The quantitative assessment of topical irritancy and its application to unphysiological pH-values

Injection of aqueous solutions of topically irritant substances intracutaneously into the skin of juvenile white mice creates a persisting oedema which can be assessed by the number of wrinkles formed upon reefing the skin at the site of injection. An algorithm is presented which transforms the number of wrinkles recorded just before as well as 2.5 h and 6 h after injection into the intensity of the oedematous reaction. The results obtained with a collective of about 10 mice per solution to be tested are reliable and reproducible; they conform to dose-response relations and hence can serve for quantifying local irritancy. The method is used to investigate the effect of pH, and a general model is developed describing the local irritancy of unphysiological pH-values.This work was encouraged and financially supported by the Swiss Federal Office of Health, Section Toxicology.     

A modified Warthin-Starry procedure at low pH for melanin

The Warthin-Starry argyrophilic silver stain peformed at pH 3.2 is a highly effective method for the demonstration of melanin in tissue sections. Eighty-five melanocytic tumors were studied using the Warthin-Starry and the Fontana-Masson procedures. The Warthin-Starry technic stained a greater number of cells in 68% of the tumors, with no loss of cellular detail. Twenty-three per cent of the tumors that appeared amelanotic on hematoxylin-and-eosin-stained sections were positive for melanin with the Warthin-Starry stain, but negative with the Fontana-Masson technic. The Warthin-Starry stain is more sensitive and specific for melanin than is the Fontana-Masson procedure.     

An assay procedure for the comparative irritancy testing of esters in the tigliane and daphnane series

A method is described for testing of diterpene esters for irritancy. The technique involves the application of acetone solutions of the toxins to the inside ears of female LACA mice. The number of mice responding per group and the log₁₀ dose data were evaluated using probit analysis with the assistance of a computer program. This evaluation has the advantage that approximations inherent with an arithmetical evaluation were eliminated, and limits may be placed upon the standard deviation of the irritant dose 50% (ID₅₀). In addition, the use of a ² test automatically eliminated results which were not attributed to random biological variation. Observations of the time to onset and the persistence of the inflammation have led to the suggestion that daphnane orthoester diterpenes may elicit their effect by means of a direct action at a receptor site in skin, whereas the tiglianeO-acyl esters may in part act by causing more general tissue damage.     

Pig skin as test substrate for evaluating topical antimicrobial activity.

The feasibility of using pathogen-contaminated pig skin as a model substrate for evaluating skin disinfectants was demonstrated. A test methodology is described that is safe, convenient to use, and adaptable to a variety of hand-washing conditions. The treatment protocol, pathogen contamination conditions, and application technique variables can all be carefully controlled to simulate clinical use conditions. The number of organisms transferred by contact was compared with the total organism count on the pig skin. The quantity of organisms transferred ranged from 10 to 60% of the total organisms, depending on the nature of the contamination conditions. The cumulative results of multiple imprint and stripping measurements were consistent with the concentration of inoculated organisms. Tests with alcohol solutions validated the methodology and clearly showed the dependence of topical antimicrobial activity on both the concentration and structure of the alcohol. Activity increased with increasing alcohol concentration and in the following order: ethanol, isopropanol, and n-propanol. All of the alcohols became less active as the severity of the test conditions was increased, i.e., higher inoculum levels for a longer incubation time before treatment. The contact imprint and stripping methods used to evaluate bacterial growth on the skin clearly showed that the alcohol treatments reduced but did not eliminate the inoculated pathogens. It was found that long lifetimes (several hours) for pathogens on the skin are possible under some environmental conditions. This observation strongly suggests that frequent hand washing is a necessary infection control practice even when opportunities for repeated pathogen contamination have not occurred.     

A method for accurate measurement of cutaneous irritancy of trichothecenes

Conventional skin irritation bioassays for trichothecenes are semiquantitative because test animals vary in sensitivity, and the intensity of cutaneous inflammation is poorly correlated with dose. A quantitative bioassay was therefore devised for toxicological studies on the irritancy of trichothecenes. A graded series of six standard solutions of T-2 toxin (10-60 micrograms/mL) in 2 microL volumes was applied to the shaved skin of young female Wistar rats. Each test sample was applied at least twice to each of five rats. After 48 hours, reactions were rated in units of equivalent concentrations of T-2 toxin, so that measurements were independent of the intensity of inflammatory reaction. Mean concentrations of replicate measurements of test solutions of T-2 toxin between 10 and 60 micrograms/mL were precise (SEM less than 1.6 micrograms/mL) and accurate (within 13% of actual concentrations).     

A sequential procedure to test the progressive decrease of rare adverse events

A program to construct the Rapid Sequential Test (R-ST), a sequential procedure to test the decrease of the rate of rare adverse events, is described. This sequential procedure is constructed with the Sequential Probability Ratio method. It relies on a model for the progressive decrease in the rate of adverse events from a given initial rate to a target rate, during a transition period. The program allows one to calculate the bounds of rejection of the R-ST, and to study its power under various types of decrease. It is shown that the R-ST gives a conclusion faster than standard sequential procedures for the same type I and II error, saving at least one quarter of the trials completed during the transition period. The use of the software to construct R-STs is discussed with respect to the specification of the model.     

A new histological procedure for re-evaluation of the serological test forHelicobacter pylori

To re-evaluate the accuracy of the serological test forHelicobacter pylori, fixation of biopsy specimens with Carnoy's solution (preserving the mucous layer in tissue preparations) followed by immunohistochemical staining (a new histological procedure) was used as the reference histological method instead of 10% formalin fixation followed by hematoxylin-eosin staining (the conventional histological procedure). Biopsy specimens (antrum and body) from 114 patients with gastritis (including non-ulcer dyspepsia) or gastric and/or duodenal ulcers were obtained by endoscopy and used for both bacteriological culture and histological examination. Serum samples were taken from all patients at the time of endoscopy. The serum levels of specific IgG and IgA antibodies forHelicobacter pylori were measured by commercial enzyme immunoassay kits. The reliability of the IgG and IgA measurements was evaluated by analyzing receiver operating characteristic curves obtained using the two histological procedures. With the conventional histological procedure as the reference, the sensitivity and specificity levels of the serological test were 87.2% and 82.1%, respectively. With the new histological procedure as reference, sensitivity and specificity were 94% and 96.7%, respectively. The insufficient accuracy reported for the serological test could be due to false-positive or false-negative results obtained when the conventional histological procedure is used as the reference. The new histological procedure used here revealed that the serological test forHelicobacter pylori is more reliable than previously thought.     

A randomized controlled clinical trial of topical insulin-like growth factor-1 therapy for sudden deafness refractory to systemic corticosteroid treatment

Background

To date, no therapeutic option has been established for sudden deafness refractory to systemic corticosteroids. This study aimed to examine the efficacy and safety of topical insulin-like growth factor-1 (IGF-1) therapy in comparison to intratympanic corticosteroid therapy.

Methods

We randomly assigned patients with sudden deafness refractory to systemic corticosteroids to receive either gelatin hydrogels impregnated with IGF-1 in the middle ear (62 patients) or four intratympanic injections with dexamethasone (Dex; 58 patients). The primary outcome was the proportion of patients showing hearing improvement (10 decibels or greater in pure-tone average hearing thresholds) 8?weeks after treatment. The secondary outcomes included the change in pure-tone average hearing thresholds over time and the incidence of adverse events.

Results

In the IGF-1 group, 66.7% (95% confidence interval [CI], 52.9-78.6%) of the patients showed hearing improvement compared to 53.6% (95% CI, 39.7-67.0%) of the patients in the Dex group (P?=?0.109). The difference in changes in pure-tone average hearing thresholds over time between the two treatments was statistically significant (P?=?0.003). No serious adverse events were observed in either treatment group. Tympanic membrane perforation did not persist in any patient in the IGF-1 group, but did persist in 15.5% (95% CI, 7.3-27.4%) of the patients in the Dex group (P?=?0.001).

Conclusions

The positive effect of topical IGF-1 application on hearing levels and its favorable safety profile suggest utility for topical IGF-1 therapy in patients with sudden deafness.

Trial registration

UMIN Clinical Trials Registry Number UMIN000004366, October 30^th, 2010.

     

Influence of topical erythromycin preparations for acne vulgaris on skin surface pH

Summary Topical erythromycin is a standard regimen for inflammatory acne vulgaris because of its action against Propionibacterium acnes. Changes in P. acnes colonization are inducible by long-lasting changes of skin surface pH. Therefore, the influence of six erythromycin preparations with approximative pH values of 7.5 (preparation A) to 10.2 (C) on the skin surface pH was evaluated in healthy volunteers using a cross-over design. Following a 14-day run-in period, a constant skin surface pH (5.0) was found. Ten subjects received single doses 2–3 days apart; 20 volunteers applied preparations A and C for 28 days. Single doses of preparations A and E (pH 8.0) increased skin pH to 6.99 and 8.61, respectively, at 15 min; it then gradually declined. The other preparations induced only a minor rise of short duration. At the end of the long-term application, the skin surface pH amounted to 5.73 (A) and 5.39 (C). There was no correlation between the effect on skin surface pH and the approximative pH of the preparations. A close relation of single-dose and long term-effects was observed, however. The skin surface pH during the application of preparation A is high enough to increase P. acnes growth about fourfold as compared with normal skin and thus may counteract the antibacterial effect. Clinical relevance should be evaluated in a controlled clinical trial comparing the efficacy of preparation A with that of preparation C.Abbreviations SGOT serum glutamate oxalacetate transaminase - SGPT serum glutamate pyruvate transaminase - gamma-GT gamma glutamyl transferase - pH-mV-meter pH milli-Volt-meter     

A standardized procedure for quantification of the ames salmonella/mammalian-microsome mutagenicity test

Factors determining the precision and variability of the Ames Salmonella test for mutagenicity were investigated. The most important source of variability in the agar-overlay method was nonuniformity in the soft-agar layer thickness. Solution of this problem resulted from application of an agar-leveling table described in this paper. Several other procedural elements also contribute to improved precision, including temperature uniformity during incubation, incubation interval, consistency of plate agar volume, completeness of mixing the soft-agar overlay, peculiarities in the interaction of mutagens and mammalian liver microsomal extract (S9), and methods of storage and controls for tester strains. When these and other effects were well-controlled, variability of the test results was reduced from 200 or 300% to only ± 10% or less. The significance of the factors affecting precision are discussed and an improved experimental protocol is presented.     

A procedure for data analysis of the rodent micronucleus test involving a historical control

No standard procedure of data analysis for rodent micronucleus tests involving historical controls has been established. In the present paper, under the presumption that the distribution of the historical control is stable and reliable, a procedure with three statistical steps is proposed to analyze the frequency of micronucleated polychromatic erythrocytes (MNPCEs). In the first step, the frequencies of MNPCEs in negative and positive control groups of a current experiment of the micronucleus test are compared with the distribution of historical negative and positive controls to examine the technical validity of the current experiment. In the second step, the frequency of MNPCEs in each treatment group is compared with the distribution of the historical negative control. In the third step, the dose-response relation is tested with the Cochran-Armitage trend test. A Monte Carlo stimulation study shows that the power of this procedure is acceptable and also this procedure is robust. An application of this procedure on real data reveals that it is effective in detecting clastogenic chemicals when the probability of a type I error is nearly .01.     

Comparison of a latex agglutination procedure with the microimmunofluorescence test for Rickettsia typhi.

Sera submitted to the Texas Department of Health for the serodiagnosis of Rickettsia typhi were tested by the microimmunofluorescent antibody technique and a new latex agglutination procedure. Results indicated that the latex agglutination test was sensitive and specific and would serve well as a first-line screening test for murine typhus.     

A rapid procedure for initial drug evaluation.

The overall aim of this work is to develop computer simulations to aid in the selection of proposed medicines and identify those most likely to succeed. One important feature is a systems approach to simulate both the target area with which the drug is designed to interact as well as the surrounding areas where feedback mechanisms may alter the expected effect. The simulation must be rapid if it is to be used to evaluate large numbers of potential drugs. Thus the procedure simplifies many of the known complex phenomena to provide a general framework and feedback mechanisms. An example of the use of the simulation to study a drug used to treat hypertension is given. A possible use of the technique is shown using the example of the effect of varying the drug dosage on the contraction of the arteriole muscle.     

A serodiagnostic test for tuberculosis.

This study describes a simple whole cell agglutination test for tuberculosis in which phenol-killed Mycobacterium tuberculosis H37Ra is used as the antigen. The test gave positive results in 59 of 63 culture-positive cases of tuberculosis due to M. tuberculosis, and in 4 of 11 culture-positive cases due to other mycobacteria. Negative results were recorded in 168 of 171 control subjects. A positive result was one in which a titre of 1/125 or more was recorded.     

A satisfactory quantitative test of lymphocyte response to phytohaemagglutinin for the definition of normal control values and recognition of immunological defects

Despite widespread doubts about the quantitative validity or clinical usefulness of lymphocyte response to phytohaemagglutinin (PHA), a satisfactory quantitative test of such responsiveness suitable for the clinical recognition of immunological defects has been developed here. This was achieved by exploring and controlling technical and other variables in the culture of lymphocytes and the quantitation of their response to phytohaemagglutinin. The aspects evaluated included intraperson as well as person-to-person variations, non-lymphocytic cell content, lymphocyte number, PHA batch, atmospheric conditions, culture duration, and quantitation of response. As a result of the information gained from these studies, together with the normal dose-response curve previously established (Fitzgerald, 1971), a satisfactory quantitative and reproducible method suitable for routine clinical use has been realized. This has been applied to the investigation of patients with suspected immunological deficiency disorders, and significant deviations from the normal have been shown. In addition, a PHA dose-response ratio derived from the responses of normal individuals and patients gives a practical quantitative expression of such defects.     

Assay of prick test inoculum volume. II. Average values and individual variability.

INTRODUCTION: The amount of reagent introduced into the skin by a prick test is critical in studies on the reproducibility and standardization of the method. OBJECTIVE: The purpose of the present study was to measure the average volume and the individual variability of the inoculum performed with a prick test standardized technique. METHODS: Two hundred forty prick tests--16 inoculations per subject--were performed on the volar sides of arms of 15 healthy volunteers. The tests were performed by a skilled tester whose technical performance complied with the criteria of good reproducibility described in international guidelines. A 1-mm tip standard device and a 50% glycerosaline solution labeled with Tc99m were used for the test. The inoculum size was calculated using a direct assay method based on the gamma camera. RESULTS: The average volume of the prick test inoculum was equal to 0.016 microliters, with a remarkable dispersion of the values around the mean (median, 15906: range, 418 to 82253 picoliters). Further, we observed great variability from one subject to another and great variability in the same subject from one skin site to another. A statistical analysis of the data shows that this variability depends on the individual characteristics of the subjects examined. A skilled tester using a standardized technique is not responsible for significant variability. CONCLUSIONS: Even when performed by a skilled operator and with standardized techniques, the prick test shows great limits of reproducibility, at least as far as the size of the inoculum volume is concerned. The variability of the inoculum depends, in a statistically significant way, on the subject's individual characteristics and therefore can be reduced only within certain limits by the standardization and perfectibility of the technique.     

A digital procedure for the computation of ultracentrifuge data.

An interactive computer system is described which allows automatic calculation of molecular weights from information obtained from an ultracentrifuge scanner. The system consists of a small (4K) mini-computer, a large display scope and a joystick. Under program control, the user can select reference points, pick the range of data to be used in his calculations etc., from the information displayed on the scope. A comparison of the results obtained using this procedure and those obtained by manual calculations is made using serum albumen.