J Cancer Res Clin Oncol (1990) 116:38M4
C~i/cer ~esearch Clinical 9 @ Springer-Verlag1990
Mucin histochemistry of virus-induced duodenal adenomas in guinea fowl I. Toshkov* and T. Kirev Institute of General and Comparative Pathology, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria
Summary. The type o f m u c o p r o t e i n s in virus-induced d u o d e n a l a d e n o m a s in guinea fowl were c o m p a r e d with those in the n o r m a l duodenal mucosa. The m u cin-producing cells in the latter contained a mixture o f acid and neutral mucins. N e u t r a l and sulphomucins prevailed in the crypts and in the lower part o f the villi, while the a m o u n t o f the sialomucins increased progressively t o w a r d the tip o f the villi. In the adenomas, goblet cells were m o r e n u m e r o u s and were unevenly distributed. In their mucin profile the deeply located t u m o r glandular structures resembled n o r m a l crypts and lower parts o f the villi and superficial portions o f the a d e n o m a s were similar to the upper part o f the villi. Qualitative changes in the mucin secretion with deviation f r o m the n o r m a l vertical distribution o f mucin types were rarely observed. The histochemical study carried out supplemented the histological characterization o f the virus-induced duodenal a d e n o m a s and contributed to the elucidation o f some aspects o f their histogenesis.
1969; G a d 1979; Listinsky and Riddell 1981; M u t o et al. 1985; R o m a g n o l i et al. 1986). In order to establish the specific significance o f such changes in t u m o r o g e n esis and their possible application in histological diagnosis and prognosis, mucin secretion was studied in h u m a n intestinal neoplasia (Culling et al. 1977; D a w son et al. 1978; Filipe and Fenger 1979; Rogers et al. 1978; M o n t e r o and Segura 1980; Reid et al. 1980; Filipe et al. 1985; I g b o a k a et al. 1986; S u n g h a r a and Jass 1986). The validity o f these findings was assessed in 1,2-dimethylhydrazine-induced colonic carcinomas in rats (Culling et al. 1981; Filipe 1975; Filipe et al. 1982). Similarly we examined the mucin profile in duodenal adenomas, induced in guinea fowl by virus strain Pts-56 (Kirev et al. 1987) and c o m p a r e d it with that o f the n o r m a l guinea fowl duodenal mucosa, in a search for quantitative and qualitative alterations that might be associated with the neoplastic transformation.
Key words: Mucins - D u o d e n a l a d e n o m a - G u i n e a
Materials and methods
fowl - Retrovirus
Osteopetrosis virus strain Pts-56 was isolated from a rooster with spontaneous bone growths and was later transmitted by in vivo passages into guinea fowls (Nedyalkov et al. 1972). The latter were commercially available, random bred, kept under common conditions and fed commercial chicken feed. No spontaneous infections were established in the guinea fowl flock. A group of guinea fowls, 1 2 days old were inoculated i. v. with 0.25 ml virus Pts-56-containing bird plasma from a previous passage of the virus. They developed bone growths, pancreatic neoplasia and duodenal adenomas (Kirev et al. 1986, 1987), Materials from duodenal adenomas of 12 of these Pts-56-infectedbirds as well as from the duodenum of 6 healthy control guinea fowls were fixed in buffered 10% formalin. After standard processing and paraffin embedding, serial 5 gm sections were stained with H & E and with the following staining procedures for histochemical demonstration of complex carbohydrates: 1. Periodic acid/Schiff (PAS) (Pearse 1968). The presence of glycogen was ruled out by prior diastase digestion or by staining with Best's carmine 2. 1% solution of Alcian blue 8GS (Fluka) in 3% acetic acid at pH 2.5, or in 0,1 M HC1 at pH 1 for 30 min (Pearse 1968)
Introduction The development o f histochemical techniques for identification o f different types o f epithelial mucins m a d e possible their characterization in various tissues and organs (Culling et al. 1981). The n u m e r o u s studies o f gastrointestinal tract m u c o p r o t e i n s have shown that they u n d e r g o histochemically distinguishable changes in certain pathological conditions (Filipe * Present address: Division of Cytopathology, Institute for Experimental Pathology, German Cancer Research Center Heidelberg Offprint requests to: I. Toshkov (A. v. Humboldt Fellow)
39 TaMe 1. Mucin staining methods and their interpretation Method"
Color
Interpretation
D/PAS
Magenta
AB pH 2.5 AB pH 2.5/PAS
Blue Blue Purple
AA pH 1.5 AA pH 3.5 AF/AB pH 2.5
Red-purple Red-purple Purple Blue Brown-black Blue Blue Negative Magenta Negative
Neutral mucins, some sialomucins Acid mucins Acid mucins Neutral and some acid mucins Sulphomucins Sialomucins Sulphomucins Sialomucins Sulphomucins Sialomucins Sulphomucins Sialomucins O-Acylated sialomucins All other mucins
HID/AB pH 2.5 AH/AB pH 2.5 PB/KOH/PAS
D/PAS, periodic acid/Schiff with prior diastase digestion; AB, Alcian blue; AA, azure A; AF, aldehyde fuchsin; HID, high-iron diamine; AH, acid hydrolysis; PB/KOH, periodate/borohydride/ saponification 3. 0.02% solution of azure A (Bayer) in phosphate/HCl buffer at pH 1.5 and in phosphate/citrate buffer at pH 3.5 for 30 min (Pearse 1968) 4. Combined staining with Alcain blue at pH 2.5 for 30 rain and PAS (Mowry 1963) 5. Combined staining with aldehyde fuchsin for 3 min and Alcian blue at pH 2.5 for 30 min (Spicer and Meyer 1960) 6. High-iron diamine/Alcian blue at pH 2.5 (modified method of Spicer 1965) with treatment in solution of 120 mg N,N-dimethyl-mphenyldiamine and 20 mg N,N-dimethyl-p-phenyldiamine (VEB Chemic Berlin) and 1.4 ml 60% FeC13 in 50 ml distilled water for 45 min to 4 h, followed by 30 min in Alcian blue 7. Acid hydrolysis of sialic acid with 0.5 M sulphuric acid at 80 ~ C for 3i h, followed by Alcian blue at pH 2.5 staining (Culling et al.
3981) 8. Periodate/borohydride/saponification/PAS(Culling et al. 1974). These methods and their interpretation are summarizedin Table 3. Sections of duodenal mucosa from control guinea fowls and of virusinduced duodenal adenomas were stained in parallel, using the same solutions and for equal time.
Results N o r m a l duodenal mucosa 1
The mucin-secreting cells in the crypts o f the normal guinea fowl duodenal mucosa were scant, of elongated form and with insignificant mucin content. In the lining epithelium of the villi they were numerous, regularly distributed and stained more intensely. In the upper portion of the villi they acquired the features of typical goblet cells (Fig. 1A). The mucin-secreting cells stained with Alcian blue and azure A. The reduction of alcianophilia after acid 1 The sections shown (Figs. 1-4) are technically not of the best because of the inferiority of photomaterials available. The authors apologise for this
hydrolysis of sialic acid was more marked in the goblet cells on the surface of the villi. PAS stained them magenta even after diastase digestion, but Best's carmine did not. The combined stainings (Alcian blue/ PAS, high-iron diamine/Alcian blue, aldehyde fuchsin/Alcian blue), distinguishing between different types of mucins, revealed that generally neutral and sulphomucins prevailed in the crypts whereas the sialomucin content grew progressively toward the tips of the villi. No O-acylated sialic acids were identified with the periodate/borohydride/KOH/PAS procedure. The brush border showed a positive reactivity for neutral mucins and partly for sialomucins. Table 2 summarizes the data from the present studies. Both acid (sulpho- and sialo-) and neutral mucins were present in the guinea fowl duodenal mucosa. These mucins were mixed in most of the cells with either type prevailing, resulting in a heterogeneous population. Duodenal adenomas
The materials from the duodenum of the Pts-56-infected guinea fowls studied included regions with focal hyperplasia of the villi, as well as tubulovillous and tubulous adenomas. Compared with the normal duodenal mucosa they showed a general increase in mucus production. This was coupled with considerable variations in the mucin content and in the mucous cell density, as well as with dissimilar mucopolysaccharide staining in adjacent regions of the adenomas, along the same structure or even in its opposite epithelial layers (Fig. 2, 4). The goblet cells were particularly numerous and formed continuous rows both in the tumor glandular structures, which resembled elongated, folded or ramified crypts, and on the surface of the enlarged and branched villi of the adenomas (Figs. 1B, 3). The density of goblet cells along the luminal face o f the polyps decreased markedly. The combined stainings revealed that there was a gradual decrease of neutral mucins and sulphomucins from the deeper zone of the adenomas toward its upper portions with a parallel increase of the sialomucins. In adenomas of considerable size and relatively abundant stalk component, however, structures with mostly or exclusively sialomucins were seen below glandular formations, in which sulphomucins prevailed. The pronounced rise in sialomucins in the adenomas was demonstrated with AB staining after acid hydrolysis, but no O-acylated sialomucins were identified. The brush border was unevenly represented in the tumor structures. In some it was intensely stained while in others it was hardly distinguishable, if at all. An increased mucinous production was observed not only in all types o f a d e n o m a s but also in seemingly unchanged villi in close proximity to them.
40
Fig. 1. A Normal duodenal mucosa. Brown-black-stained sulphomucins prevail in the crypts (a) and in the lower part of the villi (b). Blue-stained sialomucins, appearing grey on the print, are in larger amounts in the goblet cells of the upper part of the villi (c). High-iron diamine/Alcian blue x 250. B Duodenal adenoma: (a) deeply located tumor glandular structures with abundant mucin-producing cells, mostly black, indicating prominance of sulphomucins; (b) luminal portion with increased number of grey sialomucin-containing goblet cells. Aldehyde/fuchsin/Alcian blue; x 250
41
Fig. 2. Distribution of mucin-producing cells in duodenal adenomas. High-iron diamine/Alcian blue; x 40 Fig. 3. Numerous and unevenly distributed goblet cells in the upper (a) und deeply located (b) gIandular structures. PAS counterstained with haematoxylin; x 125
42
Fig. 4. Deviation from the normal distribution of mucin types in duodenal adenoma: (a) adjacent glandular structures with mostly sialomucincontaining cells (appearing grey) in the right one and sulphomucins in the left one (appearing dark); (b) Distended glandular structure in the base of a tubulovillous adenoma with different mucin types in the opposite layers. High-iron diamine/Alcian blue; x 250
Table 2. Gross subjective assessment of mucus content and mucin profile of normal duodenal mucosa and of virus-induced duodenal adenomas in guinea fowl
Type of mucins
Neutral Acid SulphoSialoO-Acylated Sialo-
Normal mucosa
Duodenal adenomas
Crypts
Base of villi
Upper part of villi
Crypts and cryptlike structures
Upper glandular structures
Luminal surface
• to + _+ to + _+ to + •
+ + + to + + + to +
+ + • +
++ _+ to + + to + + _+ to +
+++ + to + + + + to + + + to + + +
0to 0to 0to 0to
0
0
0
0
0
0
to to to to
+ ++ + ++
+ ++ + ++
0, negative reaction; _+, slight reaction; + , positive reaction; + + , marked reaction; + + + , strongly positive reaction
The results of the histochemical studies of duodenal adenomas (Table 2) indicated that mostly quantitative and partially qualitative changes in the mucin secretion were found in comparison with that of the normal duodenal mucosa. Discussion
Despite the numerous histochemical studies of gastrointestinal epithelial mucins, we were not able to find data concerning birds in the available literature. The
results of Sheahan and Jervis (1976), who have studied mucin secretion in man and ten mammalian species, correspond to our finding that guinea fowl duodenum normally produces mixtures of neutral mucins, sulphomucins and sialomucins. In the current studies we established that the specificity of the mucin stainings used was relative, as also mentioned by other authors (Lapertosa et al. 1984). Nevertheless, the simultaneous processing of sections from normal duodenal mucosa and from virus-induced adenomas was suitable for histochemical as-
43 sessment of the mucin secretion alterations occurring as a result of tumorogenesis. Many authors who have studied materials from human gastro-intestinal tumors and from chemically induced experimental colonic neoplasia established changes in the mucin secretion: increased or decreased glycoprotein production; altered proportions of different mucins; substitution of one type of mucin with another (Filipe 1975; Dawson et al. 1978; Montero and Segura 1980; Reid et al. 1980; Jass and Filipe 1981; Filipe et al. 1985). Our studies showed that a general increase in the number of mucinproducing cells and in their mucoprotein content was observed in the duodenal adenomas as compared with normal mucosa. The irregular distribution of goblet cells is a piece of evidence in support of the neoplastic character of the virus-induced polypous growths. The resemblance of their mucin secretion pattern to that of the normal duodenal mucosa and the absence of large-bowel metaplasia or gastric metaplasia, however, confirmed the initial histological diagnosis that they were adenomas rather than malignant neoplasia (Kirev et al. 1987). A shift from the normal vertical distribution of mucin types was found in prominent tumors with an abundant stalk component only. Comparable data were reported by others, who studied normal gastrointestinal mucosa and benign epithelial tumors (Goldman and Ming 1968; Filipe and Fenger 1979). Besides supplementing the histological characteristics of the virus-induced duodenal adenomas, the present studies contributed to the elucidation of some aspects of their histogenesis. Our findings supported the assumption that the growths were formed as a result of crypt elongation and hyperplasia as well as of enlargement and unification of the villi. There was also evidence for impaired differentiation of the epithelium of the glandular structures manifest by irregular distribution of mucin-producing cells. The cells with scanty mucopolysaccharides, observed in some tubulous structures, resembled the undifferentiated and regenerating goblet cells described by Wattel et al. (1977). The unevenly formed brush border in the individual glandular structures was also indicative o f the dysplastic character o f the growths. Similar findings were observed in familial polyposis coli (Romagnoli et al. 1986) and could hardly be attributed in all cases to the direction of the section. In addition to frank adenomas the alterations in mucin secretion were established in zones of duodenal mucosa hyperplasia. In this respect, the latter could be considered as a possible step in the development of the adenomas. Certain differences in the mucin production were observed in the visibly unchanged villi adjacent to the adenomas. The presence of such "transi-
tional mucosa" with histochemical features resembling those of the tumor was reported by many authors (Filipe 1969; Sheahan and Jervis 1976; Shamsuddin et al. 1982). The preneoplastic significance of these changes, however, is disputed, as they occur also in non-neoplastic conditions (Romagnoli et al. 1986). The problem will remain open until sensitive methods differentiating reactive from neoplastic processes are developed. The observed alterations in mucin secretion of the duodenal adenomas and of the adjacent mucosa could be related to tumor development rather than to virus infection only, since they were not established in inoculated guinea fowls without duodenal growths. Despite the differences between the mucin profile in particular portions of the gastrointestinal tract and between separate animal species, our results are consistent with the established alterations in mucin secretion patterns of human and experimental intestinal turnouts and should be considered when the diagnostic value o f such histochemical abnormalities is discussed.
References Culling CFA, Clay MG, Dunn WL (1974) The histochemieal demonstration of O-acylated sialic acid in gastrointestinal mucins. Their association with the potassium hydroxide-periodic acid Schiffeffect. J Histochem Cytochem22:826-832 Culling CFA, Reid PE, Worth AJ, Dunn WL (1977) A new histochemical technique of use in the interpretation and diagnosis of adenocarcinoma and villous lesions in the large intestine. J Clin Pathol 30:1056-1062 Culling CFA,Reid PE, Dunn WL, Freeman HJ (1981)The relevance of the histochemistry of colonic mucins based upon their PAS reactivity. HistochemJ 13:889403 Dawson PA, Patel J, Filipe MI (1978) Variations in sialomucins in the mucosaof the large intestine in malignancy:a quantimet and statistical analysis. HistochemJ 10:559-572 Filipe MI (1969) Value of histochemical reactions for mucosubstances in the diagnosis of certain pathologicalconditions of the colon and rectum. Gut 10:577-586 Filipe MI (1975) Mucoussecretion in rat colonicmucosaduring carcinogenesis induced by dimethylhydrazine. A morphological and histochemical study. Br J Cancer 31:60-77 Filipe MI, Fenger C (1979) Histochemicalcharacteristics in the small intestine. A comparative study of normal mucosa, benign epithelial tumours and carcinoma. HistochemJ 11:277~87 Filipe MI, Scurr JH, ElIis H (1982) Effects of fecal stream on experimental colorectal carcinogenesis. Morphologicaland histochemical changes. Cancer 50:2859-2865 Filipe MI, Potet F, Bogomoletz WV, Dawson PA, Fabiani B, Chauveinc P, Gazzard B, Goldfain D, Zeegen R (1985) Incomplete sulphomucin-secreting intestinal metaplasia for gastric cancer. Preliminary data from a prospective study from three centres. Gut 26:1319-1336 Gad A (1979) Mucin in histochemistryand questions in gastroenterology. Scand J Gastroenterol (supp114) 54:94-98 Goldman H, Ming SC (1968) Mucins in the normal and neoplastic gastrointestinal epithelium: histochemical distribution. Arch Patho185:58(~586
44 Igboaka GUA, Habib N, Dawson PA, Wood C, Williamson R, Bradfield D (1986) Mucin histochemistry in metaplastic and adenomatous polyps and surrounding colonic mucosa. J Pathol 149:212A-213A Jass JR, Filipe MI (1981) The mucin profiles of normal gastric mucosa, intestinal metaplasia and its variants and gastric carcinoma. Histochem J 13:931 939 Kirev TT, Toshkov IA, Mladenov ZM (1986) Virus-induced pancreatic cancer in guinea fowl: a morphologic study. J Natl Cancer Inst 77:713-720 Kirev TT, Toshkov IA, Mladenov ZM (1987) Virus-induced duodenal adenoma in guinea fowl. J Natl Cancer Inst 79:1117-1122 Lapertosa G, Fulcheri E, Aquarone M, Filipe MI (1984) Mucin profiles in the mucosa adjacent to large bowel non-adenocarcinoma neoplasias. Histopathology 8:805-811 Listinsky CM, Riddell RH (1981) Patterns of mucin secretion in neoplastic and non-neoplastic diseases of the colon. Hum Pathol 12:923 929 Montero C, Segura D (1980) Retrospective histochemical study of mucosubstances in adenocarcinomas of the gastrointestinal tract. Histopathology 4:281-292 Mowry R (1963) The special value of methods that colour both acidic and vicinal hydroxyl groups in the histochemical study of mucins with revised directions for colloidal iron stain, the use of alcian blue 8GX and their combination with PAS reaction. Ann NY Acad Sci 106:402~423 Muto T, Kamija J, Sawada T, Agawa S, Morioka Y, Utsunomija J (1985) Mucin abnormality of colonic mucosa in patients with familial polyposis coli a new tool for early detection of the carrier. Dis Colon Rectum 28:147 149 Nedyalkov S, Mladenov Z, Ivanov A, Kirev T (1972) Experimentally induced osteopetrosis (Osteodysplasia leucosarcomatosa gallinarum) in guinea fowl (Numida meleagris). I. Susceptibility of guinea fowl-poults to isolate from osteopetrosis-affected cock,
pathomorphology and ultrastructure of the induced changes. Bulg Acad Sci Bull Inst Gen Comp Pathol 14:51-71 Pearse AGE (1968) Histochemistry theoretical and applied. Churchill, London Reid PE, Culling CFA, Dunn WL, Ramey WC, Magil AB, Clay MG (1980) Differences between the O-acylated sialic acids of the epithelial mucins of human colonic tumours and normal controls: A correlative chemical and histochemical study. J Histochem Cytochem 28:217522 Rogers CM, Cooke KB, Filipe MI (1978) The sialic acids of human large bowel mucosa: O-acylated variants in normal and malignant states. Gut 19:587-592 Romagnoli P, Nardi F, Tonelli F, Bechi P (1986) Histochemistry of small intestinal dysplasia in familial polyposis coli. Virchows Arch B 52:117 128 Shamsuddin A, Phelps P, Trump B (1982) Human large intestinal epithelium: light microscopy, histochemistry and ultrastructure. Hum Pathol 13:790-803 Sheahan DG, Jervis HR (1976) Comparative histochemistry of gastrointestinal mucosubstances. Am J Anat 146:103-132 Spicer SS (1965) Diamine methods for differentiating mucosubstances histochemically. J Histochem Cytochem 13:211 234 Spicer SS, Meyer D (1960) Histochemical differentiation of acid mucopolysaccharides by means of combined aldehyde fuchsin-alcian blue staining. Am J Clin Pathol 33:453460 Sunghara K, Jass JR (I986) Colorectal goblet cell sialomucin heterogenity: its relation of malignant disease. J Clin Pathol 39:10881095 Wattel S, Genze J, deRooij D (1977) Ultrastructural and carbohydrate histochemical studies on the differentiation and renewal of mucous cells in the rat gastric fundus. Cell Tissue Res 176:445462 Received 8 June 1989/Accepted 10 October !989
C~i/cer ~esearch Clinical 9 @ Springer-Verlag1990
Mucin histochemistry of virus-induced duodenal adenomas in guinea fowl I. Toshkov* and T. Kirev Institute of General and Comparative Pathology, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria
Summary. The type o f m u c o p r o t e i n s in virus-induced d u o d e n a l a d e n o m a s in guinea fowl were c o m p a r e d with those in the n o r m a l duodenal mucosa. The m u cin-producing cells in the latter contained a mixture o f acid and neutral mucins. N e u t r a l and sulphomucins prevailed in the crypts and in the lower part o f the villi, while the a m o u n t o f the sialomucins increased progressively t o w a r d the tip o f the villi. In the adenomas, goblet cells were m o r e n u m e r o u s and were unevenly distributed. In their mucin profile the deeply located t u m o r glandular structures resembled n o r m a l crypts and lower parts o f the villi and superficial portions o f the a d e n o m a s were similar to the upper part o f the villi. Qualitative changes in the mucin secretion with deviation f r o m the n o r m a l vertical distribution o f mucin types were rarely observed. The histochemical study carried out supplemented the histological characterization o f the virus-induced duodenal a d e n o m a s and contributed to the elucidation o f some aspects o f their histogenesis.
1969; G a d 1979; Listinsky and Riddell 1981; M u t o et al. 1985; R o m a g n o l i et al. 1986). In order to establish the specific significance o f such changes in t u m o r o g e n esis and their possible application in histological diagnosis and prognosis, mucin secretion was studied in h u m a n intestinal neoplasia (Culling et al. 1977; D a w son et al. 1978; Filipe and Fenger 1979; Rogers et al. 1978; M o n t e r o and Segura 1980; Reid et al. 1980; Filipe et al. 1985; I g b o a k a et al. 1986; S u n g h a r a and Jass 1986). The validity o f these findings was assessed in 1,2-dimethylhydrazine-induced colonic carcinomas in rats (Culling et al. 1981; Filipe 1975; Filipe et al. 1982). Similarly we examined the mucin profile in duodenal adenomas, induced in guinea fowl by virus strain Pts-56 (Kirev et al. 1987) and c o m p a r e d it with that o f the n o r m a l guinea fowl duodenal mucosa, in a search for quantitative and qualitative alterations that might be associated with the neoplastic transformation.
Key words: Mucins - D u o d e n a l a d e n o m a - G u i n e a
Materials and methods
fowl - Retrovirus
Osteopetrosis virus strain Pts-56 was isolated from a rooster with spontaneous bone growths and was later transmitted by in vivo passages into guinea fowls (Nedyalkov et al. 1972). The latter were commercially available, random bred, kept under common conditions and fed commercial chicken feed. No spontaneous infections were established in the guinea fowl flock. A group of guinea fowls, 1 2 days old were inoculated i. v. with 0.25 ml virus Pts-56-containing bird plasma from a previous passage of the virus. They developed bone growths, pancreatic neoplasia and duodenal adenomas (Kirev et al. 1986, 1987), Materials from duodenal adenomas of 12 of these Pts-56-infectedbirds as well as from the duodenum of 6 healthy control guinea fowls were fixed in buffered 10% formalin. After standard processing and paraffin embedding, serial 5 gm sections were stained with H & E and with the following staining procedures for histochemical demonstration of complex carbohydrates: 1. Periodic acid/Schiff (PAS) (Pearse 1968). The presence of glycogen was ruled out by prior diastase digestion or by staining with Best's carmine 2. 1% solution of Alcian blue 8GS (Fluka) in 3% acetic acid at pH 2.5, or in 0,1 M HC1 at pH 1 for 30 min (Pearse 1968)
Introduction The development o f histochemical techniques for identification o f different types o f epithelial mucins m a d e possible their characterization in various tissues and organs (Culling et al. 1981). The n u m e r o u s studies o f gastrointestinal tract m u c o p r o t e i n s have shown that they u n d e r g o histochemically distinguishable changes in certain pathological conditions (Filipe * Present address: Division of Cytopathology, Institute for Experimental Pathology, German Cancer Research Center Heidelberg Offprint requests to: I. Toshkov (A. v. Humboldt Fellow)
39 TaMe 1. Mucin staining methods and their interpretation Method"
Color
Interpretation
D/PAS
Magenta
AB pH 2.5 AB pH 2.5/PAS
Blue Blue Purple
AA pH 1.5 AA pH 3.5 AF/AB pH 2.5
Red-purple Red-purple Purple Blue Brown-black Blue Blue Negative Magenta Negative
Neutral mucins, some sialomucins Acid mucins Acid mucins Neutral and some acid mucins Sulphomucins Sialomucins Sulphomucins Sialomucins Sulphomucins Sialomucins Sulphomucins Sialomucins O-Acylated sialomucins All other mucins
HID/AB pH 2.5 AH/AB pH 2.5 PB/KOH/PAS
D/PAS, periodic acid/Schiff with prior diastase digestion; AB, Alcian blue; AA, azure A; AF, aldehyde fuchsin; HID, high-iron diamine; AH, acid hydrolysis; PB/KOH, periodate/borohydride/ saponification 3. 0.02% solution of azure A (Bayer) in phosphate/HCl buffer at pH 1.5 and in phosphate/citrate buffer at pH 3.5 for 30 min (Pearse 1968) 4. Combined staining with Alcain blue at pH 2.5 for 30 rain and PAS (Mowry 1963) 5. Combined staining with aldehyde fuchsin for 3 min and Alcian blue at pH 2.5 for 30 min (Spicer and Meyer 1960) 6. High-iron diamine/Alcian blue at pH 2.5 (modified method of Spicer 1965) with treatment in solution of 120 mg N,N-dimethyl-mphenyldiamine and 20 mg N,N-dimethyl-p-phenyldiamine (VEB Chemic Berlin) and 1.4 ml 60% FeC13 in 50 ml distilled water for 45 min to 4 h, followed by 30 min in Alcian blue 7. Acid hydrolysis of sialic acid with 0.5 M sulphuric acid at 80 ~ C for 3i h, followed by Alcian blue at pH 2.5 staining (Culling et al.
3981) 8. Periodate/borohydride/saponification/PAS(Culling et al. 1974). These methods and their interpretation are summarizedin Table 3. Sections of duodenal mucosa from control guinea fowls and of virusinduced duodenal adenomas were stained in parallel, using the same solutions and for equal time.
Results N o r m a l duodenal mucosa 1
The mucin-secreting cells in the crypts o f the normal guinea fowl duodenal mucosa were scant, of elongated form and with insignificant mucin content. In the lining epithelium of the villi they were numerous, regularly distributed and stained more intensely. In the upper portion of the villi they acquired the features of typical goblet cells (Fig. 1A). The mucin-secreting cells stained with Alcian blue and azure A. The reduction of alcianophilia after acid 1 The sections shown (Figs. 1-4) are technically not of the best because of the inferiority of photomaterials available. The authors apologise for this
hydrolysis of sialic acid was more marked in the goblet cells on the surface of the villi. PAS stained them magenta even after diastase digestion, but Best's carmine did not. The combined stainings (Alcian blue/ PAS, high-iron diamine/Alcian blue, aldehyde fuchsin/Alcian blue), distinguishing between different types of mucins, revealed that generally neutral and sulphomucins prevailed in the crypts whereas the sialomucin content grew progressively toward the tips of the villi. No O-acylated sialic acids were identified with the periodate/borohydride/KOH/PAS procedure. The brush border showed a positive reactivity for neutral mucins and partly for sialomucins. Table 2 summarizes the data from the present studies. Both acid (sulpho- and sialo-) and neutral mucins were present in the guinea fowl duodenal mucosa. These mucins were mixed in most of the cells with either type prevailing, resulting in a heterogeneous population. Duodenal adenomas
The materials from the duodenum of the Pts-56-infected guinea fowls studied included regions with focal hyperplasia of the villi, as well as tubulovillous and tubulous adenomas. Compared with the normal duodenal mucosa they showed a general increase in mucus production. This was coupled with considerable variations in the mucin content and in the mucous cell density, as well as with dissimilar mucopolysaccharide staining in adjacent regions of the adenomas, along the same structure or even in its opposite epithelial layers (Fig. 2, 4). The goblet cells were particularly numerous and formed continuous rows both in the tumor glandular structures, which resembled elongated, folded or ramified crypts, and on the surface of the enlarged and branched villi of the adenomas (Figs. 1B, 3). The density of goblet cells along the luminal face o f the polyps decreased markedly. The combined stainings revealed that there was a gradual decrease of neutral mucins and sulphomucins from the deeper zone of the adenomas toward its upper portions with a parallel increase of the sialomucins. In adenomas of considerable size and relatively abundant stalk component, however, structures with mostly or exclusively sialomucins were seen below glandular formations, in which sulphomucins prevailed. The pronounced rise in sialomucins in the adenomas was demonstrated with AB staining after acid hydrolysis, but no O-acylated sialomucins were identified. The brush border was unevenly represented in the tumor structures. In some it was intensely stained while in others it was hardly distinguishable, if at all. An increased mucinous production was observed not only in all types o f a d e n o m a s but also in seemingly unchanged villi in close proximity to them.
40
Fig. 1. A Normal duodenal mucosa. Brown-black-stained sulphomucins prevail in the crypts (a) and in the lower part of the villi (b). Blue-stained sialomucins, appearing grey on the print, are in larger amounts in the goblet cells of the upper part of the villi (c). High-iron diamine/Alcian blue x 250. B Duodenal adenoma: (a) deeply located tumor glandular structures with abundant mucin-producing cells, mostly black, indicating prominance of sulphomucins; (b) luminal portion with increased number of grey sialomucin-containing goblet cells. Aldehyde/fuchsin/Alcian blue; x 250
41
Fig. 2. Distribution of mucin-producing cells in duodenal adenomas. High-iron diamine/Alcian blue; x 40 Fig. 3. Numerous and unevenly distributed goblet cells in the upper (a) und deeply located (b) gIandular structures. PAS counterstained with haematoxylin; x 125
42
Fig. 4. Deviation from the normal distribution of mucin types in duodenal adenoma: (a) adjacent glandular structures with mostly sialomucincontaining cells (appearing grey) in the right one and sulphomucins in the left one (appearing dark); (b) Distended glandular structure in the base of a tubulovillous adenoma with different mucin types in the opposite layers. High-iron diamine/Alcian blue; x 250
Table 2. Gross subjective assessment of mucus content and mucin profile of normal duodenal mucosa and of virus-induced duodenal adenomas in guinea fowl
Type of mucins
Neutral Acid SulphoSialoO-Acylated Sialo-
Normal mucosa
Duodenal adenomas
Crypts
Base of villi
Upper part of villi
Crypts and cryptlike structures
Upper glandular structures
Luminal surface
• to + _+ to + _+ to + •
+ + + to + + + to +
+ + • +
++ _+ to + + to + + _+ to +
+++ + to + + + + to + + + to + + +
0to 0to 0to 0to
0
0
0
0
0
0
to to to to
+ ++ + ++
+ ++ + ++
0, negative reaction; _+, slight reaction; + , positive reaction; + + , marked reaction; + + + , strongly positive reaction
The results of the histochemical studies of duodenal adenomas (Table 2) indicated that mostly quantitative and partially qualitative changes in the mucin secretion were found in comparison with that of the normal duodenal mucosa. Discussion
Despite the numerous histochemical studies of gastrointestinal epithelial mucins, we were not able to find data concerning birds in the available literature. The
results of Sheahan and Jervis (1976), who have studied mucin secretion in man and ten mammalian species, correspond to our finding that guinea fowl duodenum normally produces mixtures of neutral mucins, sulphomucins and sialomucins. In the current studies we established that the specificity of the mucin stainings used was relative, as also mentioned by other authors (Lapertosa et al. 1984). Nevertheless, the simultaneous processing of sections from normal duodenal mucosa and from virus-induced adenomas was suitable for histochemical as-
43 sessment of the mucin secretion alterations occurring as a result of tumorogenesis. Many authors who have studied materials from human gastro-intestinal tumors and from chemically induced experimental colonic neoplasia established changes in the mucin secretion: increased or decreased glycoprotein production; altered proportions of different mucins; substitution of one type of mucin with another (Filipe 1975; Dawson et al. 1978; Montero and Segura 1980; Reid et al. 1980; Jass and Filipe 1981; Filipe et al. 1985). Our studies showed that a general increase in the number of mucinproducing cells and in their mucoprotein content was observed in the duodenal adenomas as compared with normal mucosa. The irregular distribution of goblet cells is a piece of evidence in support of the neoplastic character of the virus-induced polypous growths. The resemblance of their mucin secretion pattern to that of the normal duodenal mucosa and the absence of large-bowel metaplasia or gastric metaplasia, however, confirmed the initial histological diagnosis that they were adenomas rather than malignant neoplasia (Kirev et al. 1987). A shift from the normal vertical distribution of mucin types was found in prominent tumors with an abundant stalk component only. Comparable data were reported by others, who studied normal gastrointestinal mucosa and benign epithelial tumors (Goldman and Ming 1968; Filipe and Fenger 1979). Besides supplementing the histological characteristics of the virus-induced duodenal adenomas, the present studies contributed to the elucidation of some aspects of their histogenesis. Our findings supported the assumption that the growths were formed as a result of crypt elongation and hyperplasia as well as of enlargement and unification of the villi. There was also evidence for impaired differentiation of the epithelium of the glandular structures manifest by irregular distribution of mucin-producing cells. The cells with scanty mucopolysaccharides, observed in some tubulous structures, resembled the undifferentiated and regenerating goblet cells described by Wattel et al. (1977). The unevenly formed brush border in the individual glandular structures was also indicative o f the dysplastic character o f the growths. Similar findings were observed in familial polyposis coli (Romagnoli et al. 1986) and could hardly be attributed in all cases to the direction of the section. In addition to frank adenomas the alterations in mucin secretion were established in zones of duodenal mucosa hyperplasia. In this respect, the latter could be considered as a possible step in the development of the adenomas. Certain differences in the mucin production were observed in the visibly unchanged villi adjacent to the adenomas. The presence of such "transi-
tional mucosa" with histochemical features resembling those of the tumor was reported by many authors (Filipe 1969; Sheahan and Jervis 1976; Shamsuddin et al. 1982). The preneoplastic significance of these changes, however, is disputed, as they occur also in non-neoplastic conditions (Romagnoli et al. 1986). The problem will remain open until sensitive methods differentiating reactive from neoplastic processes are developed. The observed alterations in mucin secretion of the duodenal adenomas and of the adjacent mucosa could be related to tumor development rather than to virus infection only, since they were not established in inoculated guinea fowls without duodenal growths. Despite the differences between the mucin profile in particular portions of the gastrointestinal tract and between separate animal species, our results are consistent with the established alterations in mucin secretion patterns of human and experimental intestinal turnouts and should be considered when the diagnostic value o f such histochemical abnormalities is discussed.
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