Sudomotor function assessment as a screening tool for microvascular complications in type 2 diabetes.

Sudoscan, a non invasive, quick, and simple method to measure sweat function, was evaluated as a screening tool for microvascular complications in type 2 diabetes. AUC of the ROC curve for detection of microvascular complication was 0.75 for an autonomic risk score, with a sensitivity of 82% and a specificity of 61%.

Screening of cardiovascular autonomic neuropathy in patients with diabetes using non-invasive quick and simple assessment of sudomotor function.

AIM: Cardiovascular autonomic neuropathy (CAN) is a common but often overlooked complication of diabetes. Sympathetic C-fibers innervating sweat glands can be impaired early on in patients with diabetes. In this study, SUDOSCAN, a new non-invasive device that assesses sudomotor function was compared to methods generally used for the investigation of CAN. PATIENTS: A total of 232 patients with diabetes were measured for heart rate variability (HRV) at rest and during moderate activity. Time and frequency domain analysis techniques, including measurement of the low-frequency (LF) domain component, were assessed during HRV testing. Ewing tests, as recommended by the French Health Authority, were also done. Electrochemical sweat conductance (ESC) was measured on the hands and feet, and a risk-score was calculated. RESULTS: Using two abnormal Ewing tests as a reference for the area under the curve (AUC) of the receiver operating characteristics (ROC) curve for SUDOSCAN, the risk-score was 0.74, with a sensitivity of 92% and specificity of 49% for a risk-score cut-off value of 35%. For the ROC curve analysis using the LF power component during moderate activity at a threshold of 90 ms(2) (first quartile) as reference, the AUC was higher for the SUDOSCAN risk-score (0.77) compared with the standard Ewing tests [E:I ratio (0.62), 30:15 ratio (0.76) and blood pressure change on standing (0.55)]. Using a cut-off value of 35%, risk-score sensitivity and specificity were 88 and 54%, respectively. CONCLUSION: SUDOSCAN, which allows quick quantitative assessment of sudomotor function, may be used for early screening of CAN in everyday clinical practice before resorting to the more sophisticated and specific, but ultimately more time-consuming, Ewing tests.

Assessment of small fiber neuropathy through a quick, simple and non invasive method in a German diabetes outpatient clinic.

INTRODUCTION: Sudomotor dysfunction is one of the earliest neurophysiologic abnormalities to manifest in distal small fiber neuropathy. SUDOSCAN® was developed to provide a non invasive, quick, simple and quantitative measurement of sweat function. The aim of this observational study was to assess sweat function in a diabetes outpatient consult clinic in Germany. METHODS: The study was conducted from February 2009 to March 2011 on patients of a diabetes outpatient clinic in Germany with type 1 and type 2 diabetes, and was conducted parallel to standard care. Sweat function was evaluated by measuring the electrochemical conductance (ESC) of the hands and feet. The method's reproducibility between 2 devices and a follow-up according to insulin administration were also assessed. RESULTS: 52 patients with type 1 diabetes and 115 patients with type 2 diabetes (69 receiving insulin) were involved in this observational study. Hand and foot conductances were lower in patients with type 2 diabetes when compared to patients with type 1 diabetes. A slight decrease in hand and foot conductances was observed in patients with type 2 diabetes without insulin, while an increase was observed in patients receiving insulin (-3.8±9.7 vs. 1.0±9.7 µS, p=0.02 for the hands and -2.2±7.5 vs. 4.1±8.8 µS, p<0.001 for the feet). Coefficient of correlation between measurements performed with the 2 different devices was 0.85 for hands and 0.93 for feet, p<0.001. No safety concern was reported and none of the subjects experienced discomfort during the tests. CONCLUSION: This preliminary study shows that the assessment of small C fiber neuropathy can be performed non invasively, quickly and effectively in standard diabetes outpatient practice with very good reproducibility. The observation that electrochemical skin conductance improves with intensified insulin treatment must be confirmed in a clinical study performed on a larger population.

Comparative efficacy of diazepam and avizafone against sarin-induced neuropathology and respiratory failure in guinea pigs: influence of atropine dose.

This investigation compared the efficacy of diazepam and the water-soluble prodiazepam-avizafone-in sarin poisoning therapy. Guinea pigs, pretreated with pyridostigmine 0.1 mg/kg, were intoxicated with 4LD(50) of sarin (s.c. route) and 1 min after intoxication treated by intramuscular injection of atropine (3 or 33.8 mg/kg), pralidoxime (32 mg/kg) and either diazepam (2 mg/kg) or avizafone (3.5 mg/kg). EEG and pneumo-physiological parameters were simultaneously recorded. When atropine was administered at a dose of 3 mg/kg, seizures were observed in 87.5% of the cases; if an anticonvulsant was added (diazepam (2 mg/kg) or avizafone (3.5 mg/kg)), seizure was prevented but respiratory disorders were observed. At 33.8 mg/kg, atropine markedly increased the seizure threshold and prevented early respiratory distress induced by sarin. When diazepam was administered together with atropine, seizures were not observed but 62.5% of the animals displayed respiratory difficulties. These symptoms were not observed when using avizafone. The pharmacokinetic data showed marked variation of the plasma levels of atropine and diazepam in different antidote combination groups, where groups receiving diazepam exhibited the lowest concentration of atropine in plasma. Taken together, the results indicate that avizafone is suitable in therapy against sarin when an anticonvulsant is judged necessary.

Effect of oleoresin capsicum (OC) and ortho-chlorobenzylidene malononitrile (CS) on ciliary beat frequency.

Tear gases are largely used to control civil unrest. Their incapacitating effects involve the eyes, skin, and respiratory tract. We aimed to evaluate the effects of ortho-chlorobenzylidene malononitrile (CS) and oleoresin capsicum (OC) on ciliary beat frequency (CBF) of mouse tracheal rings. Addition of 0.05% OC or 0.01% CS induced a progressive decrease in CBF, from 11.5+/-0.5 to 4+/-0.1 Hz (P<0.05) and from 12.5+/-0.5 to 2.5+/-0.1 Hz (P<0.05), respectively, 30 min after exposure to the tear gas. Addition of exogenous ATP inhibited the effect of OC, suggesting that ATP could be used to counteract these adverse effects on CBF. However, ATP was inefficient against CS. Methylene blue and H7 inhibited the effects of OC, whereas indomethacin had no effect. None of these drugs affected the inhibitory action of CS. These results suggest that the inhibitory effect of OC is mediated through the guanylate cyclase-dependent pathway or protein kinase C-dependent phosphorylation. Another mechanism is probably involved in CS-induced inhibitory effect. Histological analysis of the trachea revealed an increase in mucus secretion after exposure to OC, and cytoplasmic vacuoles in epithelial cells after exposure to CS.

Plethysmography for the assessment of pneumococcal pneumonia and passive immunotherapy in a mouse model.

The increasing prevalence of resistance to antibiotics of Streptococcus pneumoniae, the main causative agent of community-acquired bacterial pneumonia, necessitates the development of both new therapeutic strategies and noninvasive methods in order to evaluate their efficacy. The efficacy of passive immunotherapy with human intravenous immunoglobulin (IVIG) or solvent alone, administered intranasally or intravenously, was evaluated in a mouse model of acute pneumonia. Lung bacterial load was also evaluated, using a classical but invasive method, as was respiratory function (minute ventilation, respiratory frequency and tidal volume) using plethysmography, a simple noninvasive method commonly used in inhalation toxicology, but not previously used to assess respiratory infection. Forty-eight hours after infectious challenge, the lung bacterial load was significantly lower in IVIG-treated mice than in untreated mice. At the same time, minute ventilation was significantly lower than reference values for untreated mice (36+/-3 versus 57+/-8 mL.min(-1), p<0.01, and 31+/-2 versus 50+/-5 mL.min(-1), p<0.01 for intranasal and intravenous administration of solvent, respectively) but not in mice treated with IVIG by either route of administration. Plethysmography therefore appears to be a simple and reliable test for the follow-up of acute respiratory infection.

Altered expression of lung cytochrome P450 3A1 in rat after exposure to sulfur mustard.

Expression of cytochromes P450 (CYP) and glutathione S-transferases in the lung may be affected by inhaled pollutants. We have investigated the effect of sulfur mustard on the expression of CYP 1A1, 2B1, 2E1 and 3A1, as well as of alpha-, micro- and pi-glutathione S-transferases in rat lung. Sulfur mustard (0.025, 0.05 or 0.1 mg/kg) or its vehicle was administered to anaesthetized animals by intratracheal injection. Expression of CYP and glutathione S-transferases was analysed 24 hr after administration of the vesicant warfare using western blotting. Preservation of airway epithelium integrity after animal exposure to sulfur mustard was confirmed by histological examination of tracheal and lung tissues from control and treated animals. Constitutive levels of CYP 2B1 and 3A1 proteins were found in lung tissue from control rats, whereas CYP 1A1 and 2E1 proteins were not detected. Animal exposure to sulfur mustard enhanced CYP 3A1 protein levels by 80 to 103%. In contrast, exposure to sulfur mustard neither modified CYP 2B1 expression, nor led to detectable expression of CYP 1A1 or 2E1. Constitutive levels of alpha-, micro- and pi-glutathione S-transferase proteins were found in lung tissue from control rats. Exposure to sulfur mustard had no effect on expression of either of the glutathione S-transferases. Our results show that intratracheal exposure to sulfur mustard selectively increases CYP 3A1 expression in rat lung. Taking into account the major role of CYP of the 3A family in the metabolism of drugs, up-regulation of CYP 3A1 by sulfur mustard might have important therapeutic consequences.

Effect of increased pressure on tracheal ciliary beat frequency.

Effects of increased ambient pressure on mucociliary clearance have been poorly investigated. The effects of increasing pressures on ciliary beat frequency (CBF) of guinea-pig tracheal rings were studied in vitro. Increased pressures of 25 and 100 kPa induced a significant and equivalent enhancement of CBF from 30 min after the pressure increase. The increase in CBF observed after a pressure increase of 50 kPa (inspiratory oxygen fraction = 21%), was significantly greater than that observed with an equivalent oxygen tension at atmospheric pressure, i.e. with a gas mixture containing 30% oxygen. Addition of N(G)-nitro-L-arginine methylester (L-NAME) inhibited the enhancement in CBF observed after the 25 kPa pressure increase. Addition of L-arginine reversed the effect of L-NAME. These results demonstrate that a pressure increase applied to tracheal rings, in vitro, induces an enhancement of ciliary beat frequency and that generation of nitric oxide may be involved in this ciliary stimulation.

Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs.

Respiratory tract lesions induced by sulfur mustard (SM), a chemical warfare agent, are characterized by epithelial damage associated with inflammatory cell infiltration. To test the potential role of matrix metalloproteinase gelatinases in these lesions, we evaluated gelatinase activity, albumin content, and total cell count in bronchoalveolar lavage fluid of guinea pigs 24 h after an intratracheal injection of 0.2 mg/kg of SM. The bronchial lavage and alveolar lavage fluids were analyzed separately. The increase in inflammatory cell content of the bronchial lavage fluid, mainly macrophages, observed in SM-intoxicated guinea pigs was accompanied by an increase in albumin and in 92-kDa gelatinase activity. There was a significant correlation between albumin content and 92-kDa gelatinase activity (r = 0.67) and between 92-kDa gelatinase and the number of macrophages. Immunohistochemistry performed on tracheal sections showed the presence of 92-kDa gelatinase at the site of intraepithelial cleavages. Zymography analysis of culture medium conditioned by guinea pig tracheal epithelial cells demonstrated that these cells produced in vitro 92-kDa gelatinase on stimulation. Culture of human bronchial epithelial cells obtained by the explant technique showed a marked increase in 92-kDa gelatinase after exposure to 5 x 10(-5) M SM that reinforced the relevance of our animal results to human exposure to SM. These results suggest that in SM respiratory intoxication, 92-kDa gelatinase of both inflammatory and epithelial cell origins could be involved in epithelial cell detachment.

Airway epithelial damage and release of inflammatory mediators in human lung parenchyma after sulfur mustard exposure.

This study was performed to evaluate the morphological effects of sulfur mustard on human lung parenchyma in vitro and to measure the metabolites of arachidonic acid which are released during acute exposure to the alkylating agent. Histological analysis of the tissue following exposure to sulfur mustard for a period of 45 min at 10 mM revealed the presence of paranuclear vacuoles in the epithelium, specifically, in the ciliated cells. The release of metabolites of arachidonic acid were determined in the bath fluids by an enzymo-immunoassay. The basal release of prostaglandin E2 (PGE2: 1.36 +/- 0.33 ng/g tissue) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha: 8.83 +/- 1.17 ng/g tissue) were not modified during tissue exposure to sulfur mustard (45 min, 0.1 mM). In addition, the basal release of cysteinyl-leukotriene E4 (LTE4: 1.55 +/- 0.44 ng/g tissue) was also not altered by challenge of the tissues with sulfur mustard. In contrast, when the human lung parenchyma was stimulated with anti human IgE (anti-IgE) only the basal release of the metabolite of the 5-lipoxygenase pathway was significantly increased (LTE4: 6.84 +/- 1.57 ng/g tissue). These data suggest that sulfur mustard may produce morphological alterations in epithelial cells and at the time point studied (45 min exposure), this effect is not associated with a release of arachidonic acid metabolites. However, the increased release of LTE4 by anti-IgE suggests that the target cells for sulfur mustard and anti-IgE in the human lung may be different.

Comparative acute toxicity of o-chlorobenzylidene malononitrile (CS) and oleoresin capsicum (OC) in awake rats.

Tear gases are largely used to control civil unrest. Their incapaciting effects involve eyes, skin and respiratory tract. This study was performed to compare acute respiratory effects of o-chlorobenzylidene malononitrile (CS), oleoresin capsicum (OC) and their respective solvents in awake rats, using an integrated system of nose-only exposure and multiple monitoring of breathing. Aerosols were generated by a Collison Nebulizer from the solutions held in tear gas sprays. The reduction of minute ventilation, observed during a 5 min exposure, was significantly more important with CS than with OC: minute ventilation represented 29+/-8 and 50+/-6% of pre-exposure minute ventilation respectively (P<0.05). The reduction of minute ventilation observed with CS and OC solvents alone was not significantly different from that observed with the tear gases themselves. The decrease in minute ventilation observed, between the second and the fifth minute of exposure, was of the same level for repeated exposure separated by 24 h. Time necessary to recover to 80% of pre-exposure minute ventilation was not significantly different between the two tear gases: 722+/-272 and 691+/-262 s for CS and OC respectively (NS). Histological analysis of the trachea, performed at the end of exposures, revealed an increase in mucus secretion after exposure to OC and cytoplasmic vacuoles in epithelial cells after exposure to CS. In the lungs, interstitial oedema was observed after exposure to OC and emphysema after exposure to CS.

Comparative toxicity of sulfur mustard and nitrogen mustard on tracheal epithelial cells in primary culture.

Sulfur mustard (SM) and mechlorethamine (HN2) are two alkylating agents. SM represents a potential chemical warfare agent and HN2 is used in cancer chemotherapy. Based on the similarities of their action, although few comparative studies of their effects have been performed on the same model, many compounds effective against HN2 side-effects have been proposed, unsuccessfully, against SM-induced lesions. We performed this study to compare the toxic effects of these two alkylating agents on rabbit tracheal epithelium in primary culture. Using neutral red uptake, we evidenced that for a time of contact of 24hr, HN2 LC(50) was significantly lower than SM LC(50) (0.034+/-0.009 and 0.132+/-0.023mm, respectively; P<0.001). On the other hand, for exposure at 10(-3)m, the time necessary to decrease the cell viability rate to 50% was shorter with SM than with HN2 (11+/-1min and 54+/-2min, respectively; P<0.0001). These two alkylating agents induced apoptosis which was evidenced by DNA ladder and by 4',6-diamidino-2-phenylindole (DAPI) DAPI staining. The apoptosis rates were time and dose dependent for the two toxics: mild doses induced apoptosis, while higher doses induced necrosis.

Airway epithelial damage induced by sulfur mustard in guinea pigs, effects of glucocorticoids.

Sulfur mustard (SM) represents a potential chemical warfare agent. In order to characterize SM-induced airway epithelial damage, we studied the effects of an intratracheal injection of 0.3 mg/kg of SM in guinea pigs, 5 h, 24 h, 14 days and 35 days after exposure. During the acute period, lesions prevailed in tracheal epithelium exhibiting intra-epithelial blisters, inflammatory cell infiltration and columnar cell shedding with exposure of basal cells. Fourteen days after intoxication, tracheal epithelium appeared disorganized and showed a significant decrease in height and cell density. Tracheal epithelium recovery was still not complete even 35 days after SM-intoxication. At day 14, in SM-intoxicated guinea pigs treated with betamethasone from day 7 to day 14, epithelium height, cell density and cell proliferation (evaluated by immunohistochemistry) were significantly increased compared to untreated guinea pigs. In conclusion, the lesions observed in SM-intoxicated guinea pigs seem to be in accordance with clinical human observations and are relevant to the study of airway epithelial damage induced by SM. This animal model could be used to illustrate tracheal epithelium regeneration mainly derived from basal cells and to show glucocorticoid effects on airway epithelial recovery after chemical aggression.

Effect of gas density variations on respiratory input impedance in humans.

The forced oscillation technique is a widely-used non-invasive method of characterizing the dynamic behaviour of the respiratory system. We used the forced oscillation technique to investigate respiratory mechanics in healthy subjects during simulated dives in dry hyperbaric chambers. We observed frequency dependence of input impedance, which was mainly density-dependent. To explain this result, we propose a model of the respiratory system, based on flow redistribution in a two-pathway circuit. This model, using the electrical analogue, is composed of two Resistance-Self Inductance-Capacitance (R-I-C) pathways set up in parallel. It allowed us to explain the dynamic behaviour of respiratory impedance under hyperbaric conditions in healthy subjects. Changes in respiratory impedance according to frequency vary with the relative importance of the inequalities of the two time constants RC and I/R between the two pathways. With low values of density, RC inequality predominates, whereas I/R inequality tends to predominate with high values of density.

Glucocorticoids inhibit sulfur mustard-induced airway muscle hyperresponsiveness to substance P.

To explore the mechanisms of airway hyperreactivity to aerosolized substance P observed in guinea pigs 14 days after intratracheal injection of sulfur mustard (SM), we studied the effects of epithelium removal and inhibition of neutral endopeptidase (NEP) activity on airway muscle responsiveness. Tracheal rings from SM-intoxicated guinea pigs expressed a greater contractile response to substance P than rings from nonintoxicated guinea pigs. After epithelium removal or incubation with the NEP inhibitor phosphoramidon, the contractile responses of tracheal rings to substance P did not differ in guinea pigs injected with SM or ethanol (SM solvent). Treatment of the guinea pigs with betamethasone for 7 days before measurement abolished the airway muscle hyperresponsiveness observed in untreated SM-intoxicated guinea pigs and partially restored tracheal epithelium NEP activity. In addition, the tracheal epithelium height and cell density of SM-intoxicated guinea pigs treated with betamethasone were significantly greater than in those without betamethasone. These results demonstrate that SM intoxication induces airway muscle hyperresponsiveness to substance P by reducing tracheal epithelial NEP activity and that glucocorticoids might inhibit this hyperresponsiveness by increasing this activity.

Acute and chronic respiratory effects of sulfur mustard intoxication in guinea pig.

Sulfur mustard (SM) has been used as a vesicant chemical warfare agent. To investigate the respiratory damages it causes, we studied the effects on guinea pigs of an intratracheal injection of 0.3 mg/kg of SM 5 h and 14 days after injection. Five hours after SM intoxication, respiratory system resistance and microvascular permeability were increased. These alterations were not prevented by pretreatment with 50 mg/kg sc of capsaicin 2 wk before SM intoxication. Histological studies showed columnar cell shedding all along the tracheal epithelium, bronchoconstriction, and peribronchial edema. Fourteen days after SM intoxication, guinea pigs demonstrated airway hyperreactivity to aerosolized substance P and histamine. Pretreatment with phosphoramidon caused a further increase in airway responsiveness to substance P. Neutral endopeptidase activity in the tracheal epithelium was decreased by twofold in SM-intoxicated guinea pigs. At this stage, the tracheal epithelium was disorganized and atrophic. These results demonstrate that in guinea pigs SM intoxication induces severe lesions to the tracheal epithelium, which might account for the airway hyperresponsiveness observed 14 days after intoxication.