j. Comp. Path. 1994 Vol. 110, 169-183

H y p e r p l a s i a and H y p e r t r o p h y of B cells in the Islets o f L a n g e r h a n s in H a m s t e r s Infected wi th the 139H

Strain of Scrapie

X. Ye, R. I. Carp, Y. Yu, R. Kozielski and P. Kozlowski New York State Institule for Basic Research in Developmenlal Disabililies, 1050 Foresl till!

Road, Staten Island, New York 10314, U.S.A.

Summary Previous studies showed that in hamsters the 139H, but not the 263K, scrapie strain caused a marked increase in pancreatic size and led to obesity, hypoglycaemia and striking hyperinsullnaemia. In the preceding paper (Ye el al., 1994), the islets o1" Langerhans in 139H-affected hamsters showed cellular atrophy, fibrosis, cytoplasmic vesicles and nuclear pathological changes. In the present study, the profiles of pancreatic islets were classified into three sizes with an image analyzer. The number and total area covered by "small" islet profiles were less in 139H-affected than in normal hamsters. In contrast, the number and the area oF"medium" and "large" islet profiles were significantly greater in 139H than in normal hamsters.

With antibodies to insulin, glucagon, somatostatin and pancreatic polypeptide, the proportions of B, A, D and F cells were determined. With somatostatin-positive cells arbitrarily given a value of" 1, the ratio of B:A:D:F cells in the islets was 27:5:l:0'04 in normal hamsters and 122:7:1:0'04 in 139H-affected hamsters. The increase in B cells would account lbr the islet enlargement and the hypoglycaemia-hyl)erinsulinaenaia seen in 139H-afl'ected hamsters.

Introduction

In the previous paper we found that hamsters affected with the 139H scrapie strain had pathological changes in the islets ofLangerhans (Ye et al., 1994). The changes included cellular atrophy, fibrosis, cytoplasmic vesicles and pyknosis, karyorrhexis and karyolysis of the nuclei. The level ofscrapie-associated protein (PrP so) and the infectivity titres in pancreas were much lower than in brain. These results suggested that the pathological changes in the islets of 139H- affected hamsters were caused by a disorder of the control centre, i.e. the central nervous system (CNS), including the hypothalamus and the pituitary. There are several cell types in the islets of Langerhans and it is possible that one such type responds to the disorder of specific areas in the CNS more readily than others. In this study, we have quantitated changes in the number and size of the pancreatic islet profiles and determined the immunostaining areas of B, A, D and F cells in the pancreatic islet profiles in both control and 139H-affected hamsters. We also discuss the relationship between PrP sc accumulation and the pathological changes in the islets of 139H-affected hamsters.

0021-9975/94/020169 + 15 $08.00/(1 © 1994. Academic Press Limited

] 70 X. Ye e t al,

Mater ia l s and Methods

Animals

Female, weanling LVG/LAK hamsters were obtained, maintained and inoculated as described previously (Ye et al., 1994).

Inocula

Normal hamster brain (NHB) preparation and the 139H scrapie strain were obtained and passaged as described by Ye et aL (1994).

Immunostaining Methods

Paraffin wax tissue sections for immunostaining were prepared and stained as described by Ye et al. (1994). The sources and concentrations of different antibodies were as follows: anti-insulin, 1 in 1 (BioGenex Laboratories, San Ramon, CA, USA); anti-glucagon, 1 in 500 (DAKO Corporation, Santa Barbara, CA, USA); anti-somatostatin, 1 in 2000 (Chemicon International Inc., Temeeula, CA, USA); anti-pancreatlc polypeptide, 1 in 2000 (ICN Biomedical Inc., IL, USA). Immunostaining was carried out by either the peroxidase anti-peroxidase (PAP) method or the supersensitive alkaline phosphatase kit (BioGenex). For the PAP method, after reaction with the primary antibody and rinses, the sections were incubated with a second antibody (sheep anti-rabbit serum, 1 in 100; Cappel Organon Teknika Co., Durham, NC, USA) for 30rain at room temperature, rinsed in phosphate-buffered saline (PBS) (pH 7.4), and incubated with rabbit PAP (sheep anti-rabbit serum, 1 in 400; Cappel Organon Teknika Co., Durham, NC, USA) for 1 h at room temperature and then with diaminobenzidine substrate for 3 rain at room temperature. With the supersensitive alkaline phosphatase kits, after reaction with the primary antibody and rinses, the sections were incubated sequentially at room temperature with biotinylated secondary antibody, alkaline phosphatase-conjugated streptavidin, and chromogen substrate (BioGenex). Three controls were included. (1) Negative control: specimens which did not contain the antigen to be stained were processed in the same way as the unknown. (2) Reagent control: sections were allowed to react with a non-immune serum instead of the immune primary antibody. (3) Neutralized control: sections were allowed to react with an antiserum that had been exposed to an excess of the specific antigen. To obtain the best staining results tbr each primary antibody, an initial pilot experiment included several dilutions, some higher and some lower than the recommended dilution.

The immunostains were made with the Histomatic Slide Stainer-Code On Series (Fisher Scientific, Porta Hood, Model 171, Pittsburgh, PA, USA), an autostainer which uses an automated and very accurate capillary action system. The numbers orB, A, D and F cells in the largest islet in pancreatic sections from each control and scrapie-affected hamsters were counted by means of a light microscope.

Assessment of Imrnunostaining

Computer-assisted analysis of the distribution and intensity of immunostalning was used. The Quantimet 970 image analyser, which has a very high degree of precision, satisfied the need for increased objectivity and accuracy. A stained slide was scanned by a scanner mounted on a microscope. After calibration with the standard optical density and the length, the measurements that can be made include cell area and optical density of stained ceils. Analysis was accomplished with the Quantimet 970 system as described previously (Ye et al., 1994).

Data were analysed with the Lotus 123 program and the CSS statistics software packages. We obtained information on the number of B, A, D and F ceils in the largest islet of each pancreas section from control and 139H-affected hamsters, the number and area of the islets, the area showing positive staining, and the optical density of the positively stained areas. This information was stored, analysed statistically, and

Pancreatic Changes in Hamsters with Scrapie 171

compared by Student's t-test or analysis of variance with the data from sections obtained from animals inoculated with normal hamster brain. The numerical data presented in this paper are expressed as the mean 4-standard error. Sections of the pancreas were stained with haematoxylin and eosin (HE) and photographed as described previously (Ye et al., 1994).

Resul ts

As reported by Ye et al. (1994), HE staining showed that the pancreatic islets of 139H-affected hamsters were larger than those of control hamsters. Islet cells in 139H-affected hamsters (Fig. l d) were hypertrophied compared with those in controls (Fig. lc). As observed by Carp el al. (1990), clear spaces in the islets of 139H-affected hamsters are probably areas of haemorrhage in which blood cells were washed away during fixation and staining processes. It was observed previously (Ye el al., 1994) that these abnormal structures were almost centrally located within islets and were surrounded by B cells, some of which were abnormally elongated; each of these structures was referred to as a "blood vessel core" (BVC). In the present study, the profiles of pancreatic islets (including the lumen areas of BVCs) were arbitrarily classified into three sizes by means of the image analyser. As shown in Fig. 2, the number of "small" (area <0'01 mm 2) islet profiles was reduced from 28.04-2.8 per 50ram 2 pancreatic area (pan) in control animals to 15"0 4- 1 '9 per 50 mm 2 pan in 139H- affected hamsters. In contrast, the number of "medium" (areas 1>0.01 mm 2 and <0'1 mm 2) islet profiles was increased significantly from 19.44-2.6 per 50 mm 2 pan in control animals to 46' 1 4- 3"7 per 50 mm 2 pan in 139H-affected hamsters. The number of "large" (areas >10.1 mm 2) islet profiles was 15.04-3.6 per 5 0 m m 2 pan in 139H-affected hamsters; no large islets were found in control animals. The total number of pancreatic islet profiles was much greater in scrapie-affected (76' 14-6"6 per 50 mm 2 pan) than in normal animals (47'44- 5'5 per 50 mm 2 pan).

The total area covered by "small" islets was less in 139H-affected hamsters (0'08 4- 0'01 mm 2 per 50 mm 2 pan) than in control animals (0' 13 4- 0'02 mm 2 per 50 mm 2 pan) (Fig. 3). In contrast, the area of"medium" islet profiles was significantly greater in 139H-affected hamsters (1"824-0"18 mm 2 per 50 mm 2 pan) than in control animals (0.42 +0'07 mm 2 per 50 mm 2 pan). For islets classified as "large", there were 2"97 4-0"77 mm 2 per 50 mm 2 pan in 139H- affected hamsters, but no "large" islets found in controls. The proportion of the area of pancreas covered by islet tissue was much greater in scrapie- affected (4.874-0.73 mm 2 per 50 mm 2 pan; 9.74 per cent of the pancreatic area) than in normal animals (0.55 4- 0"08 mm 2 per 50 mm ~ pan; 1' 1 per cent of the pancreatic area).

The use of antibodies against insuiin, glucagon, somatostatin and pancreatic polypeptide enabled the immunostaining areas of B, A, D, and F cells to be measured by means of the Quantimet 970 image analyser system. Figs 4a and 4b show the pancreatic sections of normal and 139H-affected hamsters immunostained with insulin (ir-In). It is noteworthy that (1) most of the islet cells were stained with insulin antibody, (2) the area of the insulin-immuno- stained cells was larger in 139H-affected than in control hamsters, (3) the B

172 x . Ye et al.

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P a n c r e a t i c C h a n g e s i n H a m s t e r s w i t h S c r a p i e 173

SMALL MEDIUM LARGE TOTAL Number of pancreatic islets. All islets were placed in three categories (small, medium and large). The number per 50 mm ~ of pancreatic section in each category is shown for control hamsters (NHB, n = 14) and for 139H-inlEcted hamsters (n=20). Small islets <0"01 mln'-'; medium islets >/0.01 mm '~ and <0'1 ram'2; large islets >10.1 mm '~. **, P value <0'01; ***, P value <0,001.

cells in 139H-affected hamsters were hypertrophied (Fig. 4b) as compared with those in control hamsters (Fig. 4a), and (4) the density of the stain in the pancreatic islets was less in 139H-affected hamsters than in controls.

Figures 4c and 4d show immunostaining with glucagon (ir-Glu) in the islets of control and 139H-infected hamsters. The ir-Glu was located in the outer rim of the islets in both groups.

Figure 5 shows the somatostatin (ir-SS) immunostaining in the islets of control and 139H-affected hamsters. The ir-SS was also located in the peripheral mantle of the islets in both control and 139H-affected hamsters.

Table 1 shows comparisons of ir-Glu and ir-SS in the pancreatic islets of control and 139H-affected hamsters. Although the ir-Glu stained areas were significantly greater in 139H-affected hamsters (0.204-0'04 mm 2 per 50 mm 2

Fig. 1. Haematoxylin and eosln (HE) stain of pancreatic islets, a. Pancreatic islets of a control hamster, x 82. b. Pancreatic islets of a 139H-affected laamster, x 82. c. Pancreatic islets of the control hamster, x 328. d. Pancreatic islets of the I39H-affected hamster. × 328. The islets in the 139H- affected hamster were considerably larger than those in the control hamster. Islet cells in 139H- afl~cted hamsters are hypertrophied (Fig. ld) as compared with those in controls (Fig. lc). Cystic areas ("blood vessel core"; BVC) are present in the islets of 139H-affected hamsters. Aci, acini; Is, islet.

174

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Fig. 4. Immunostalned insulin and glucagon in islets, a. Pancreatic islet of a control hamster (insulin immunostained, HE counterstain, x 245). b. Pancreatic islet of a 139H-affected hamster (insulin immunostained, HE counterstain, x 245). Note that most of the islet cells stained with insulin antibody; the area o£1nsulin-immunostained ceils in the 139H-affected hamster is larger than in the control hamster; B cells in 139H-affected hamsters are hypertrophied (4b) as compared with those in control hamsters (4a); the intensity of immunosta ined insulin (Jr-In; arrowhead) is less in the islets of the 139H-affected hamster than in those of the control hamster, c. Pancreatic islet oF a control hamster (glucagon immunostaincd, HE counterstain. × 245). d. Pancreatic islet of a 139H- affected hamster (glucagon immunostained, HE counterstain, x 245).

The immunostained glucagon (ir-Glu, arrowhead) is located in the outer rim of both the control (4c) and the 139H-affected hamsters (4d). In the control hamster, the ir-Glu cells form a circle in this section, and many of the cells are parallel to the mantle (4c). However, in 139H-aII~cted hamsters, the ir-GIu cells are scattered in the peripberal mantle and are not parallel to the mantle (4d). Small arrow shows that the orientation of the islet ceils is changed in the 139H-affected hamster. Also, there is marked hyperplasia and hypertrophy orthc islet cells and marked swelling of the cell nuclei in the 139H-aflhcted hamster.

Pancreatic Changes in Hamsters with Scrapie ] 75

Fig. 5. Immunostained somatostatin in islets, a. Pancreatic islet of a control hamster, x 82. b. Pancreatic islets of a 139H-affected hamster, x82. c. Pancreatic islet of the control hamster, x 328. d. Pancreatic islet of the 139H-affected hamster, x 328.

The immunostained somatostatln (ir-SS, arrowhead) is located in the outer rim of the islets of both the control and the 139H-affected hamsters. In the control hamster, many of the ir-SS cells are parallel to the mantle (Fig. 5a and c}, but in tile 139H-affected hamster, the ir-SS cells are scattered in the peripheral mantle and are not parallel to the mantle (Fig. 5b and d).

Pancreatic Changes in H a m s t e r s w i t h S c r a p i e 177

Table I Analys is o f i m m u n o r e a c t i v e g lucagon and somatostatln in the pancrea t ic islets of control and

139H-affected hamsters

ir-Glu ir-SS

lnoculum PA PA % PA PA %

NHB 0'094-0.02 17-54-2"4 0"024-0.00 3'24-0"6 (15) (15) (16) (16)

139H 0.20 4- 0.04* 5"6 4- 0.8" ** 0.03 4- 0.01 1.0 4- 0.2' * (20) (20) (1~) (18)

Numbers of animals given in parentheses. PA, positively stained area (mm~/50 ram2); PA%, percentage of positively stained area in total islets. *, P<0"05; **, P<0.01; ***, P<0'001.

pan) than in control animals (0.09 +0'02 mm 2 per 50 mm 2 pan), the percent- age of total islet area stained with ir-Glu was significantly less in 139H-affected hamsters (5'6 4- 0"8 per cent) than in control animals (17.5 4- 2'4 per cent). No significant difference in the areas stained with ir-SS was noted between control and 139H-affected animals. However, the percentage of the total islet area stained with ir-SS was significantly less in 139H-affected hamsters (1'04-0'2 per cent) than in control animals (3"2 4- 0"6 per cent). These differences in percentages were a function of the dramatic increase in the number of B cells in the islets.

Immunostaining for pancreatic polypeptide (ir-PP) in islets of a control and a 139H-affected hamster is shown in Fig. 6. The areas stained with ir-PP did not differ significantly in control (0'0006-4-0"0003 mm 2 per 50 mm 2 pan) and 139H-affected hamsters (0.0011 + 0"0005 mm 2 per 50 mm 2 pan). In addition, the percentages of total islet area stained with ir-PP did not differ significantly.

In Fig. 7, the data on positively stained areas of B, A, D and F cells are summarized. The areas immunostained with insulin and glucagon were significantly greater in 139H-affected than in control animals. The areas immunostained with somatostatin and pancreatic polypeptide did not differ significantly between the scrapie-affected and control groups. The proportion of B, A, D and F cells was also determined. With somatostatin-positive cells arbitrarily given a value of 1, the ratio of B:A:D:F cells in the islets of normal hamsters was 27:5:1:0"04, whereas in the islets of 139H-affected hamsters, the ratio was 122:7:1:0"04. Table 2 shows comparisons of B, A, D and F cell numbers in the largest islet profile of each pancreas section from control and 139H-affected hamsters. The average number of B cells in the largest islet profiles of 139H-affected hamsters ( 1123" 7 4- 102-9) was significantly increased as compared with the average number in control hamsters (439'74-37'2). However, there were no significant differences in the numbers of A, D and F cells between control and 139H-affected hamsters.

As shown in Fig. 8, the average optical density per pancreatic area (OD/ pan) of ir-In was less in 139H-at-tkcted hamsters than in control animals. The OD/pan ofir-In "small" islets was significantly less in 139H-affected hamsters (0.33 4- 0"02) than in control animals (0"43 4- 0'03). Similarly, the OD/pan of the it-In "medium" islets was significantly less in the 139H-affected hamsters

178 X. Ye e t al .

Fig. 6. Immunostained pancreatic polypeptide in islets, a. Pancreatic islet of a control hamster, x 328. b. Pancreatic islets of a 139H-affected hamster, x 328. The immunostained pancreatic polypeptide (ir-PP, arrowhead) is located in the outer rim of the islets of both control and 139H-affected hamsters, Is, islet.

(0.314-0.02) than in control animals (0.404-0.03). The OD/pan of ir-In "large" islets in the 139H-affected hamsters was very low (0'234-0"03). The total OD/pan of Jr-In was significantly less in 139H-affected hamsters (0.294- 0'01) than in control animals (0'42 4- 0.01). The results suggest that in islets of 139H-affected hamsters either the concentration of insulin in B granules was decreased, or the number of B granules was decreased, or both.

P a n c r e a t i c C h a n g e s in H a m s t e r s w i t h Scrapie 179

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Table 2 Number of B, A, D and F cells in the largest islet profile of each pancreatic

section in control (n = 14) and 139H-affected (n = 18) hamsters

Number of cells in

Control 139H-affected P-value (,ell hamsters hamsters

B 439.7±37.2 1123'7±102'9 <0'0001 A 65'5±5'3 77,1~7'2 0'22 D 22.6±2.4 16.4±2.2 0.07 F 0'3~0.1 0,4~0,1 0,63

The OD/pan of ir-Glu did not differ significantly between control animals (0"25 4-0.01) and 139H-affected hamsters (0'24 4-0"02), nor did the OD/pan of ir-SS (0" 17 4-0"02 for control and 0'21 4- 0"03 for 139H-affected hamsters). The OD/pan ofir-PP also did not differ significantly between control (0'26 4- 0"03) and 139H-affected hamsters 0"28 4- 0'02).

Discussion

Histopathological examination in the present study showed that islets from 139H-affected hamsters were markedly enlarged, demonstrating both hyper-

180 X. Y e e t al .

Stained cells' OD of ir-ln in NHB/139H

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The average optical density per pancreatic area (OD/pan) of lr-In fi'om 139H-affected hamsters (n= 20) and control hamsters (NHB, n= 14). Small islets <0'01 mm~; medium islets t>0'0I mm ~ and <0.1 ram2; large islets >f0.1 iron"; average ol" optical density of stained cells per pancreatic area. **, P value <0.01; ***, P value <0.001.

plasia and hypertrophy of the islet ceils (Carp et al., 1990; Ye et al. 1994). Dispersion of the endocrine tissue of the pancreas in the form of small islets within a much larger exocrine gland greatly complicates quantitative studies. However, changes in the number and size of islet profiles in a given area of pancreatic tissue can be quantitated precisely by the Quantimet 970 image analyzer.

The islets of Langerhans are composed of four different cell types and comprise 1-2 per cent of total pancreatic mass in normal animals (Granner, 1988). As shown in the present study, the proportion of the area of pancreas covered by islet tissue was much greater in scrapie-affected (4-87 4- 0.73 mm 2 per 50 mm 2 pan; 9"74 per cent of the pancreas area) than in control animals (0'55 +0.08 mm 2 per 50 mm 2 pan; 1.1 per cent of the pancreas area). The proportion of the area orB, A, D and F cells was determined by the Quant imet 970 computer image analysis system. With D cells arbitrarily given a value of 1, the ratio of B:A:D:F cells in the islets was 27:5:1:0"04 in normal hamsters and 122:7:1:0"04 in 139H-affected animals (see Fig. 7). The immunostaining area of B cells in 139H-affected hamsters was approximately 7.7 times greater than in control animals. Since increases in immunostaining area may represent

Pancreatic Ghanges in Hamsters with Scraple 181

either an increase in the size of individual cells, or in the number of cells, or both, we counted the number of B, A, D and F cells fi'om the largest islet found in each pancreatic section of all animals. To standardize the counting process, only the largest islet profile in each section was assayed. The average number of B cells in the largest islet fi'om pancreatic sections of 139H-affected hamsters was approximately 2'5 times greater than in control samples (Table 2). There were no significant differences in the number of A, D, and F cells between control and 139H-affected hamsters. Visual evidence indicated that the B cells were also hypertrophied (Figs 4c and 4d). Numerical evaluation also suggests that there was B cell hypertrophy: as noted above there was a 7'7 fold increase in the total B cell immunostained area. A portion of this increase was a function of the B cell hyperplasia noted above (2'5-fold increase in B cell number in 139H-infected animals). Another part of this increase was related to the increased number of islet profiles per 50mln ') section (76.1 in 139H- affected animals and 47.4 in controls). These factors would yield an increase in B cell area of approximately four-fold. The remaining portion of the incre- ment in immunostained area (approximately two-fold) was probably a ihne- tion of the enlargement orB cells observed in 139H-affected hamsters. For A cells, the significant increase in the immunostaining area but not in the number of cells suggests cellular hypertrophy (see Figs 4c and d). The results indicate that the increase in the number and size of B cells plays a major role in the enlargement of pancreatic islets, and also in the hypoglycaemia-hyperinsuli- naemia seen in hamsters infected with the 139H scrapie strain.

It is clear that paracrine and autocrine regulation occurs within the islets of Langerhans, i.e. the secretory product of each islet cell type can regulate the activities of neighbouring cells of another cell type, or can regulate the cell itseli: According to Samols el al. (1983), if the A, B and D cells are visualized as the vertices of an equilateral triangle, there are three potential interactions by short (intra-islet) or long (systemic circulation) loops: (1) insulin may inhibit glucagon secretion; (2) glucagon may stimulate insulin and somatostatin secretion; (3) somatostatin may inhibit insulin and glucagon secretion. Thus, there is a potential positive-negative glucagon-insulin feedback, a potential positive-negative glucagon-somatostatin feedback, and a potential negative somatostatin-insulin feedback (Samols et al., 1983). These systems are affected by many factors such as islet architecture, islet cell composition, blood flow, extracellular fluid flow, cellular shape and orientation, extracellular deposi- tions, receptor domains, cell-to-cell contacts and islet microenvironment (Ye et al., 1994). The changes in the number and ratio orB, A, D and F cells in the islets of 139H-affected hamsters would certainly affect the potential interac- tions among these cells within the islets.

Marked hyperinsulinaemia in 139H-affected hamsters has been reported previously; the concentration of immunoreactive insulin in the plasma of scrapie hamsters was as much as 49 times higher than that seen in control hamsters (Carp et al., 1990). It is possible that the effect of autocrine regulation of insulin on B cells might be decreased in 139H-affected hamsters. The reduced effect may be explained by down-regulation of receptors, which is a general response to a high concentration of circulating hormone. Prolonged

182 X. Ye et al.

exposure of tissues to a high concentration of insulin would decrease insulin receptors on cell membranes, and consequently the autocrine effect of insulin might be decreased.

A further interesting question concerns the optical density of insulin immunostaining. Two very useful tools were used in our immunostaining studies: (1) immunostaining with the Histomatic Slide Stainer-Code On Series (Fisher Scientific) technique, which standardizes the immunostaining inten- sity, and (2) the Quantimet 970 analyser, which accurately quantitates the optical density of objects. The human visual system cannot effectively quanti- tate optical density of immunostains and the background density of tissue sections.

Density analyses showed that the optical density of insulin-immunostaining cells was less in the islets of 139H-affected hamsters than in control animals. This suggests that either the concentration of insulin in B granules was decreased or the number of B granules was decreased, or both. Because hamsters infected with the 139H scrapie were only moderately hypoglycaemic but showed marked hyperinsulinaemia as measured by immunoreactivity, it is possible that B cells release insulin molecules more quickly in 139H-affected hamsters than in control animals. These molecules might not have enough time for post-translational modification. That is to say, B cells may release insulin molecules prematurely in 139H-affected hamsters; therefore, the bioactivities of these insulin molecules might be affected. In normal individuals, proinsulin (Pin) levels are 5-15 per cent of immunoreactive insulin (IRI). The levels of Pin can be substantially elevated in patients with non-insulin-dependent diabetes mellitus (NIDDM), islet cell tumours or chronic renal failure. Recently, Saad et al. (1990) reported that Pin accounted for 50 per cent of the total IRI in some NIDDM patients with severe hyperglycaemia. Current insulin radio-immunoassays possess high cross-reactivity to P in and thus over- estimate true insulin values. A specific assay for the measurement of true insulin concentration is now available (Linco Research, Inc., St Louis, MO, USA). The results of our studies suggest that the percentage of true insulin in total IRI is less in 139H-affected hamsters than in control animals. By means of assays for true insulin and for total insulin, we intend to assess this possibility.

The development of an obesity syndrome in mice surviving the effects of intracerebral infection with mouse-adapted canine distemper virus (CDV), a morbillivirus related to measles, has been described (Nagashima et al., 1992). The syndrome included pronounced hyperplasia and moderate hypertrophy of fat cells; reduced brain, ovary, uterus and prostate weights; and increased liver, kidney and pancreas weights, with greatly enlarged pancreatic islet tissue. These obese animals were also hyperinsulinaemic. The authors suggested that the loss of critical populations of hypothalamic neurons as a result of earlier CDV infection ultimately led to the development of morbid obesity. Many recent studies of transmissible spongiform encephalopathies have suggested that PrP sc may play an important role in disease pathogenesis (Taraboulos et al., 1992; Xi et al., 1992). Our finding in this study raises a key question, namely, whether the pathological changes in the islets of Langerhans are caused directly by PrP ~ itself or by changes in the central nervous system. As

Pancreatic Changes in Hamsters with Scrapie 183

an approach to resolving this issue, we compared the levels of PrP sc in the pancreas, spleen and brain of the 139H-affected hamsters (Ye et al., 1994). Our studies indicated that the level ofPrP sc as well as infectivity (Carp el al., 1990) in pancreas were at least 1000 times less than in the brain. The implications of this finding were explored in detail by Ye et al. (1994); in brief, it appears unlikely that the scrapie agent directly causes pathological changes in organs outside the central nervous system. It is most likely that the pathological changes noted in the islets of Langerhans in 139H-affected hamsters are a function of the specific targeting by scrapie of hippocampal, hypothalamic and/or pituitary cells that are involved in endocrine function (Carp el al., 1990; Ye et al., in preparation).

Acknowledgments

We thank Ms Jennifer DeKolf for editing and excellent assistance in preparation of this manuscript, and Ms Sharon Callahan, Dr Judy Shek and Mr R. Weed for their collaboration. The authors appreciate the excellent assistance of Ms Mary Ellen Cafaro with the layout of figures and Ms Sharon Mathier for graphics preparation. This work was supported by the New York State Department of Mental Hygiene and NIH grant AG0912. Dr Ye was supported by a fellowship from the CSI/IBR Center for Developmental Neuroscience.

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~ Received, June 18th, 1993 '] Accepted, Oclober 21st, 1993]