Applicability of an exaggerated forearm wash test for efficacy testing of two corticosteroids, tacrolimus and glycerol, in topical formulations against skin irritation induced by two different irritants.

BACKGROUND/PURPOSE: Alternatives to corticosteroids in the treatment of irritant contact dermatitis (ICD) are needed and may include glycerol and topical immunomodulators like tacrolimus. Because the efficacy of different treatments in experimentally induced ICD may vary depending on the irritant applied, we tested the efficacy of four anti-irritant compounds using the two different irritants sodium lauryl sulfate (SLS) and nonanoic acid (NON). METHODS: In a randomized, double-blind, controlled trial, healthy volunteers were exposed to 5% SLS and 50% NON (the right and the left forearm, respectively) in a cumulative wash test. Induction of ICD was obtained by three daily washings for 7 days, followed by a maintenance phase with two daily washings for 12 days. Treatment (triamcinolone acetonide, clobetasol propionate, tacrolimus and glycerol ointment) was started at day 7 and applied immediately after washing. Vehicle and no treatment served as the control. Reactions were evaluated clinically and instrumentally. RESULTS: No treatments were significantly better than the other treatments and controls. There was a tendency toward a dose-dependent response to corticoid treatment, and a trend toward worsened irritancy by tacrolimus on SLS-irritated skin. Explained variance in the experiment by anova revealed a very small effect of treatments compared with an immense and significant subject effect. CONCLUSION: No claims of effective anti-irritant properties for any of the ointments can be maintained. Application of the present wash test as a tool for anti-irritant efficacy testing may be complicated by the small observed variance explained by treatment.

The irritant potential of n-propanol (nonanoic acid vehicle) in cumulative skin irritation: a validation study of two different human in vivo test models.

BACKGROUND/PURPOSE: Human in vivo cumulative irritation tests with low-grade irritants simulate real-life exposure to skin irritants. The test outcome depends not only on the substance tested but also on the design of the assay. More than one experimental irritant is usually used because chemicals have diverse mechanisms of action on the skin. We used sodium lauryl sulfate (SLS) and nonanoic acid (NON) in three different concentrations plus their vehicles, water and n-propanol, respectively, to validate our test models and to optimize test concentrations. METHODS: Healthy volunteer forearm skin was exposed in two different cumulative test models: a repeated open model (ROAT) and an exaggerated wash test model. ROAT: 10-min daily exposures for 5+4 days (no irritation on weekend) to SLS 0% (water), 0.5%, 1.0% and 2.0% on the right arm and NON 0% (n-propanol neat), 10%, 20% and 30% on the left arm. Wash test: induction of irritation by three daily washings for 6 days and maintenance of the dermatitis by two daily washings for 12 days with SLS 0%, 5%, 10% and 15% or NON 0%, 30%, 40% and 50%. Reactions were evaluated clinically and instrumentally (transepidermal water loss, colorimetry and hydration) at sequential time points. Additionally, for the wash test, subjective pain scores were obtained from the volunteers. RESULTS: In the ROAT, n-propanol exhibited irritation potential at the level of SLS 1.0% and, by visual scoring, was only found to be significantly different from the two highest concentrations of NON (20% and 30%). In the wash test, n-propanol was much less irritating than SLS, and it could only be distinguished statistically from NON (any concentration) by visual reading. For both test models, n-propanol, by instrumental measurements, was not significantly different from any NON concentration. CONCLUSION: In cumulative irritation test assays, n-propanol appears to be quite irritant itself and may thus be a significant contributor to NON irritation. Moreover, n-propanol was more irritant in the ROAT compared with the wash test.

The hairless guinea-pig as a model for treatment of acute irritation in humans.

BACKGROUND: The effect of six skin care formulations on experimentally induced acute irritation was studied in hairless guinea-pigs (HLGP) and in human volunteers (HV). The formulations were a basic cream, a carbomer cream and four modifications of the carbomer cream, containing either 10% isopropyl palmitate (IPP cream), 10% glycerol (glycerol cream), 19.5% canola oil (canola oil cream) or 0.5% (-)-alpha-bisabolol (bisabolol cream). METHODS: Acute irritation was induced by occlusive tests with 1% sodium lauryl sulfate aq. in both HLGP and HV, and in HV also by using nonanoic acid in n-propanol (NON) 20%. The irritant reactions were treated twice daily with the formulations from the time of removal of the patches. Evaluation of skin irritation and efficacy of treatments was performed daily for 4 days using clinical scoring, evaporimetry (transepidermal water loss), hydration measurement and colorimetry. RESULTS: The glycerol cream was the only product showing effects potentially better than no treatment in HV. CONCLUSION: The HLGP was too sensitive an animal model as a predictor for effect in humans. There was no difference in efficacy of the formulations against the two different irritants in HV.

The hairless guinea-pig as a model for treatment of cumulative irritation in humans.

BACKGROUND: The effect of six skin-care formulations (SCFs) on experimentally induced cumulative irritation was studied in hairless guinea-pigs (HLGPs) and in human volunteers (HVs). The formulations were a basic cream, a carbomer cream and four modifications of the carbomer cream, containing either 10% isopropyl palmitate (IPP cream), 10% glycerol (glycerol cream), 19.5% canola oil (canola oil cream) or 0.5% (-)-alpha-bisabolol (bisabolol cream). METHODS: In HLGP, irritant dermatitis was induced with 30 min daily exposure for 4 days to 0.5% sodium lauryl sulfate aq. (SLS). In HVs, irritant dermatitis was induced with 10 min daily exposure for 5+4 days (no irritation on weekends) to 3% SLS aq. on the right and 30% nonanoic acid (NON) in n-propanol on the left volar forearm. Clinical scoring was performed daily; evaporimetry (total epidermal water loss (TEWL)), hydration and colorimetry were measured at baseline (day 0) in the middle and at the end of treatment. Treatments were applied twice daily. The basic cream and the IPP cream were excluded from testing in HLGP because they were known from previous studies to be irritant in HLGP, while all formulations were known to be equally and well tolerated locally in humans. RESULTS: All formulations worsened the skin irritation in HLGP: the glycerol cream the least, the canola oil cream the most, while the bisabolol cream and the carbomer cream were indistinguishable. In humans, the glycerol cream was better than 'No Treatment' after cumulative irritation with both SLS and NON. The basic cream was better tolerated in humans than was expected from previous testing in HLGPs. CONCLUSION: In conclusion, the results from the studies in HLGPs and HVs are in agreement with regard to ranking of the SCFs. Further, the glycerol cream showed a positive treatment effect on both SLS- and NON-irritated skin in HVs.

Effect of hemodiafiltration and sepsis on chemotaxis of granulocytes and the release of IL-8 and IL-10.

BACKGROUND: Extracorporeal circulation, such as cardiopulmonary bypass and hemodialysis, has been associated with an activation of the immune system. Continuous veno venous hemodiafiltration (CVVHD) is used in critically ill septic patients. During CVVHD, cytokines are excreted in ultrafiltrate. When the membranes, used in CVVHD, are incubated with leukocytes in vitro a slight production of cytokines is observed. Due to the underlying disease it is difficult to investigate the effect of CVVHD in septic patients. We therefore studied the separate effect of CVVHD on the chemotaxis of granulocytes, the proliferation of lymphocytes and the release of IL-8 and IL-10 in healthy pigs compared to an endotoxin and a control group. METHODS: Thirty-one pigs were anesthetized and mechanically ventilated. CVVHD was performed in 10 pigs. Eleven pigs received an infusion of Escherichia coli endotoxin 30 microg/kg, and 10 pigs served as a control group. The chemotaxis of granulocytes was measured in an assay chamber, and the cytokines IL-8 and IL-10 with an enzyme-linked immunosorbent assay. The adhesion molecules CD18 and CD62 on lymphocytes were measured using monoclonal antibodies, and the lymphocyte proliferation was measured without stimulation and in response to mitogens. RESULTS: CVVHD was accompanied by lymphocytopenia and increased spontaneous lymphoproliferative response, but no change in adhesion molecules on lymphocytes or cytokine levels in plasma, and no decrease in the chemotaxis of granulocytes. Following endotoxin we observed a pronounced lymphocytopenia and an increased secretion of IL-8 and IL-10, a decrease in the expression of CD18 on lymphocytes and in the stimulated lymphocyte proliferation and in the chemotaxis of granulocytes. CONCLUSION: CVVHD does not, in contrast to endotoxin-induced sepsis, influence chemotaxis of granulocytes, the production of IL-8 and IL-10 or the proliferation of lymphocytes.

Cardiopulmonary bypass elicits a pro- and anti-inflammatory cytokine response and impaired neutrophil chemotaxis in neonatal pigs.

BACKGROUND: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response and organ dysfunction, especially in children. Plasma concentration of inflammatory markers are increased in response to the trauma of cardiac surgery and CPB. The aim of the present study was to investigate whether the CPB procedure in itself elicits increased levels of inflammatory markers in neonatal pigs. METHODS: The inflammatory response was measured in piglets undergoing sternotomy alone (sham group, n=13) or sternotomy and CPB (n=14). Inflammatory mediators were measured at baseline and at fixed time-points during and after CPB. IL-8, IL-10 and TNF-alpha levels and C-reactive protein (CRP) concentrations were measured in plasma samples. Polymorphonuclear neutrophils (PMN) chemotaxis was measured ex vivo, and CD-18 expression using an immunofluorescence technique. RESULTS: Immediately after the CPB procedure increased IL-8 levels were found in the CPB group, but not in sham operated animals (P=0.005). Simultaneously, a marked IL-10 response was measured in the CPB group. Concurrently, PMN chemotaxis decreased in CPB animals but not in the sham group (P=0.04). CD-18 expression and CRP levels were not significantly different between groups and TNF-alpha showed no changes in either group. The chemotactic response did not correlate with plasma IL-8 or IL-10, nor with CD-18 expression. CONCLUSION: The CPB procedure elicited a systemic inflammatory response in terms of significantly elevated plasma levels of IL-8 and IL-10. Furthermore, a temporary and simultaneous decrease in PMN chemotaxis was observed immediately after CPB.

L-selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase.

The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L-selectin on the neutrophil surface enhances the adhesive function of beta(2)-integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L-selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules.

Characterization of the priming effect by pituitary canine growth hormone on canine polymorphonuclear neutrophil granulocyte function.

In this report, we demonstrate that canine growth hormone (cGH) is capable of priming canine polymorphonuclear neutrophil granulocytes (PMN) in a manner resembling that of human PMN. The cGH influences important functions that are involved in the process of recruitment of PMN, i.e., shape change, chemotaxis, CD11b/CD18 expression, adhesion, and subsequent transendothelial migration. Also, intracellular O(2)(-) production was evaluated. We investigated the priming effect by incubating PMN with purified pituitary cGH at various concentrations (10 to 800 microg/liter). The capacity for shape change was significantly (P < 0.05) enhanced, whereas the chemotactic response under agarose was significantly (P < 0.05) reduced. The chemotactic migration in Boyden chambers (10-microm-thick polycarbonate filter; lower surface count technique) was significantly (P < 0.05) enhanced, presumably due to cGH-induced hyperadhesiveness to the lower surface of the filters. The adhesion in albumin-coated microtiter plates and adherence to canine pulmonary fibroblasts were significantly (P < 0.05) increased, and the increased adhesion resulted in a significant (P < 0.01) increase in transendothelial migration using canine jugular vein endothelial cells. The increase in adhesion was associated with a significant increase in CD11b/CD18 expression. Furthermore, intracellular O(2)(-) production was significantly enhanced in response to both phorbol myristate acetate (P < 0.01) and opsonized zymosan (P < 0.05). In the absence of a PMN-stimulating agent, cGH did not influence the effector functions investigated except for an increased expression of CD11b/CD18.

Determination of the adhesive capability of canine polymorphonuclear neutrophil granulocytes using a fluorometric microtiter plate cellular adhesion assay.

A rapid fluorometric cellular adhesion assay in microtiter plates was evaluated for canine polymorphonuclear neutrophil granulocytes (PMN) using the fluorescent indicator calcein acetoxymethyl ester (Calcein AM). Optimum adhesive responsiveness occurred in bovine serum albumin (BSA)-coated plates after 30 min incubation at 37 degrees C with 5 x 10(5) PMN in 100 microl per well using phorbol myristate acetate (PMA, 100 ng/ml) as stimulant. The adhesive responsiveness for 15 laboratory beagle dogs was determined and the mean percentage of adherence for unstimulated (2.9%) and stimulated (74.3%) PMN and corresponding 95% confidence intervals was determined. The mean intra-assay coefficient of variation (CVintra) for unstimulated and stimulated adhesion in the assay was: CVintra = 12.9% and CVintra = 4.1%, respectively. The mean inter-assay coefficient of variation (CVinter) for unstimulated and stimulated adhesion, respectively, in the assay was: CVinter = 22.9% and CVinter = 5.6%. The assay was highly reproducible and labelling with Calcein AM neither reduced PMN viability nor activated the PMN.

The long-term biological variability of fasting plasma glucose and serum fructosamine in healthy Beagle dogs.

The aim of this study was to estimate the long-term (month-to-month) between-dog, within-dog and analytical components of variance for fasting plasma glucose and serum fructosamine in healthy dogs to assess the usefulness of a single measurement of these analytes in a single dog. Fasting plasma glucose and serum fructosamine were measured in blood samples collected every month for 9 months from 23 clinically healthy dogs, and the results were subjected to nested analysis of variance. The between-dog variation, the within-dog variation, and the analytical variation were 3.8%, 9.5% and 3.7%, respectively, for plasma glucose and 4.2%, 11.1% and 2.8%, respectively, for serum fructosamine. The maximum allowable analytical imprecision, analytical inaccuracy and difference between analytical methods were 4.8%, 2.6% and 3.2%, respectively, for plasma glucose and 5.6%, 3.0% and 3.7%, respectively, for serum fructosamine. The index of individuality, 2.7 for both analytes, indicated that the test results from single dogs can be compared usefully to the corresponding population-based reference intervals. The number of samples required to estimate the true individual mean value +/-5% for a single dog was 16 for fasting plasma glucose and 20 for serum fructosamine. The one- and two-sided critical differences expressing the difference needed for two serial results from the same dog to be significantly different at a 5% level was 24% and 28%, respectively, for plasma glucose and 27% and 32%, respectively, for serum fructosamine.

Evaluation of the urinary cortisol: creatinine ratio in the diagnosis of hyperadrenocorticism in dogs.

The diagnostic accuracy of the urinary cortisol:creatinine ratio (CCR), with the cortisol being measured by ELISA, was evaluated by subjecting data from 18 dogs with and 20 dogs without hyperadrenocorticism to receiver operating characteristic (ROC) curve analysis. The area under the ROC curve (W 0.93, SE(w) 0.044) was much higher than 0.5, indicating that the CCR did distinguish between dogs with and without hyperadrenocorticism. A cutoff value of about 60 x 10(-6) was associated with the highest sensitivity (1.0) and specificity (0.85). At the disease prevalence rate of the present study (0.47), the positive and negative predictive values were 0.87 and 1.0, respectively. These numbers indicate that canine hyperadrenocorticism may be safely excluded when the CCR is below 60 x 10(-6) but that a test of higher specificity (eg, the ACTH stimulation test) should be used to confirm the diagnosis of canine hyperadrenocorticism when the CCR is above 60 x 10(-6).

Heparin-induced increase in serum levels of aminotranferases. A controlled clinical trial.

Sixty-four patients over the age of 40 years, undergoing elective surgery of at least one hour's duration, were randomized to treatment with either a thromboembolic deterrent ( TED ) stocking (Kendall Co.) or subcutaneous low-dose heparin 5 000 IU every 12 hours. Serum levels of alanine aminotransferase (S-ALAT), aspartate aminotransferase (S-ASAT), gamma-glutamyl transpeptidase (S-gamma-GT) and alkaline phosphatase (S-ALP) were measured. S-ALAT increased significantly on the 5th and 10th postoperative day, from 27 +/- 2 (x +/- SE) to 40 +/- 4 (p less than 0.01) and 55 +/- 7 U/l (p less than 0.001), respectively, in the heparin group and was significantly higher in the heparin than in the TED group both on the 5th (p less than 0.01) and 10th (p less than 0.05) postoperative day. S-ASAT and S-gamma-GT increased significantly during heparin treatment, but did not differ significantly from the values of the TED group. No change in S-ALP was registered in either group. It is concluded that prophylactic treatment with low-dose heparin induces a significant increase in S-aminotransferase levels, especially in S-ALAT. The phenomenon has profound differential diagnostic implications in conditions such as pulmonary embolism and acute myocardial infarction.

Urinary retention during i.m. and extradural morphine analgesia.

In the postoperative period fifty-six healthy patients undergoing cholecystectomy or operations for duodenal ulcer, received, in a randomized order, i.m., "high-level" or "low-level" extradural morphine. Thirty-five per cent in the i.m. group, 33% in the high-level group and 50% in the low-level group suffered urinary retention, in all cases within the first 24 h. The mean cumulative dose of morphine necessary for pain relief was in the same range (13.4-16.5 mg) during the first 2 h of therapy for all groups, while the amounts after 24 and 48 h were twice to four times with the i.m. route compared with the extradural route. A peripheral effect of morphine on the urinary bladder is possible and the mechanism of action is discussed.

Postoperative analgesia and lung function: a comparison of intramuscular with epidural morphine.

Thirty healthy patients subjected to cholecystectomy or operation for duodenal ulcer were allocated randomly for postoperative analgesic treatment with morphine i.m. or epidurally. Morphine was given only at the request of the patients and only as much was given as was needed to obtain satisfactory pain relief. Patients in the epidural group were given morphine exclusively by epidural injection. In the epidural group a lower incidence of radiological changes in the lungs was found postoperatively - 21% as against 67%. Compared with the i.m. group, there was a higher arterial oxygen tension and a slower increase in alveolar-arterial oxygen difference. It is concluded that epidural morphine analgesia reduces the degree of postoperative lung dysfunction compared with conventional i.m. morphine treatment.