Evidence for a Humoral Mechanism which Prevents Growth of Dermatophytes1

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é??ç¸?vidence for a Humoral Mechanism which Prevents Growth of Dermatophytes1

In view of the ability of dermatophytes to thrive on even extremely simple nutrient substrates and under relatively wide ranges of temperature, pH, illumination, and oxygen tension, it is of interest to ask why they should fail to invade living tissues below the waterelectrolyte barrier of the skin. The observations of others pertinent to this question, notably those of Saeves, Kogoj, Smolka, Jadassohn, Sulzberger, and Newcomer, Wright, and Sternberg have been cited in a previous communication (1). That this failure to invade living tissue does not result from lack of essential nutrients or of sufficient oxygen is indicated by the often demonstrated ability of these fungi to thrive in serum as well as on all sorts of nonkeratinous internal tissues after death including the corium of human skin even when kept at low oxygen tensions.The true religion on sale question of the existence of some type of circulating antibody in the blood serum which is fungistatic against dermatophytes has been the subject of considerable controversy. In 1934, Ayers and Anderson (2) reported that serum taken from patients with dermatophytids when added in ten per cent concentration to Sabouraud's medium completely inhibited the growth of dermatophytes isolated from such patients, whereas serum from normal individuals had no such effect. They further stated that this antibody was rapidly destroyed at room temperature. Lewis and Hopper (3) on attempting to repeat this work were unable to confirm it and found only irregular partial inhibition of the growth of dermatophytes in the presence of serum taken from individuals with dermatophytosis. Furthermore, such inhibition was also noted by them in a few instances where serum from persons without dermatophytosis was used. Attempts to demonstrate agglutinins, precipitins, or complement fixing antibodies in the sera of patients with superficial mycoses have uniformly failed (4).This present report concerns some of our investigations on the nature of the defense mechanisms possessed by living tissues which so strictly keep dermatophytes from invading them.MATERIALS AND METHODSTrichophyton mentagrophytes was the representative dermatophyte used in all of the experiments. Inocula consisted of either pinhead size bits of fungal growth teased from a fresh colony grown on Sabouraud's agar, or opalescent suspensions in sterile distilled water prepared from relatively powdery colonies by shaking mycelial fragments with glass beads.Millipore filter chambers of the HA type were prepared as described by Algire, Weaver and Prehn (5) and these were sterilized prior to use by immersion in 70 per cent alcohol for fifteen minutes followed by three rinses with sterile distilled water. The largest borse bottega veneta online pores of these filters are about 0.3 microns in diameter and thus they exclude the passage of cells but do not interfere with the free penetration of proteins and tissue fluids.Dialysis bags were prepared from links of london stores Visking cellulose casing 1.0 cm. in diameter and were sterilized by boiling for fifteen minutes in water. After such boiling these bags were demonstrated to essentially retain their impermeability to serum proteins by the following experiment. A series of such bags boiled and nonboiled were filled with 3.0 ml. quantities of human serum and suspended in separate tubes in 20.0 ml. volumes of distilled water for three days. At the end of this period maximum protein loss from any of the bags was less than 0.4 per cent as determined by biuret assay.In order to avoid contamination of the outsides of dialysis bags with fungal inocula the following procedure was followed in some of the in vitro16THE JOURNAL OF INVESTIGATIVE DERMATOLOGYexperiments. Glass melting point tubes were filled with the opalescent fungal suspension and then sealed by flaming their ends. These sealed tubes were placed into concentrated nitric acid for fifteen minutes and subsequently rinsed several times with sterile sodium bicarbonate solutions and distilled water. They were then with the use of sterile technic slipped into the sterile dialysis bags. After tying the bags, the thin glass tubes were broken to release their contents inside the bags.The mice used in these experiments were <a href="http://www.photo2video.co.uk/truereligion/">true religion on sale</a> normal adult animals of either dba or Swiss albino strain.EXPERIMENTAL PROCEDURES, RESULTS, AND COMMENTSInitially we sought to establish whether primarily cellular or humoral mechanisms were involved in preventing the growth of T. mentagrophytes in living tissues. In one experiment, millipore chambers containing bits of T. mentagrophytes colonies were implanted intraperitoneally into three mice. After three weeks these filters were removed from the mice and opened. In none was there the slightest evidence of growth of the original inoculum. Similar control chambers were placed on Sabouraud's medium and from all <a href="http://www.photo2video.co.uk/giuseppezanottisneakers/">giuseppe zanotti sneakers women</a> vigorous growth of T. mentagrophytes was obtained.In a second experiment, six mice were given inoculations of T. mentagrophyte suspension into the anterior chambers of their eyes. After two to six weeks observation in none of the eyes did grossly visible fungal growth or cellular infiltration appear. One of the mice was killed two weeks after inoculation and cultures of both eyes readily yielded colonies of T. mentagrophytes on Sabouraud's medium even though there was no grossly visible fungal growth.In both of these experiments striking fungistatic effect on T. mentagrophytes was demonstrated in vivo in areas out of contact with direct cellular defense mechanisms.In order to obtain further information about the nature of the noncellular dermatophytesuppressing mechanism in question the following further experiment was giuseppe zanotti sneakers women undertaken. Small inocula of T. mentagrophytes were placed inside sterilized dialysis bags which were then implanted intraperitoneally into ten mice. At intervals of one, two, three, four, five and twelve weeks, respectively one, one, three, one, two, and two of the bags were removed and examined. There was no evidence of severe tissue reaction around any of the bags and in none of them was there the slightest evidence of growth of the fungal inocula. Similar control dialysis bags prepared at the start of woolrich outlet the experiment and placed into either Sabouraud's medium or normal human serum all showed vigorous fungal growth in less than two weeks. Even after as long as the twelve week stay in the peritoneal cavities of mice, when the original inocula were removed from the dialysis bags and planted on Sabouraud's medium, vigorous growth of the fungus occurred. This experiment indicated that the humoral factor involved in the suppression of dermatophytic growth in the living tissues of the mouse was dialyzable and hence not ordinary protein antibody either natural or acquired.At this point, two explanations for these observations involving a dialyzable humoral factor still seemed possible. One was the existence in tissue <a href="http://www.barrioroma.it/bottegavenetaoutlet/">borse bottega veneta online</a> fluids during life of a dialyzable, fungistatic substance. The other was the possible production by the fungus of a dialyzable, autocatalytic material necessary for fungal growth which was too rapidly removed by excretory or metabolic mechanisms of the living host. This latter rather unlikely possibility was eliminated by an experiment in which it <a href="http://www.photo2video.co.uk/linksoflondon/">links of london stores</a> was shown that wide variations in volume of liquid Sabouraud's medium kept continuously stirred failed to cause variations in the rate at which equal small inocula of T. mentagrophytes confined inside of dialysis bags grew out. Thus, growth in a bag suspended in 25 mis. of such constantly stirred medium appeared exactly equally and as rapidly as in bags in 150 and 600 mis. of medium.Attempts were next made to demonstrate a dialyzable antifungal factor in human serum. It was felt that such antifungal material must be relatively unstable in view of the eventual growth of T. mentagrophytes in human serum.A series of dialysis bags containing suspensions of T. mentagrophytes was prepared by the glass melting point tube technic already described and placed into a series of ten small sterile test tubes. Sterile serum was obtained from a healthy adult donor and 1.5 mis. were placed into each of the tubes. In five of the tubes the serum was removed daily and replaced with fresh serum from the same donor. All tubes were kept on a shaker to keep their contents in circulation. After three to four days, fungal growth appeared in all five tubes where the serum was not changed. In the five tubes where the serum was changed daily, growth failed toEVIDENCE FOR A HUMORAL MECHANISM 17occur in three tubes even after three weeks while in two it appeared on the fifth day and was slow and inhibited for another three days.This experiment was repeated with serum from a second normal donor but 5.0 ml. amounts of serum were used in each <a href="http://www.biennalecarrara.it/spacciowoolrichbologna/">woolrich outlet</a> tube. In three control tubes where the serum was not changed, growth appeared grossly within three days, whereas in two tubes where the serum was replaced with fresh serum each day, growth was delayed for three additional days.