Molecular detection of Mycoplasma pneumoniae in adults with community-acquired pneumonia requiring hospitalization.

Mycoplasma pneumoniae infection was diagnosed in 18 (12.5%) of 144 adults hospitalized with community-acquired pneumonia. The infection was demonstrated by PCR in 15 patients and by serology, using two methods, in 10 patients. The mean age of the 8 patients with positive M. pneumoniae PCR and negative serology was significantly higher than that of the 10 patients with positive serology.

Intravenous and inhalation toxicokinetics of sarin stereoisomers in atropinized guinea pigs.

We report the first toxicokinetic studies of (+/-)-sarin. The toxicokinetics of the stereoisomers of this nerve agent were studied in anesthetized, atropinized, and restrained guinea pigs after intravenous bolus administration of a dose corresponding to 0.8 LD50 and after nose-only exposure to vapor concentrations yielding 0.4 and 0.8 LCt50 in an 8-min exposure time. During exposure the respiratory minute volume and frequency were monitored. Blood samples were taken for gas chromatographic analysis of the nerve agent stereoisomers and for measurement of the activity of blood acetylcholinesterase (AChE). In all experiments, the concentration of (+)-sarin was below the detection limit (<5 pg/ml). The concentration-time profile of the toxic isomer, i.e., (-)-sarin, after an intravenous bolus was adequately described with a two-exponential equation. (-)-Sarin is distributed ca. 10-fold faster than C(-)P(-)-soman, whereas its elimination proceeds almost 10-fold slower. During nose-only exposure to 0.4 and 0.8 LCt50 of (+/-)-sarin in 8 min, (-)-sarin appeared to be rapidly absorbed. The blood AChE activity decreased during the exposure period to ca. 15 and 70% of control activity, respectively. There were no effects on the respiratory parameters. A significant nonlinearity of the toxicokinetics with dose was observed for the respiratory experiments.

Aerosolized endotoxin is immediately bound by pulmonary surfactant protein D in vivo.

Collectins are carbohydrate binding proteins that are implicated in innate host defense. The lung collectins, surfactant proteins A and D (SP-A and SP-D), bind a variety of pathogens in vitro and influence phagocytosis by alveolar macrophages. In this report we show that SP-D binds endotoxin (lipopolysaccharide, LPS) in vivo in a rat model of acute respiratory distress syndrome (ARDS). Intratracheal aerosolization of LPS in rats resulted in the typical features of human ARDS. Total amounts of SP-D, as well as the carbohydrate binding properties of SP-D were measured in lung lavage as a function of time. The amount of SP-D did not change during 24 h. Interestingly, SP-D in lung lavage isolated from rats during the first 2 h after LPS treatment, was not able to bind to carbohydrate. Further analysis revealed that the carbohydrate binding sites of SP-D were occupied by LPS, suggesting that SP-D is an LPS scavenging molecule in vivo. Electron microscopic analysis indicated that, 1 h after LPS aerosolization, aggregates of SP-D with LPS were found in lysosomal structures in alveolar macrophages. We conclude that the lung collectin SP-D binds inhaled endotoxin in vivo, which may help to protect the lung from endotoxin-induced disease.

Efficacy of Curosurf in a rat model of acute respiratory distress syndrome.

Curosurf, a natural lung surfactant, is considered a potential candidate for improving the treatment of acute respiratory distress syndrome (ARDS). To investigate this in a rat model of early-stage ARDS, Curosurf (62.5, 125 or 250 mg x kg(-1)) was administered by intratracheal bolus at 10 or 24 h following an intratracheal lipopolysaccharide (LPS; 1.6 mg x kg(-1)) challenge. Survival, respiratory frequency (fR), lung wet weight (LWW), total protein and cell differentiation in bronchoalveolar lavage fluid (BALF) were assessed. Curosurf treatment at 10 h after LPS challenge resulted in 100% survival at both 62.5 and 125 mg x kg(-1); at a dose of 250 mg x kg(-1) administered at 10 h after LPS, 1 out of 6 animals died. At a dose of 125 mg x kg(-1) Curosurf administered at 24 h after LPS, 1 out of 6 animals died. In contrast, only 35% of animals survived when not treated with Curosurf. Curosurf treatment resulted in an improved fR and in a significantly decreased LWW, total protein and number of polymorphonuclear cells in BALF. In conclusion, Curosurf treatment improved respiratory frequency and decreased mortality, pulmonary oedema and inflammation. As the decreased mortality was observed in spontaneously breathing nonoxygenated animals, the results cannot be extrapolated to human artificially ventilated acute respiratory distress syndrome patients with the expectation of a decreased mortality. The results suggest, however, that Curosurf may be an important therapeutic measure in early-stage acute respiratory distress syndrome.

Inhalation toxicokinetics of soman stereoisomers in the atropinized guinea pig with nose-only exposure to soman vapor.

The toxicokinetics of the four stereoisomers of the nerve agent C(+/-)P(+/-)-soman were studied in anesthetized, atropinized guinea pigs for nose-only exposure to soman vapor. During exposure the respiratory minute volume (RMV) and respiratory frequency (RF) were monitored. Blood samples were taken for chiral gas chromatographic analysis of the concentrations of nerve agent stereoisomers and for measurement of the progressive inhibition of acetylcholinesterase (AChE). The animals were exposed for 4-8 min to 0.4-0.8 LCt50 of C(+/-)P(+/-)-soman. Concentrations of the P(-)-isomers increased rapidly during exposure, up to several nanograms per milliliter of blood. Mathematical equations describing the concentration-time courses of the P(-)-isomers were obtained by nonlinear regression. The kinetics were mathematically described as a discontinuous process, with a monoexponential equation for the exposure period and a two-exponential equation for the postexposure period. The absorption phase of C(+)P(-)-soman lagged behind that of the C(-)P(-)-isomer, presumably due to preferential covalent binding at as yet unidentified binding sites. The terminal half-life observed after nose-only exposure is longer than that observed after an equitoxic iv bolus administration, which suggests the presence of a depot in the upper respiratory tract from which absorption continues after termination of the exposure. Two types of nonlinearity of the toxicokinetics were observed, i.e., with dose and with exposure time. The AChE activity was rapidly inhibited during exposure to the nerve agent vapor. There were no soman-related effects on RMV and RF. The toxicokinetics of the soman stereoisomers observed for nose-only exposure are compared with those determined for iv bolus and sc administration.

Evaluation of PCR, culture, and serology for diagnosis of Chlamydia pneumoniae respiratory infections.

We prospectively studied 156 patients with a diagnosis of community-acquired pneumonia requiring admission. Several respiratory specimens were obtained for the detection of Chlamydia pneumoniae by cell culture and PCR. Three serum samples were obtained from each patient. Serological diagnosis of a C. pneumoniae infection was determined by the microimmunofluorescence (MIF) test, the complement fixation (CF) test, and recombinant lipopolysaccharide (LPS) enzyme-linked immunosorbent assay (ELISA; referred to as the rDNA LPS ELISA). Twenty-three patients (15%) had serological results compatible with acute C. pneumoniae infection; nine (39%) of these subjects were C. pneumoniae PCR positive. Twenty-two patients (14%) had positive PCR results without serological evidence of an acute C. pneumoniae infection. An attempt was made to calculate the sensitivities and specificities of the MIF test, rDNA LPS ELISA, and PCR for the diagnosis of chlamydial community-acquired pneumonia. Several "gold standards" were defined. Generally, the sensitivities of the rDNA LPS ELISA and MIF were comparable, while the sensitivity of the CF test was shown to be very low. Independent of the gold standard used, the best PCR results were obtained with nasopharyngeal specimens. However, the predictive value of a positive C. pneumoniae PCR result for patients with community-acquired pneumonia remains unknown and may be low. Although a widely accepted gold standard is still lacking, the rDNA LPS ELISA may currently be the preferred tool for diagnosing acute respiratory Chlamydia infections in routine clinical practice. However, the MIF test remains the method of choice for determining the prevalence of C. pneumoniae infections in a given community.

Toxicokinetics of sulfur mustard and its DNA-adducts in the hairless guinea pig.

In order to provide a quantitative basis for pretreatment and therapy of intoxications with sulfur mustard (SM) the toxicokinetics of this agent as well as its major DNA-adduct were studied in male hairless guinea pigs for the intravenous, respiratory and percutaneous routes. The study comprised measurement of the concentration-time course of SM in blood and measurement of the concentrations of intact SM and its adduct to guanine in various tissues at several time points after administration of, or exposure to SM. SM was analyzed in blood and tissues by gas chromatography with automated thermodesorption injection and mass-spectrometric detection. DNA-adducts were measured via an immuno-slot-blot method. In contrast with nerve agents of the phosphofluoridate type, SM partitions strongly to various organs, especially the lung, spleen, liver and bone marrow. The respiratory toxicity of SM appears to be local, rather than systemic. Surprisingly, the maximum concentration of SM in blood upon percutaneous exposure to 1 LCt50 (10,000 mg.min.m-3, estimated) is approximately 6-fold higher than that for nose--only exposure to 3 LCt50 (2,400 mg.min.m-3). Pretreatment of hairless guinea pigs with the potential scavengers N-acetyl cysteine or cysteine isopropyl ester did not significantly increase the LCt50-value for nose--only exposure to SM vapor.

New generic approach to the treatment of organophosphate poisoning: adenosine receptor mediated inhibition of ACh-release.

Current treatment of acute organophosphate (OP) poisoning includes a combined administration of a cholinesterase reactivator (oxime), a muscarinic receptor antagonist (atropine) and an anticonvulsant (diazepam). This treatment is not adequate since it does not prevent neuronal brain damage and incapacitation. Here, as in a recent review it is stated that other therapeutic approaches may improve protection. Former studies on the "direct effects" of oximes led to the conclusion that drug-induced inhibition of acetylcholine (ACh)-release shortly (1 min) after the acute OP-intoxication, could prevent and counteract convulsions and improve survival. In general, the accumulation of ACh in the synaptic cleft is considered to be responsible for the symptoms that ultimately lead to death. Therefore, prevention or suppression of this excessive accumulation of ACh could be a generic approach to antagonize OP-poisoning. Preliminary evidence for this concept has been put forward. Evaluation of drugs that would be able to prevent and counteract ACh accumulation, led to the conclusion that adenosine receptor agonists could be promising candidates. Pilot experiments demonstrated that intramuscular administration of the adenosine receptor agonists NECA (5'-N-ethylcarboxamido-adenosine) or CPA (N6-cyclopentyl adenosine) 1 min following a subcutaneous soman poisoning (1.5-2LD50) in rats, resulted in (1) prevention or postponement of chewing, salivation, convulsive activity, and respiratory distress (cholinergic symptoms), (2) improvement of survival rate (24 h), (3) a low level of extracellular brain ACh, as opposed to high levels of extracellular brain ACh in untreated animals. It is concluded that (1) adenosine agonists protect acutely soman-poisoned rats without the need of additional treatment with atropine, oxime or diazepam, (2) prevention of ACh accumulation in this way may be a new generic approach in the treatment of OP-poisoning.

Toxicologic evaluation of pepper spray as a possible weapon for the Dutch police force: risk assessment and efficacy.

The efficacy and possible health risks of pepper spray were evaluated. In a number of countries, pepper spray is being used by police forces to aid in arresting aggressive individuals. Pepper spray is commercially available as a canister filled with Capsicum extract, which contains capsaicin as the active component. When applied in the form of a spray, it causes an acute inflammation, and humans involuntarily close their eyes, experience a burning feeling on the skin, and are usually rapidly incapacitated. Use by the U.S. police was successful in subduing aggressive individuals in 90% of cases, and a reduction of injury to both police and arrested individuals was noted. In general, pepper spray appeared to be a relatively safe weapon with small risk of causing acute physical harm. Despite this evidence, a number of fatalities were reported in the United States following the use of pepper spray. However, it was concluded that it was not the pepper spray but rather other factors such as drugs and hog-tying that contributed to the cause of death. In only 1 case, that of an asthmatic man, was it concluded that the use of pepper spray contributed to the cause of death. Much attention has been paid to possible genotoxic effects of Capsicum extract such as mutagenicity and carcinogenicity. It was concluded that the risk of long-term health effects is negligible. Because pepper spray may induce bronchoconstriction, people suffering from chronic obstructive lung disease may be hypersensitive to it. Although the results of one study indicate that asthmatics do not develop additional bronchoconstriction following inhalation of capsaicin, the number of experimental data are too few to draw sound conclusions.

Diagnosis of Chlamydia pneumoniae infection in patients with chronic obstructive pulmonary disease by micro-immunofluorescence and ELISA.

The incidence of Chlamydia pneumoniae infection was determined in patients with chronic obstructive pulmonary diseases (COPD) by prospective serial serology. Chlamydia-specific IgG, IgM and IgA antibodies were detected with a recombinant DNA lipopolysaccharide (LPS) ELISA as well as with a micro-immunofluorescence (MIF) assay with C. pneumoniae elementary bodies. From 271 consecutive COPD patients who visited the outpatient clinic of the department of pulmonary diseases (211 males, 60 females, age range 34-88 years, mean age 66 SD 10 years), blood samples (n = 1058) were taken every 2-7 months; the observation period ranged from 3 to 19 months (mean 15 SD 4). The prevalence of chlamydial IgG was 72% with the MIF and 53% with the rDNA LPS ELISA. More than 90% of the COPD patients had no significant changes in their chlamydia-specific IgG, IgA and IgM titres in either test during the observation period. Seven (3%) patients had MIF results indicating acute C. pneumoniae infection during their surveillance period, of whom five were confirmed by rDNA LPS ELISA. Eleven (4%) additional patients were infected during observation, as determined by rDNA LPS ELISA only. These patients had significantly elevated C. pneumoniae-specific IgG and IgA MIF titres, as compared with the patients without infection. All 18 patients with serological evidence of acute infection during their surveillance period were re-tested in a commercial MIF test that can distinguish between C. pneumoniae, C. trachomatis and C. psittaci LPS-specific antibodies, but no evidence of C. trachomatis or C. psittaci infection was found. The incidence of chlamydial infection was 2.2 and 5.3/100 person-years, when diagnosed by MIF and rDNA LPS ELISA, respectively. It is concluded that the rDNA LPS chlamydia assay may currently be the most sensitive serological tool for diagnosing recent respiratory chlamydia infections and that C. pneumoniae infection occurs frequently in COPD patients.

Intratracheal aerosolization of endotoxin (LPS) in the rat: a comprehensive animal model to study adult (acute) respiratory distress syndrome.

The aim of the study was to extend existing evidence that intratracheal aerosolization of LPS may serve as a very relevant model to study ARDS. The authors investigated the sequence of pathogenic events reflected by changes in levels of tumor necrosis factor alpha (TNF alpha), surfactant-associated protein A (SP-A) in BAL fluid, in addition to cell count, edema formation, and respiratory function. Within 24 h following intratracheal aerosolization of LPS in the rat, ARDS could be diagnosed according to the lung injury score for patients. This score includes the extent of the inflammatory density on chest X-rays, the severity of hypoxemia, the decline in lung compliance, and the level of PEEP (positive end expiratory pressure). In addition, other typical features of human ARDS appeared to be present in this model: (1) increased microvascular permeability reflected by edema, elevated levels of protein and of LDH, and increased numbers of PMNs in BAL fluid; (2) high levels of TNF alpha in BAL fluid preceding the appearance of PMNs; (3) changes in breathing pattern and a gradual development of respiratory failure with decreased compliance. SP-A levels in BAL fluid doubled within one hour after LPS administration, suggesting that this collectin may play a role in the immediate inflammatory response. Taken together, the findings presented here suggest that intratracheal LPS administration mimics the clinical development of ARDS very closely.

Pharmacological effects of oximes: how relevant are they?

The increased international concern about the threat of military and terroristic use of nerve agents, prompted us to critically consider the expected value of the currently available oxime treatment of nerve agent poisoning. Although oximes have been designed to reactivate the inhibited acetylcholinesterase (AChE), clinical experience has indicated that they are not always very effective as reactivators and at this very moment none of them can be regarded as a broad-spectrum antidote. In spite of this drawback, oximes are worth further investigating, since recent data derived from soman or tabun lethally intoxicated non-human primates suggest that the oxime HI-6 may exert a pharmacological effect that is not related to reactivation of inhibited AChE, but still leads to survival. This pharmacological effect causes recovery of neuronal transmission in the respiratory centres of the brain and recovery of neuromuscular transmission in the diaphragm. These findings have stimulated research to reveal the pharmacological basis of these effects in order to find drugs which could be more effective and less toxic than the available oximes. Since cholinergic drugs were able to exert this effect, a new concept for further treatment is suggested: maintenance of neuronal transmission in spite of continued AChE-inhibition by pharmacological manipulation of the cholinergic receptor. This should renew interest in the diverse pharmacological effects of oximes to reach a more effective treatment in the future.

Effects of low doses of cholinesterase inhibitors on behavioral performance of robot-tested marmosets.

To investigate at which dose levels undesirable effects started, behavioural performance and several physiological parameters were measured in marmosets (Callithrix jacchus) after soman (1.75 and 3.5 micrograms/kg), sarin (3 and 6 micrograms/kg), physostigmine (10 and 20 micrograms/kg), and pyridostigmine (200 and 400 micrograms/kg). Effects on performance were investigated with a discrete-trial, two-choice visual discrimination task and a hand-eye coordination task. The former test appeared more sensitive to disruption than the hand-eye coordination task. "Motor speed" was not disrupted by any of the four compounds. However, "choice time" as well as "no attempts" increased and were clearly more disturbed by soman and physostigmine than by sarin and pyridostigmine. All effects had disappeared after 24 h. Except for a small effect of sarin on heart rate and blood pressure, none of the cholinesterase (ChE) inhibitors affected a number of physiological parameters at behavioural effective does that caused a profound ChE inhibition in blood. Take together, these results strongly suggest that both soman and physostigmine may interfere with higher CNS functions at low dose levels. These effects may go undetected because physical signs are absent.

The functional role of molecular forms of acetylcholinesterase in neuromuscular transmission.

The severity of poisoning following acetylcholinesterase (AChE) inhibition correlates weakly with total AChE activity. This may be partly due to the existence of functional and non-functional pools of AChE. AChE consists of several molecular forms. The aim of the present study was to investigate which of these forms will correlate best with neuromuscular transmission (NMT) remaining after partial inhibition of this enzyme. Following sublethal intoxication of rats with the irreversible AChE inhibitor soman, diaphragms were isolated after 0.5 or 3 h. It appeared that at 3 h after soman poisoning the percentage of G1 increased, while those of G4 and A12 decreased. NMT was inhibited more strongly than in preparations obtained from the 0.5 h rats with the same level of AChE inhibition, but with a normal ratio of molecular forms. NMT correlated positively with G4 as well as with A12, but inversely with G1. In vitro inhibition with the charged inhibitors DEMP and echothiophate resulted in higher levels of total AChE, relatively less G1 and more G4 and A12 than after incubation with soman, but led to less NMT. Treatment of soman-intoxicated rats with the reactivating compound HI-6 resulted in preferential reactivation of A12, persisting low levels of G1 and concurrent recovery of NMT as compared with saline-treated soman controls with equal total AChE activity. Apparently, in rat diaphragm G4 and A12 are the functional AChE forms.

Non-reactivating effects of HI-6 on hippocampal neurotransmission.

Effects of the oxime HI-6, unrelated to reactivation of acetylcholinesterase (AChE), on field potentials in the dentate gyrus of the rat hippocampus following AChE inhibition, were investigated both in vitro and in vivo. In hippocampal slices, AChE inhibition decreased the perforant path evoked population spike amplitude (PSA). This effect could be prevented by pre-incubation of the slices with atropine (0.1-1 microM) or with the M1 muscarinic receptor antagonist pirenzepine (1 microM). A similar preventive effect was found after pre-incubation with the GABAA antagonist picrotoxin (20 microM), suggesting that the effects of AChE inhibition in vitro may be due to an enhancement of GABAergic inhibitory activity via activation of M1-muscarinic receptors. The effects of AChE inhibition in vivo were variable; both increases and decreases of the PSA were found. Following AChE inhibition, HI-6 increased the PSA dose-dependently, both in the in vivo and in the in vitro hippocampus. At higher oxime doses the perforant path stimulation elicited multiple population spikes. The effects of the oxime were presumably not mediated by an antagonism of cholinergic receptors, since they could not be mimicked with cholinergic antagonists like atropine, mecamylamine or gallamine. Further testing of the nature of the HI-6 effect in hippocampal slices in vitro, using a paired antidromic-orthodromic stimulation protocol, showed that HI-6 may interfere with GABAergic inhibition.

Search for a therapy against soman-intoxication.

This mini-review mainly describes a part of the pharmacological research carried out in our laboratory during the past decades, aimed at finding a therapy against intoxication by cholinesterase-inhibiting organophosphates, in particular against the nerve agent soman. In particular soman, because this is one of the nerve agents that consistently appears to be very resistant to treatment. Various experimental approaches are described. Yet, even after all these years of research an adequate (pre)treatment against poisoning by soman is still not available.

Comparison of the therapeutic effects and pharmacokinetics of HI-6, HLö-7, HGG-12, HGG-42 and obidoxime following non-reactivatable acetylcholinesterase inhibition in rats.

The oximes HI-6, HLö-7, HGG-12, HGG-42 and obidoxime were used in a previously developed rat model to evaluate the therapeutic effects of oximes other than acetylcholinesterase (AChE) reactivation (so-called "non-reactivating effects"). To test this anaesthetized, atropinized and artificially ventilated rats (n = 8 or 16) were poisoned with a three times LD50 dose of the potent AChE-inhibitor crotylsarin (CRS, i.v.). CRS-inhibited rat AChE dealkylates instantaneously, thereby excluding AChE reactivation by the oximes. Five minutes after poisoning the rats were treated (i.v.) with an oxime or saline and 10 min later artificial ventilation was terminated. Survival times were determined. Saline-treated animals died within 15 min. In comparison, treatment with HI-6, HLö-7, HGG-12, HGG-42 or obidoxime resulted in a significant prolongation of survival time. In the groups treated with HLö-7, HI-6 or HGG-12, 12-37% of the animals survived more than 24 h. It was investigated whether differences in therapeutic effectiveness are caused by differences in pharmacokinetics of the oximes. The plasma half-lives of HI-6, HLö-7, HGG-12, HGG-42 and obidoxime amounted to 67, 63, 27, 55 and 179 min, respectively. At doses of 75 or 150 mumol/kg, all oximes could be detected in brain and medulla oblongata in similar amounts (6-10 nmol/g tissue). In vitro, all oximes were effective in restoring failure of neuromuscular transmission (NMT) caused by CRS, albeit with varying potency. All oximes bound with affinities in the micromolar range to rat brain muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Therapeutic efficacy of HI-6 in soman-poisoned marmoset monkeys.

The therapeutic efficacy of the oxime HI-6 against intoxication with the irreversible cholinesterase (ChE) inhibitor soman was tested in marmoset monkeys. Five out of six marmosets, intoxicated with 5 x LD50 soman and treated immediately with diazepam (0.2 mg.kg-1 iv) and 15 sec later with atropine (0.5 mg.kg-1 im) and HI-6 (50 mg.kg-1 im), survived for more than 24 hr. One of these animals died after 4 days. In the HI-6-treated marmosets blood ChE activity was inhibited at a rate slower than that in three animals treated similarly but with saline instead of HI-6. The latter marmosets died within 8 min after soman. HI-6 achieved its plasma peak 5 min after injection and was eliminated with a t1/2 of about 40 min. In a second experiment similarly treated marmosets were euthanized at 5 min (three saline-treated animals) or at 10 min (three HI-6-treated animals) after the soman intoxication to enable the determination of acetylcholinesterase (AChE) activities in diaphragm and brain tissue. In addition, in these animals blood AChE and butyrylcholine esterase (BuChE) activities were determined. Low AChE activities were encountered in diaphragms and brains. These levels were not significantly different between saline- and HI-6-treated marmosets. In vitro treatment with HI-6 at 40 min after soman still led to an increase of the AChE activity, which was significant in diaphragm, suggesting that postmortem AChE inhibition had occurred. The ratio of AChE to BuChE in blood was significantly enhanced in HI-6-treated animals, indicating that HI-6 preferentially reactivated AChE. It is concluded that (i) HI-6 is an effective treatment against soman poisoning in marmosets and (ii) AChE reactivation or protection by HI-6 contributed to the survival of the animals.