At a higher dose, PDTC (10 mg/kg) also prolonged survival time and improved haemodynamics in LPS-treated WKY rats. In the ex vivo study, it was noted that the relaxation elicited by ACh was significantly (P < 0.05) attenuated in LPS-treated WKY rats. This attenuation of the ACh-induced relaxation by LPS in WKY rats was significantly reversed by pretreatment with 10 mg/kg PDTC. 6. In conclusion, PDTC prolongs survival time in rats with endotoxaemia and improves the septic shock syndromes both in vivo and ex vivo. Thus, we propose that PDTC may be of use in septic patients.     

An infant with 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase deficiency presenting with typical neonatal hepatitis syndrome: the first Japanese case

We investigated the in vivo effects of thalidomide on the production of tumor necrosis factor-alpha (TNF-alpha). An in vivo systemic release of TNF-alpha occurred after the injection of lipopolysaccharide (LPS) in male ddY mice, and the TNF-alpha serum levels reached 652.2 +/- 75.7 pg/ml 90 min after the injection of LPS (0.3 mg/kg, i. p.). When thalidomide (1, 3, or 6 mg/kg) was administered intraperitoneally 3 h before the injection of LPS (0.3 mg/kg, i. p.), thalidomide markedly enhanced LPS-induced TNF-alpha release in a dose-dependent manner. The TNF-alpha serum levels at 90 min were 640 +/- 58.6, 1985 +/- 132.6, and 2795 +/- 203.5 pg/ml, respectively, compared to 628.6 +/- 64.4 pg/ml in mice treated with LPS-alone. Pretreatment with a single injection of thalidomide (1, 3, or 6 mg/kg, i. p.) dose-dependently increased the subsequent mortality caused by a challenge with LPS (15 mg/kg, i. p.), a dose that caused death in 10% of the control mice. We conclude that thalidomide enhances in vivo TNF-alpha secretion and the lethality of LPS in mice.     

A comparison of the abilities of chlorpromazine and molindone to interact adversely with guanethidine

Chlorpromazine and molindone were tested for their abilities to impair conditioned avoidance behavior of rats. Chlorpromazine was effective within the dose range of 0.3 to 7.0 mg/kg (ID50approximately 2.0 mg/kg); molindone was effective within the range of 0.3 to 5.0 mg/kg (ID50 approximately 0.6 mg/kg). Behaviorally relevant doses of chlorpromazine and molindone were then tested for their effects on blood pressure and on adrenergic mechanisms. When given intravenously to anesthetized, hypertensive animals, both drugs (1.0 mg/kg) produced significant but transient vasodepression. When given intraperitoneally to anesthetized or to conscious hypertensive rats, the drugs did not produce significant effects on blood pressure. Both drugs (1.0 mg/kg) blocked responses to an alpha agonist (methoxamine), but chlorpromazine was significantly more potent than molindone. In addition, chlorpromazine produced a dose-dependent (1.0-10.0 mg/kg) inhibition of 3H-l-norepinephrine uptake into heart, but molindone at the same doses produced no inhibition of uptake. In related experiments, it was found that guanethidine (50 mg/kg) was an effective agent for lowering blood pressure of hypertensive rats. When chlorpromazine (3-10 mg/kg) was administered concomitantly with guanethidine, the blood pressure lowering properties of guanethidine were diminished or abolished. When molindone (1-10 mg/kg) was administered concomitantly with guanethidine, there was no loss of blood pressure control. It is concluded that molindone is an important drug, because it is an antipsychotic agent that does not interact adversely with guanethidine.     

Nimesulide prevents lipopolysaccharide-induced elevation in plasma tumor necrosis factor-alpha in rats

The present study was undertaken to test the hypothesis of possible inhibitory effect of nimesulide (4-nitro-2-phenoxymethane-sulfoxide) on plasma TNF-alpha level. Male Sprague-Dawley rats were injected intraperitoneally (i.p.) with E. coli lipopolysaccharide (LPS; 1 mg/kg), which resulted in a dramatic increase in plasma TNF-alpha level peaked 60 min post injection (3890+/-280 pg/ml, compared to undetectable values in the control group). Nimesulide (30 mg/kg) injected i.p. 60 min prior to LPS, prevented LPS-induced elevation in plasma TNF-alpha. Nimesulide alone did not alter circulating levels of TNF-alpha. It appears that the anti-inflammatory properties of nimesulide may in part be attributed to its inhibitory effect on TNF-alpha production.     

Mechanisms of insulin resistance during acute endotoxemia

We characterized the mechanisms underlying acute endotoxin-induced alterations in glucose metabolism and determined the extent to which catecholamines mediate these changes. Acute endotoxemia was induced in chronically catheterized awake rats by a bolus injection of lipopolysaccharide (LPS; 1 mg/kg; LD10). Basal glucose turnover (Rt; infusion of [5-3H]glucose), in vivo insulin action on overall glucose utilization (euglycemic clamp), glycolysis, and glycogen synthesis were determined in four groups of rats. These groups received 1) LPS (LPS rats; n = 6), 2) saline (control rats; n = 6), 3) LPS and alpha beta-blockade (alpha beta-blockade and LPS rats; n = 9), or 4) saline and alpha beta-blockade (alpha beta-blockade control rats; n = 9). In the basal state, LPS induced hypotension and transient hyperglycemia. These changes were associated with glycogen depletion in both skeletal muscle and liver, and increased Rt. During hyperinsulinemia, whole body glucose disposal was 37% decreased (105 vs. 166 mumol/kg.min; P < 0.01). This whole body insulin resistance was characterized by decreased glycogen synthesis and glycogen synthase activity, but not by altered whole body glycolysis. alpha beta-Blockade abolished transient hyperglycemia, increased Rt, and accelerated basal liver glycogen depletion (45 vs. 105 mmol/kg dry, LPS and alpha beta-blockade rats vs. LPS rats; P < 0.05), but inhibited muscle glycogenolysis. alpha beta-Blockade did not reverse the insulin resistance induced by endotoxin. These data suggest that catecholamines counteract the LPS-induced increase in basal glucose turnover and stimulate muscle glycogenolysis during acute endotoxemia. These effects might explain the better preservation of hepatic glycogen in the absence than in the presence of alpha beta-blockade and serve as a defense mechanism against hypoglycemia. Catecholamines do not seem to be the immediate causes of insulin resistance during acute endotoxemia.     

Pretreatment of young pigs with vitamin E attenuates the elevation in plasma interleukin-6 and cortisol caused by a challenge dose of lipopolysaccharide

The effect of a short-term, high-dose intramuscular injection of d-alpha-tocopherol was studied in pigs given a challenge dose of lipopolysaccharide (LPS). Twenty-four pigs surgically fitted with jugular catheters were used in a 2 x 2 factorial design. Pigs received either 0 or 600 mg d-alpha-tocopherol by intramuscular injection for 3 d before receiving an intraperitoneal injection of saline containing either 0 or 5 microgram/kg body weight Escherichia coli LPS. Blood was collected from indwelling jugular catheters at 0, 1, 2, 4, 6, 8, 12 and 24 h after injection of LPS. Plasma alpha-tocopherol levels were 13-fold greater (P < 0.01) at time 0 in pigs pretreated with 600 mg d-alpha-tocopherol (9.9 +/- 1.3 mg/L) than in those not treated with d-alpha-tocopherol (0.74 +/- 0.09 mg/L). Injection of LPS increased (P < 0.05) plasma levels of interleukin-6 (IL-6) and cortisol at 2-h postinjection, regardless of vitamin E treatment. However, pigs that received alpha-tocopherol before the LPS challenge had substantially lower (P < 0.05) peak levels of IL-6 and cortisol than pigs not receiving alpha-tocopherol. These results suggest that supplementation with a surfeit level of vitamin E reduces the response of pigs to endotoxin.     

In vivo nitrate tolerance is not associated with reduced bioconversion of nitroglycerin to nitric oxide

BACKGROUND: In vitro data suggest that reduced bioconversion of nitroglycerin (NTG) to nitric oxide (NO) contributes to the development of vascular and hemodynamic tolerance to NTG. We examined the in vivo validity of this hypothesis by measuring NTG-derived NO formation by in vivo spin-trapping of NO in vascular tissues from nitrate-tolerant and -nontolerant rats. METHODS AND RESULTS: Five groups (n = 6 to 8 each) of conscious chronically catheterized rats received NTG (0.2 or 1 mg/h IV) for 72 hours (nitrate-tolerant groups). Four other groups received either NTG vehicle (placebo, for 72 hours) or were left untreated (control). Nitrate tolerance was substantiated by a reduced (55% to 85%) hypotensive response to NTG in vivo and a reduced relaxation to NTG in isolated aortic rings. NTG-derived NO formation in aorta, vena cava, heart, and liver was measured as NOFe(DETC)2 and NO-heme complexes formed in vivo during 35 minutes combined with ex vivo cryogenic electron spin resonance spectroscopy. NO formation was significantly (P < .05) increased in all tissues in nitrate-tolerant rats in an NTG dose-dependent manner. Furthermore, the amount of NO formed from a bolus dose of NTG (6.5 mg/kg over 20 minutes) was similar in nitrate-tolerant and -nontolerant rats. CONCLUSIONS: The results suggest that vascular and hemodynamic NTG tolerance occurs despite high and similar rates of NO formation by NTG in tolerant and nontolerant target tissues. This finding is compatible with the assumption that reduced biological activity of NO, rather than reduced bioconversion of NTG to NO, contributes to in vivo development of nitrate tolerance.     

Differential expression of apoptosis receptors on diffuse and intestinal type stomach carcinoma

1. A transient two fold increase in the cyclic GMP content was observed in rat freshly isolated glomeruli 6 to 9 h after a single subcutaneous injection of 20 mg kg-1 cyclosporine A (CsA) in conscious animals. 2.In vitro stimulation with endothelin 3 (ET-3) of isolated glomeruli obtained from CsA-untreated rats resulted in a dose-dependent increase in cyclic GMP content. The increase observed with 10 nM ET-3 was similar to that observed in glomeruli isolated 9 h after in vivo CsA administration. 3. The rise in glomerular cyclic GMP content after in vivo CsA injection was prevented by in vivo treatment with L-NAME (10 mg kg-1) or by in vitro calcium deprivation of the incubation medium. 4. The stimulating effects of CsA on glomerular cyclic GMP content were inhibited by in vivo administration of the ETB receptor antagonist BQ-788 (2 mg kg-1) but not by the ETA receptor antagonist BQ-123 (2 mg kg-1). 5. The maximum increase in glomerular cyclic GMP content induced in vitro by acetylcholine (100 microM) and by ET-3 (100 nM) was slightly lower (approximately by 20-25%; P < 0.05) in glomeruli from CsA-treated rats than in glomeruli from untreated rats. In contrast, the maximum increase achieved with 1 microM sodium nitroprusside was similar in both groups. 6. A single subcutaneous injection of CsA did not significantly alter the glomerular mRNA expression of constitutive endothelial NO synthase (eNOS), as evaluated by RT-PCR, whereas the mRNA expression of the inducible NO synthase (iNOS), which follows pretreatment with lipopolysaccharide, was prevented. 7. These results indicate that in vivo administration of a single dose of cyclosporine A transiently increases the cyclic GMP content of freshly isolated glomeruli, and that activation of ETB receptors and stimulation of the NO pathway are involved in this process. Furthermore, a single administration of CsA does not impair eNOS mRNA expression and only slightly reduces NO-dependent glomerular cyclic GMP production.     

Calcitriol modulates in vivo and in vitro cytokine production: a role for intracellular calcium

Calcitriol modulates in vivoand in vitro cytokine production: A role for intracellular calcium. Background. Several immunomodulatory properties of calcitriol are currently known, however, only little information is available regarding the in vivo and in vitro effects of calcitriol on cytokine production in chronic renal failure. Methods. To study the in vitro effect of calcitriol on lipopolysaccharide (LPS)-induced cytokine production, peripheral blood mononuclear cells (PBMC, 2.5 ml/ml) from 12 chronic dialytic (HD), 15 undialyzed chronic renal failure (CRF) patients and 10 normal subjects (N) were incubated at 37 degrees for 12 hours with 100 ng of LPS (E. coli and P. maltofilia). Increasing doses of calcitriol from 10-10 to 10-9 M were added and cell associated TNF-alpha and IL-1beta were determined by immunoreactive tests after three freeze-thaw cycles. The intradialytic TNF-alpha and IL-1beta production were evaluated in vivo in 12 HD patients before and after three months of intravenous calcitriol treatment (6 microgram/week). Intracellular calcium [Ca++]i was determined on PBMC with a cytofluorimetric assay using FLUO-3 AM as the indicator. Results. In vitro, TNF-alpha increased from 3.6 +/- 1.9 pg/cell to 1797 +/- 337 in N, from 4.5 +/- 1.7 to 1724 +/- 232 in CRF and from 3.4 +/- 2.3 to 1244 +/- 553 in HD after the LPS stimulus. The production of TNF-alpha was inhibited by calcitriol in a dose-dependent manner [LPS + Vit.D3 100 ng, 2.9 +/- 2.1 in N, 3.7 +/- 1.9 in CRF and 3.4 +/- 1.7 in HD; LPS + Vit.D3 50 ng, 263 +/- 296 (N), 6.73 +/- 11 (CRF), 38 +/- 28 (HD); LPS + Vit.D3 25 ng = 873 +/- 583 (N), 325 +/- 483 (CRF), 588 +/- 507 (HD); LPS + Vit.D3 12.5 ng, 954 +/- 483 (N), 912 +/- 510 (CRF), 875 +/- 527 (HD)]. Comparable data were observed on IL-1beta production. In vivo, the intradialytic TNF-alpha increase (from 8.5 +/- 2.3 to 19 +/- 5.6 pg/2.5 x 106 cell) during hemodialysis was markedly reduced after calcitriol therapy (from 6.6 +/- 3.1 to 11 +/- 4.7). [Ca++]i decreased from 105 +/- 25 to 72 +/- 18 nM (P < 0.05) and a positive correlation between cytokine levels and [Ca++]i was found (r = 0.79; P < 0.001). Conclusions. The in vitro increase of cell-associated cytokine after LPS challenge was inhibited by calcitriol in a dose-dependent manner. These data suggest a possible in vivo modulatory effect of calcitriol therapy on cytokine production in hemodialysis.     

Bacterial translocation is inhibited in inducible nitric oxide synthase knockout mice after endotoxin challenge but not in a model of bacterial overgrowth

BACKGROUND: Studies have shown that nitric oxide (NO) and NO synthase (NOS) inhibitors injure and protect organs after endotoxin (lipopolysaccharide [LPS]) challenge. OBJECTIVE: To test the hypothesis that LPS-induced gut injury and bacterial translocation (BT) are mediated through activation of inducible NOS (iNOS). DESIGN: A randomized, controlled study using genetically altered, iNOS gene knockout mice. SETTING: University research laboratory. METHODS: Forty-five wild-type (iNOS+/+) or homozygous mutant (iNOS-/-) mice weighing 25 to 35 g were challenged with Escherichia coli LPS or saline (10 mg/ kg) intraperitoneally (n = 8/group). In a second set of experiments, a bacterial overgrowth model of BT (E coli monoassociation) was tested (n = 6-7/group). The mesenteric lymph nodes and cecums were cultured, and liver, ileal, and blood nitrite and nitrate levels measured 24 hours after LPS or E coli monoassociation. RESULTS: After LPS challenge, 87.5% of the iNOS+/+ mice but 0% of the iNOS-/- mice had BT to their mesenteric lymph nodes (P < .01; chi 2 analysis). Nitrite and nitrate levels of the liver, ileum, and blood were higher in the iNOS+/+ mice (P < .05). In the E coli overgrowth model, BT to mesenteric lymph nodes occurred in 100% of iNOS-/- and iNOS+/+ mice. CONCLUSIONS: In this limited study, LPS-induced BT did not occur in iNOS-deficient mice, suggesting that LPS induction of increased iNOS activity is necessary for LPS-induced BT to occur. In contrast, iNOS activation does not seem to be necessary in a bacterial overgrowth model of BT.     

Anesthesia with sodium pentobarbital enhances lipopolysaccharide-induced cardiovascular dysfunction in rats

Lipopolysaccharide (LPS)-induced hypotension and impaired aortic contraction to norepinephrine (NE) are thought to be consequent to induction of nitric oxide synthase (iNOS). Anesthesia is often employed in studies of the mechanisms mediating LPS-induced cardiovascular dysfunction in rats. Since sympathetic nervous system activity and compensatory mechanisms can be altered by anesthesia, this study was designed to determine a) if the cardiovascular dysfunction associated with LPS (5 mg/kg, i.v.)-induced endotoxin shock is enhanced in anesthetized compared with conscious male Wistar rats, and b) the potential role of iNOS in these responses to LPS. Arterial pressure and heart rate were continuously measured via a femoral arterial cannula. Six hours after LPS, conscious rats had a stable mean arterial pressure (MAP) and were tachycardic, while anesthetized rats showed a significant decrease in MAP without tachycardia. Small mesenteric arterioles (200-300 microns) were isolated, and the endothelium was removed six h after LPS. Intraluminal diameter was continuously recorded while vessels were maintained at a constant intraluminal pressure of 40 mmHg. Norepinephrine-induced contraction and oscillations/min were impaired to a greater extent in arterioles from LPS-treated anesthetized rats than in those from conscious rats. Calcium-dependent and -independent nitric oxide formation, reflected as cGMP accumulation, were also determined in aortic rings treated with a chelator of Ca2+, EGTA, or the inhibitor of nitric oxide synthase activity, L-NAME. In rings from saline-treated conscious and anesthetized rats, cGMP accumulation was significantly reduced by EGTA and L-NAME, indicating calcium-dependent constitutive (cNOS) activity. However, in aortic rings from LPS-treated conscious and anesthetized rats, cGMP accumulation was not affected by EGTA and was significantly greater in rings from anesthetized vs. conscious rats. These results suggest that cardiovascular dysfunction is more prominent in LPS-treated anesthetized vs. conscious rats. This effect may be related to increased induction of iNOS in the presence of anesthesia.     

The effect of endotoxin on in vivo rat alveolar macrophage phagocytosis

This study was performed to explore whether alveolar macrophage (AM) phagocytosis would be impaired during endotoxemia. Therefore, we characterized in vivo AM phagocytic function in rats following either intravenous (i.v.) or intratracheal (i.t.) administration of lipopolysaccharide (LPS). The i.v. administration of LPS to rats at dosages of 0, 1, 2, and 5 mg/kg showed that increasing LPS doses were significantly associated with increased AM phagocytosis of 198Au colloid (P < .01), decreased recovery of AMs in bronchoalveolar lavage (BAL) (P = .017), no significant differences in neutrophil recovery by lavage (P = .15), or in the concentration of albumin in BAL (P = .14). Across the dosages of LPS administered i.t. (i.e., 0, 1, 5, and 10 mg/kg), there was no difference in AM phagocytosis (P = .29), a significant decrease in AM recovery (P = .002), a significant increase in neutrophil number (P = .01), and little effect on the concentration of albumin (P = .06). Thus, we found that the administration of endotoxin to rats did not impair in vivo AM phagocytic function. In fact, our findings suggest that the i.v. administration of LPS may increase AM phagocytosis of 198Au.