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Lipids in Leprosy 2

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Lipids in Leprosy 2 }N'J,.: HN;\" IONAL JOU!lN;\L OF 1 .EI'ROSY Volume ~8, Number ~ Printed in tilt: U .S.A. Lipids in Leprosy 2. Histochemistry of Lipids in Human Leprosyl,2 Isamu Sakurai and Olaf K. Skinsnes 3 It is well known that lepra cell s contain stain (:1 1). 10 per cent neutralized formalin "large amounts of lipid substances, especial­ for various stains useful in the identification ly phospholipids and fatty acids. Opinions of lipids, and formol -calcium solution for as to the origin of these lipids have, howev­ Baker's and Klti ver-Ba lTcra's methods for ,er, been varied ( 1.8, H. 1(1 . 17, 2S- :l2) . phospholipids. This progressive accumulation of fatty Each of the tissues fix ed in Hic1ley's solu­ ~ ub s tan ces in lepromatous macrophagcs tion was transferred to 70 per cent ethyl and the absence of such accumulation in alcohol, dehydrated in graded alcohols, tuberculoid leprosy warrants continuing in ­ embedded in paraffin and cut at four mi­ vestigation with respect to its relationship crons. Tissues fi xed in either 10 per cent to the apparent defect in cellular immunity neutrali zed formalin or formol -calcium so­ that is characteristic of lepromatous lepro­ lutions were embedded ,in carbowax (poly­ sy. The following obscrvations are a contin­ ethylene glyeol, Union Carbide Corp. ), uation of prcliminary investi gations initi­ which docs not interfere with the various ~ ted in this area. lipid stains (2~) . They wcre cut at 4 to 8 microns in a low humidity, air-conditioned MATERIALS AND METHODS room at a temperature of approximately Skin biopsics from 21 Hong Kon g and 22°C. The sections were then placed in a Taiwan Chinese patients with leprosy were flotation bath made of 40 volumes of di­ divided into three groups: ( 1 ) 14 cases of ethylene glycol, 50 volumes of distilled histologically lepromatous leprosy, either water and 10 volumes of 40 per cent untreated or treated with diaminodiphenyl formalin to dissolve the embedding mcdi­ sulfone or Giba-1906, (2) two cases of um and to spread the sections. They were histologically tuberculoid leprosy, and ( 3) then picked up on glass slides precoated fi ve instances of leprosy treated with B.663 vvith a thin £1m of gelatin eonsisting of a , for from two months to over a year. The mixture of JO gm. gelatin, 60 ml. distilled latter group included two cases of lepro­ water, 50 m1. glycerine and 1 ml. phenol. matous and three cases of intermediate The mixture had been preheated to dis­ ( dimorphous) leprosy. The B.663-treated solve the gelatin, and filtered. cases were regarded as a distinct group Histochemical procedures were per­ because reRections of cellular lipid metabo­ formed mainly in accord with the methods lism in these p atients have characteristics detailed by Pearse (]9) and Okamoto et differing from that in the other groups. al. (18). The paraffin-em bedded tissues The spccimens were immcdiately fi xcd were sta in ed with hematoxylin and eosin, in three different fi xatives: Ridley's periodic acid-Schiff, Ziehl-Neelsen's stain, modified Zenker-formol solution for Triff Triff stain (:H) , Sudan black B stain eom­ bined with lipid extraction by ethanol, pyr­ I R ccei,'cd for publication 9 April 1970, idine. or chloroform-methan01 (2: 1 ) mix­ ~ This work was supportcd by Grant AI·088i8 ture for firmly bound lipids and ceroid. from the 01a li oll al Inslitute of All ergy and Infec· ti ous Diseases, National Institutes of H ealih, Be· Sudan black B stain together with extrac­ Ihesda, ,\Iaryl:lIld ~OO I 4. tion by acidified chloroform-methanol mix­ :1 I. Sak urai. M.D., SeD., Assi stant R esearcher. ture ( 1% conc. hydroehloric acid in chloro­ and O. K. Sk illSn es. M.D ., Ph, D., Departmen t of Pa thology, Uni"crsity of Hawaii School of Medicine. form -methanol, 2: 1, mixture ) was used as a Leahi Hospital. 3675 Kilauea Avenue. Honolulu, negati ve control for the latter procedure. Ilawaii 96816. (D r. Sa/ntraj's presenl nrlrll'ess: Dc· partment of Pathology, N ihon University Sc hool of The earbowax-embedded tissues were sub­ ,Medicine, ltabashi.ku, Tokyo, Japan. jected to the following battelY of pro- 389 390 Internati01U11 J oumal of Lepmsy 1970 Sudan III & Nile Blue Neutral Sulfate No . of Fat Red No . of Fat ~ases cases orange- pink-blue Lepromatous 14 10 yellow or blue -"'+ Tuberculoid 2 trace 2 blue - mostly mostly B.663-treated 5 5 red red t--v-H+- FIG. 1. Histochemistry of neutral lipid in human leprosy. cedures: Sudan III stain (Daddi's meth­ than neutral fat such as cholesterol es ters, od) for lipid in general; fett rot stain for phospholipids and fatty acids may bc lipid in general; Nile blue sulfate method stain ed orange-yellow, and glycolipids arc for neutral fat and lipoid; Fischl er's method not generally stain cd with Sudan III. Neu­ for fatty acids together with lipid extraction tral fat should stain red, cholesterol s are by pyridine as a negative control; Okamo­ pale red and oHier lipoid substances such to, Shimamoto and Sonoda's methods for as phospholipids are blue by means of Nile cholesterin and its esters; Okamoto, Ueda, blue sulfate method (18, 19) . As shown in Kusumoto and Hashimoto's m ethod Figure 1, lepra cells in lepromatous leprosy (modified Molisch reaction ) for glycolip­ are stained orange-yellow by Sudan III and ids; Okamoto, Shimamoto, Ueda, Kusumoto fett rot stains, and blue or pink-blue by and Shibata's mercury diphenylcarbazone Nile blue sulfate, suggesting that some lep­ method for phospholipids and glycolipids; ra cell s contain a small amount of neutral Baker's acid hematein method for phospho­ fat, but the main components are probably lipids, together with Baker's pyridine ex­ lipoid substances as first pointed out by traction technic as a negative control; M itsuda (16) . In contrast to lepromatous Kluver and Barrera's phthalocyanin method leprosy epithelioid cells in lesions of tuber­ for phospholids excluding sphingomyelin, culoid leprosy present only trace of color in and probably glycolipids. both Sudan III and fett rot stains, and are stained light blue by Nile blue sulfate. RESULTS Tuberculoid leprosy lesions contain only Neutral fat (Fig. 1). Three methods were small amounts of lipids, which can scarcely employed for the identification of neutral be demon strated by histochemical proce­ fat as well as lipids in general. These dures. consisted of the Sudan III, fett rot and Four of fi ve cases treated with B.663 Nile blue sulfate stains. There are no spe­ showed red reaction in lepra cells by the cific histochemical methods available for Nile blue sulfate method, and were stained neutral fat. The Sudan and fett rot dyes red by Sudan III and fett rot stains, in­ stain not only neutral fat but also other stead of orange-yellow as seen in untreated lipids. The presence of neutral fat in tis­ lepromatous leprosy. One of these five sues, however, may be suggested by a red cases yielded almost similar results to that color reaction with Sudan III and fett rot found in untreated lepromatous leprosy. as well as by metachromasia (pink-red) in This case had been treated for the shortest the Nile blue sulfate method. Other lipids period among five cases. Most macrophages 38, 4 Sakurai & Skinsnes: Lipids in Leprosy. 2. 391 Number Fischler's of Leprosy bacillus cases method Lepromatous 14 -H+ many Tubercul oid 2 -- ,-...../ + none or few B.663-treated 5 - -.,J + few FIG. 2. Histochemistry of fatty acid in human leprosy. (lepra cell s) in B.663-treated cases have themselves stained with this meth od and larger vacuoles or globules in their cyto­ th e bacilli had bcen destroyed and digcsted plasm which proba bly contain la r ger in the tuberculoid lesions. Largc globi in amounts of neutral fat th an in untreated lepromatous lesions stain ed dark blue. lepromatous tissues. B.663-treated cases varied in rcacti on to One exception was found in the un­ fatty acid staining. Two of five cases were treated lepromatous group. These sections slightly positive and the other three were reveal ed nonspecific panniculitis in reac­ negative. Positive cases presentcd granular tional phase. The color reaction in lipid form s of bacilli which are stain able with staining was peculiar in this case, differing this method. The positive results were from that in the other cases of lepromatous reconfirmed by pyridine-extraction. leprosy. Some macrophages had a great Glycolipid. Okamoto, Ueda, Kusumoto deal of neutral fat, demonstrating as bright and Hashimoto's modified Molisch reaction red in Sudan III and fett rot stains, and as was employed for the identification of gly­ pink-red in the Nile blue sulfate method. colipids. All cases in the three groups . Such neutral fat-laden cells are commonly studied were negative by this method . seen in the deep corium and subcutis, or Phospholipid (Fig. 4). Phospholipids were around the dermal appendages. Besides evaluated by three different methods con­ lepromatous changes, this case showed lep­ sisting of Okamoto et oZ. mercury di­ ra reaction manifested as nonspecific pan­ phenylcarbazone method, Baker's acid he­ niculitis, vasculitis and less bacilli. Neutral matein method, and Khi ver and Barrera's fat deposition in macrophages in this case copper phthalocyanin method. The results may have resulted from phagocytosis of are shown in Figure 4. necrotic fat cells in the subcutaneous tissue. Baker's method seemed to be the most Free cholesterol and cholesterol ester. sensitive. Positive reaction was clearly Sections were strained with the Oka­ shown as dark blue against a yellowish moto et aZ.
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