Detection of Systemic Amyloidosis in the Pig-Tailed Macaque (<I>Macaca

Detection of Systemic Amyloidosis in the Pig-tailed Macaque (Macaca nemestrina)

Renee R Hukkanen,1,2,* H Denny Liggitt,1,2 David M Anderson,1,2 and Stephen T Kelley1,2

Secondary amyloidosis is a progressive systemic disease for which there is no reliable diagnostic assay, preventive measure, or treatment. In an attempt to elucidate an antemortem diagnosis, 30 female pig-tailed macaques (Macaca nemestrina) at the Washing- ton National Primate Research Center were surveyed for amyloidosis. Amyloid was demonstrated histologically in 47% (14 of 30) of the animals. The distribution and severity of amyloid deposition was variable. Affected animals had a mean age (±1 standard deviation) of 13.2 ± 4.9 y, which was signifi cantly greater than the mean age of unaffected animals (9.3 ± 4.1) y. Twelve tests were evaluated for detection of amyloidosis; the diagnostic value of each was determined through comparison of histologically positive and histologically negative animals. Diagnostic tests evaluated were endoscopic examination and biopsy of the stomach and colon, abdominal ultrasonography, hepatic radiology, serum amyloid A (SAA), endothelin 1, alpha-fetal protein, aspartate aminotransferase (AST), alanine aminotransferase, gamma-glutamyltransferase (GGT), alkaline phosphatase, cholesterol, blood urea nitrogen, total bilirubin, C-reactive proteins, and erythrocyte sedimentation rate. Amyloidotic animals demonstrated a distinctive serologic profi le: elevated SAA, GGT, and AST in combination with decreased total protein and albumin. Radiology demonstrated hepatomegaly in animals with hepatic amyloid deposition. In the absence of known infection or trauma, an amyloidotic serologic profi le and radiologic hepatomegaly are consistent with systemic amyloidosis in M. nemestrina.

Abbreviations: AA, secondary amyloidosis; AFP, alpha fetoprotein; AL, primary amyloidosis; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; C/B length, crux to base length; CRP, C-reactive protein; ELISA, enzyme-inked immunosorbent assay; ESR, erythrocyte sedimentation rate; ET-1, endothelin 1; GI, gastrointestinal; GGT, gamma- glutamyltransferase; PCR, polymerase chain reaction; SAA, serum amyloid A; SRV, simian retrovirus; STLV, simian T-lymphotropic virus type 1; TDP, Tissue Distribution Program; VD, ventrodorsal; WaNPRC, Washington National Primate Research Center

Amyloid is a protein which, when folded incorrectly, aggregates birefringence with polarization. Amyloid fi bril type may be dis- extracellularly. First described in 1854 by Rudolph Virchow,44 tinguished through 1 of 2 methods that exploit biochemical dif- amyloid fi brils were ultrastructurally delineated in 1959.53 There ferences in the fi brils: potassium permanganate oxidation or the are currently 20 recognized clinical syndromes associated with Strasburg method. Potassium permanganate oxidation demon- individual forms of amyloid. Diseases in which amyloid plays a strates that AL fi bers retain Congo Red staining properties post- pathogenic role are classifi ed as ‘amyloidotic;’ examples of which oxidation whereas AA fi brils do not.17,52 The Strasburg method include Alzheimer disease, adult-onset diabetes, transmissible demonstrates that AA fi bers retain electrophoretic and antigenic spongiform encephalopathy, lichen dermatitis, senile cardiovas- properties after formalin fi xation whereas AL fi bers do not. cular disease, reticulum cell sarcoma, plasma cell dyscriasis, and Nonhuman primates (NHP) are models for multiple amyloid multiple myeloma.28,29 Amyloidosis has historically been classifi ed diseases.31 Published models of cerebral amyloidosis include the based on clinical presentation; however, classifi cation is now based crab-eating macaque (Macaca fascicularis), rhesus macaque (M. on biochemical composition. Primary amyloidosis (AL) is due to mulatta),33,34 squirrel monkey (Saimiri sciureus),49 gray mouse le- overproduction of the immunoglobulin light chain and may be mur (Microcebus murinus),7 and common marmoset (Callithrix neoplastic or genetic in origin. Secondary, or reactive amyloidosis jacchus).3 Reactive amyloidosis has been described in the Patas (AA), is due to an accumulation of serum amyloid A (SAA)—an monkey (Erythrocebus patas),32 squirrel monkey (S. sciureus),4 acute phase protein associated with inflammation.43 Although mandrill (Papio sphinx),56 chimpanzee (Pan troglodytes),23 and bar- the mechanisms and exact cause of secondary amyloidosis are bary ape (M. sylvanus).9 Endocrine disorders of the pancreas have incompletely understood, it may be precipitated by long-standing been associated with amyloid deposits in the baboon (Papio spp., infection or malignancy. Genetic, infectious, and environmental Mandrillus leucophaeus),21,24 Macaca nigra,22 and Formosan rock components may all play a role in fi bril accumulation. macaque (M. cyclopis).21 Although histologic examination is the ‘gold standard’ for di- In biomedical research, naturally occurring diseases may have agnosis of amyloid, both forms (AA and AL) are ultrastructurally deleterious effects on experimental outcomes.37 Animals with similar. Both AA and AL fi bers bind Congo Red and exhibit green severe amyloid deposition are poor research and anesthesia can- didates because of altered hepatic, renal, and gastrointestinal (GI) Received: 12 Oct 2005. Revision requested: 28 Nov 2005. Accepted: 2 Jan 2006. function. Amyloidosis may present clinically as cachexia,6,20 in- 1 2 Department of Comparative Medicine and Washington National Primate Research Center, 6,10,43 6,12 University of Washington School of Medicine, Seattle, Washington. tractable diarrhea, or arthralgia in macaques (M. mulatta, *Corresponding author. Email: [email protected] M. nemestrina, and M. arctoides).

119 Vol 56, No 2 Comparative Medicine April 2006

Currently, there is no way to screen for subclinical amyloidosis. pentobarbital sodium overdose (100 mg/kg; Nembutal, Ovation In an attempt to establish an antemortem diagnostic test or panel Pharmaceuticals, Deerfi eld, IL), consistent with recommendations of tests for detection of amyloidosis, a colony survey of Macaca of the 2000 Report of the American Veterinary Medical Associa- nemestrina was performed at the Washington National Primate tion Panel on Euthanasia.1 Research Center (WaNPRC). For a period of 12 mo, from April Blood collection. Blood was obtained via the femoral vein by us- 2003 to March 2004, animals selected for removal from the colony ing 22-gauge needles and phlebotomy tubes. Blood was collected were sampled and imaged. Diagnostic tests included: endoscopic into serum separator and ethylenediamine tetraacetic acid tubes, examination and biopsy of the stomach and colon, ultrasonog- which were gently agitated; samples in serum separator tubes raphy of the liver, hepatic radiology, serum amyloid A (SAA) were allowed to clot for 15 min. All tubes were spun at 1000 × g for enzyme-linked immunosorbent assay (ELISA), endothelin 1 (ET- 15 min to obtain serum and plasma. Aliquots of each were frozen 1) ELISA, alpha-fetoprotein (AFP) ELISA, tissue histology (the at –70 °C for immunoassays. gold standard of diagnosis), and an expanded chemistry panel Serology. Expanded serum chemistry panels were performed including C-reactive protein (CRP), erythrocyte sedimentation on fresh serum and plasma by the Department of Laboratory rate (ESR), albumin, blood urea nitrogen (BUN), bilirubin, total Medicine, University of Washington Medical Center, by using an cholesterol, total protein, gamma-glutyltransferase (GGT), ala- LX-1 Synchron (Beckman Coulter, Brea, CA) automated labora- nine aminotransferase (ALT), aspartate aminotransferase (AST), tory system. alkaline phosphatase (ALP), and AFP. The results of each assay Hepatic radiography. Ventrodorsal (VD) and right lateral views were compared with the histologic amyloid status (positive or were obtained for each animal. Standard lead scales were used to negative) of each animal by the Wilcoxon signed rank test. Sen- determine magnifi cation and normalize measurements on each sitivity, specifi city, and predictive values were determined where fi lm. Three parameters were calculated from each fi lm set: 1) the applicable. number of vertebrae to which the liver shadow extended (lumbar extension) on a VD view, 2) the length (cm) of the liver shadow Material and Methods from crux to base (C/B length), measured to the right of the midline, perpendicular to the crux on a VD view, and 3) the extension Animals. For a period of 12 mo, 48 female M. nemestrina mon- of the liver shadow below the sternum, measured in intercostal keys that were unsuitable for research or breeding applications spaces on a right lateral. All animals were fasted for a minimum were removed from the WaNPRC colony and assigned to this of 16 h prior to imaging, to ensure gastric emptying. project through the Tissue Distribution Program50 (TDP; located Ultrasonography. Images of the liver, spleen, and kidneys were at the WaNPRC in Seattle); complete data sets (necropsy, serology, taken on an Ultramark 4 (Advanced Technology Laboratories, and imaging) were available on 30 of these animals. These ani- Bothell, WA) machine using 1 of 2 transducers (5 MHz curved mals originated from 1 of 2 WaNPRC breeding colonies (Tulane, or linear array). Printed images were obtained for comparison LA and Bogor, Indonesia). The breeding colony in Tulane consists of relative echogenicity of the spleen, kidney, and liver. Hepatic of captive-reared, group-housed (outdoor open-air gang cages) parenchymal echogenicity, homogeneity, artifact formation, and animals of known health, genetics, and geneology. The Indone- perivascular connective tissue were examined and compared be- sian breeding colony consists of animals with minimal medical tween animals. and genetic information. Animals arrived at the Seattle facility Endoscopy. Animals were intubated and prepared with 3 variable amounts of time (3 to 87 mo) prior to euthanasia. Ani- warm-water enemas. Endoscopic guided stomach (glandular and mals were screened for viral pathogens (SRV-2, simian T-lympho- aglandular portions) and colonic biopsies were performed using tropic virus type 1 (STLV), and simian immunodefi ciency virus) a Pentax FG-24X (Pentax Medical Company, Montvale, NJ). Upon prior to entry into the Seattle colony, and biannually (SRV-2 and recovery, animals were given ketoprofen (5 mg/kg; Ketofen, Fort STLV) thereafter. Animals positive for virus by polymerase chain Dodge Animal Health, Fort Dodge, IA) intramuscularly once and reaction (PCR) assay were removed from the colony. then orally at 12-h intervals for the next 24 h. In cases of overt gas- Animal housing in the Seattle colony consisted of individual trointestinal bleeding after biopsy, sucralfate (250 mg/kg; TEVA metal cages with rotating environmental enrichment toys.51 Ani- Pharmaceutical Industries, North Wales, PA) was administered mals were fed commercial monkey biscuits (Purina, Lab Diet, by gastric lavage prior to extubation. Animal Specialties, Hubbard, OR), supplemented with a variety Necropsy and histopathology. Histologic evaluation was per- of fruits, vegetables, and novel dietary enrichment items. Waste formed on 30 animals. Sections of the GI tract were collected from was collected in absorbent paper bedding (Absorption CareFresh, all animals: stomach (glandular and aglandular portions), small International Absorbents, Ferndale, WA) in individual waste pans intestine (jejunum midway from stomach to cecum and ileum under each cage. Water was available ad libitum. All animal re- at ileocecal junction), and large intestine (cecum at the cecocolic lated activities were performed in accordance with guidelines junction and colon at rectum–colon transition). Three sections of from the National Institutes of Health under a protocol approved the liver were collected from all animals (distal tip of the right by the Institutional Animal Care and Use Committee of the Uni- medial lobe including the gall bladder, left lateral lobe, and papil- versity of Washington. lary process of the caudate lobe). When available from the TDP, Anesthesia. Blood collection, radiology, and ultrasonography samples from the duodenum, spleen, pancreas, kidneys, and ad- were performed while animals were sedated with ketamine HCl renals were obtained and evaluated. Tissues were placed into 10% (10 mg/kg; Ketaject, Phoenix Pharmaceuticals, St Joseph, MO) neutral buffered formalin, processed conventionally, embedded and atropine sulfate (0.04 mg/kg; American Pharmaceutical Part- in paraffi n, cut, and stained with hematoxylin and eosin.45 Tissues ners, Schaumberg, IL). Endoscopy was performed while animals were examined by light microscopy. On the basis of the work of were sedated with teletamine–zolazepam (5 mg/kg; Telazol, Fort Ganowiak and others,18 amyloid was graded from 0 to 4+, accord- Dodge Animal Health, Fort Dodge, IA) and atropine. Animals ing to the amount of deposition and percentage of tissue involved were sedated with ketamine–atropine prior to euthanasia with 120 Detection of amyloidosis in pig-tailed macaques

Table 1. Summary of amyloid deposition in amyloid-positive animals Animal Liver Stomach Small intestine Large intestine Adrenals Spleen Kidney Reason for euthanasia 1 ++ + + + + +Systemic disease: hepatitis 2 ++ + + + + +Degenerative condition: osteoarthritis 3 ++ + + NT + – Systemic disease: bacterial enteritis 4 ++ + – ++– Nonassignable 5 + – + – +++Systemic disease: renal failure 6 ++ – ++– + Chronic diarrhea 7 + –– + –NT+ Chronic diarrhea 8 + –– – + NT NT Nonassignable 9 + –– – + – + Systemic disease: anemia, hemosiderosis 10 – ++ + – + – Systemic disease: vasculitis 11 – – + – – NT – Nonassignable 12 – – – – + – – SRV-2-positive 13 – – – – + – – SRV-2-positive 14 – – – – – – + Reproductive concern: dystocia +, amyloid present; –, amyloid absent; NT, no tissue available. in the most severely affected section. Histologic fi ndings were Histology. The incidence of amyloidosis among animals sur- confirmed by 3 independent pathologists. Sections containing veyed was 47% (14 of 30 monkeys). Amyloid was present in 30% histologic evidence suggesting amyloid deposition were stained (9 of 30) of the livers and 30% (9 of 30) of the GI tissue examined with Congo Red.17 (Table 1). Most (78%, 7 of 9) of the animals with hepatic amyloid SAA ELISA. Serum Amyloid A ELISA kits for human diagnos- also had GI amyloid and vice versa (78%, 7 of 9). Of the 9 animals tic use (Anogen, Ontario, CA and TriDelta Diagnostics, Morris with GI amyloid, deposits occurred in multiple regions of the GI Plains, NJ) were performed on previously frozen (−70 °C) sam- tract in 78% (7 of 9) of the animals. The small intestine was the ples according to the manufacturers’ directions. most common site of deposition (78%, 7 of 9), followed by the AFP ELISA. An ELISA kit for human diagnostic use was ob- stomach (67%, 6 of 9) and colon (67%, 6 of 9). Amyloid was pres- tained (Panomics, Redwood City, CA) and performed on banked ent in 38% (9 of 24) of the adrenal glands examined, 25% (7 of 28) blood (−70 °C) according to the manufacturer’s directions. Sam- of the kidneys, and 24% (6 of 25) of the spleens (Table 1). All 9 ples from macaques with hepatocellular carcinoma were not macaques with hepatic amyloid, all 6 animals with splenic amy- available for use as controls. loid, and most (89%, 8 of 9) cases of GI amyloid demonstrated ET-1 ELISA. An ELISA kit for human diagnostic use (Parameter additional organ deposition. Isolated organ deposition occurred Human Endothelin-1 Immunoassay, R&D Systems, Minneapolis, in the kidneys (1 animal), the adrenals (2 animals), and the small MN) was used on previously frozen (−70 °C) samples according intestine (1 animal). to the manufacturer’s directions. Liver. In mild cases, amyloid deposition was noted between Statistical analysis. The 30 animals were grouped retrospec- hepatocytes and endothelial cells (space of Disse) of the sinu- tively, in light of presence (general) and location (hepatic or GI) of soids (Figure 1A), close to central veins; periportal regions were amyloid deposition. Three divisions consisting of paired group- spared. In moderate cases, amyloid fibers were compact and ings were established to evaluate the signifi cance of antemortem dense, surrounding hepatocytes and separating one from an- diagnostic tests in predicting amyloid status. As the distribu- other. In severe cases, amyloid replaced as much as 90% of the tion of data was not uniform, the Wilcoxon signed rank test, a lobar parenchyma. In such cases, acinar architecture was obliter- nonparametric alternative to the paired t test, was used. When ated by amyloid. group mean values were compared, both 1- and 2-tailed Wilcoxon GI tract. Amyloid deposits within the GI tract varied in severity signed rank tests were considered where applicable (95% confi - between animals and within segments of the GI tract. Mild gastric dence). A chi-square analysis was applied to the data in the form amyloid deposits occurred in subepithelial tissue within the glan- of a 2-by-2 table to determine sensitivity, specifi city, and predictive dular stomach. In moderate cases, deposits were present deep values. The threshold for statistical signifi cance was set at P = 0.05 within the mucosa, dividing and separating glands, obliterat- for all analyses. ing normal architecture (Figure 1B) The aglandular stomach was spared in mild and moderate cases. Of the 30 animals studied, Results 27 (90%) had concomitant mild to moderate lymphoplasmacytic gastritis with gastric-associated lymphoid tissue proliferation. Gross pathology. Mild cases of amyloidosis, regardless of organ, Gastritis was graded from minimal to severe in light of distri- were not detected at gross necropsy. Moderate to severe amyloid bution within the mucosa, herniation of gastric-associated lym- deposition endowed a pale tan to yellow color to the liver and phoid tissue, edema, and presence of lymphoplasmacytic and kidneys. Moderate to severe amyloid deposits altered renal pa- polymorphonuclear cells. There was no association between de- renchymal composition, imparting a fi rm texture to the medulla gree of gastritis and gastric amyloid deposition. Small intestinal and pelvis. The range of gross liver lesions varied from small tan amyloid presented clinically with diarrhea and weight loss due foci near lobule margins to diffuse tan-red mottling of all lobes. to villar distention with amyloid and loss of absorptive capacity. Severely affected livers bulged on cut section and were extremely Enteric parasitism (encysted nematode larvae within the stomach friable with indistinct acinar morphology. and intestinal submucosa and serosa) was an incidental fi nding 121 Vol 56, No 2 Comparative Medicine April 2006

ABC

DEF

Figure 1. Histologic amyloidosis. (A) Hepatic amyloidosis. Mild amyloidosis with maintenance of sinusoids, hepatocellular plates, and cell–cell connec- tions. Loosely arranged, pale eosinophilic amyloid fi brils are present within the space of Disse (indicated by arrows). Fibrils line sinusoids and surround hepatocytes. Hematoxylin and eosin stain; magnifi cation, ×400; bar, 100 μm. Inset: Photomicrograph demonstrating the zonal pattern of amyloidosis. Pale regions represent amyloid deposits. Hematoxylin and eosin stain; magnifi cation, ×20; bar, 1 mm (B) Gastrointestinal amyloidosis. Moderate amyloid deposition within the glandular stomach. Pale eosinophilic amorphous material is present within the lamina propria, dividing and separating mucosal glands, obliterating normal architecture. Hematoxylin and eosin stain; magnifi cation, ×200; bar, 100 μm. (C) Congo Red-stained sample showing moderate large intestinal amyloidosis. The lamina propria is distended by eosinophilic amyloid fi brils which replace normal cellularity. Magnifi cation, ×400; bar, 100 μm. (D) Splenic amyloidosis. Splenic amyloid replaces more than 60% of the parenchyma, radiating from follicles into surrounding parenchyma. Hematoxylin and eosin; magnifi cation, ×40; bar, 500 μm. Inset: Photomicrograph demonstrating the eosinophilic glassy amyloid fi brils. Hematoxylin and eosin stain; magnifi cation, ×200; bar, 100 μm. (E) Adrenal amyloidosis. The pale zone (indicated by arrow) between the cortex and medulla is composed of compact eosinophilic amyloid fi brils. Hematoxylin and eosin stain; magnifi cation, ×100; bar, 200 μm. (F) Renal amyloidosis. Amyloid fi brils fi ll the intersititium of the renal pelvis, separating and surrounding tubules. Hematoxylin and eosin stain; magnifi cation, ×40; bar, 1 mm. in 30% (9 of 30) of the animals, with similar distribution among circumferential, separating the medulla from the cortex with sig- amyloid-positive (29%, 4 of 14) and amyloid-negative (31%, 5 of nifi cant cortical infi ltration. Severity of deposition is summarized 16) animals. Large intestinal amyloid deposits extended into the in Table 2. mucosa, obliterating normal architecture through separation and Kidney. Renal amyloid occurred predominantly within the reduction of glands (Figure 1C). pelvic interstitium (Figure 1F), surrounding and compressing Spleen. In mild to moderate cases, amyloid fi brils surrounded tubules; severity of deposition is summarized in Table 2. Glomer- follicles, extending from the white pulp into the red pulp. In se- ular amyloidosis (expansion of the mesangial matrix with pale vere cases, amyloid obliterated normal architecture (Figure 1D). eosinophilic fi brillar material) occurred only in cases of severe Severity of deposition is summarized in Table 2. pelvic amyloidosis. Lymphoplasmacytic interstitial nephritis and Adrenal gland. Amyloid deposits uniformly occurred at the cor- tubular lesions (degeneration, regeneration, and ectasia) were ticomedullary junction, extending into and compressing the zona represented equally between amyloid-positive and -negative ani- reticularis (Figure 1E). Mild to moderate cases displayed multifo- mals, occurring in 89% (24 of 27) of the kidneys examined. Mild cal to coalescing deposits, whereas deposits in severe cases were to moderate glomerular disease (mesangioproliferative glomeru- 122 Detection of amyloidosis in pig-tailed macaques

Table 2. Histologic grading of amyloida Discussion Organ 0 1+ 2+ 3+ 4+ Amyloidosis is a disorder of protein metabolism; increased Liver 21 0153 production and reduced destruction of precursor proteins leads 47 GI: stomach 24 1221 to accumulation. Among macaques, amyloid deposition varies GI: small intestine 23 0025 greatly. Rhesus monkeys (M. mulatta) display generalized and 6,10,33 GI: large intestine 24 3111 cerebral amyloidosis; pig-tailed macaques (M. nemestrina) display GI and hepatic amyloidosis;16,40,43 crab-eating macaques Spleen 19 0033 (M. fascicularis) display pancreatic, vascular, and cerebral amyloi- Adrenals 19 1332 dosis;34,42 stump-tailed macaques (M. arctoides) display GI amy- Kidney 21 2320 loidosis;39 and Celebes (or crested black, M. nigra) and Formosan a Percentage of the parenchyma replaced by amyloid in the most affected section ex- rock (M. cyclopis) macaques both display pancreatic amyloido- amined: 0, 0%; 1+, less than 40%; 2+, 40% to 80%; 3+, 80% to 90%; 4+, 90% to 100%. sis.10,12 Amyloidosis in NHP has been associated with rheumatoid arthritis,9,10,17 viral infection (SRV-2), parasitism,2,5 chronic losclerosis) was present equally between animals with (86%, 6 of catheterization,14 and enterocolitis.6,10,12,20,36,39,40,43,48 Amyloidosis 7) and without (86%, 12 of 14) renal amyloid. secondary to enterocolitis, historically attributed to Shigella sp., Age. When evaluated according to amyloid status, age was has been described in multiple macaque species (M. radiata, M. found to be a signifi cant (P < 0.05) indicator of amyloid for all nemestrina, M. fascicularis, and M. mulatta).20,25 forms of amyloid deposition (systemic, hepatic, and GI). Statistics The cohort of animals selected for analysis was a representative (P values, range, and mean age) for animals with and without sample of M. nemestrina that were removed from the WaNPRC amyloid deposition are summarized in Tables 3, 4, and 5. The colony for various reasons (Table 1). Although animals were not overall mean age of the cohort of 30 animals was 11.1 y. euthanized due to amyloidosis, animals often had 1 or more clini- Imaging. Hepatic radiology. Abdominal radiology demonstrated cal conditions (renal failure, viral status, parasitism, and diarrhea) significant (P < 0.05) differences in sternal extension and C/B that have been correlated with amyloid deposition. The incidence length between animals with hepatic deposition and those with- of amyloidosis was 47% (14 of 30) among the animals surveyed. out. Lumbar extension was not signifi cant (Table 6). Amyloid deposition occurred in the liver, GI tract, and spleen, Ultrasonography. Ultrasound examination was nondiagnostic either alone or in combination, in 79% of amyloid-positive ani- for hepatic, adrenal, splenic, and renal amyloidosis. mals. The remaining 21% of amyloid-positive animals had single Endoscopy. Endoscopic evaluation did not demonstrate visual organ deposition in the kidney, intestine, or adrenal gland. In a GI tract lesions (hyperemia, ulceration, and so forth). Tissues previous survey of M. nemestrina at the WaNPRC, amyloid depo- from amyloid-positive animals were subjectively more friable sition occurred in the liver, GI tract, and spleen, either alone or in and prone to mucosal separation (biopsy-induced trauma) with combination, in 75% (186 of 248) of amyloid-positive animals.43 endoscopic manipulation. Among amyloid-positive M. mulatta, a lower incidence of hepatic Gastrointestinal biopsy. The presence of amyloid within biopsy amyloid (36.8%6 and 40%36) and a higher incidence of intestinal tissues directly correlated with fi ndings from necropsy tissues in amyloid (100%)6,36 has been described. 27% (3 of 11) of the animals. Independently, gastric biopsy was Consistent with previous studies,12,36,43,48 the incidence of amy- 50% sensitive and 71% specifi c, with a 33% positive predictive loid deposition increased with age among the animals surveyed. value and an 83% negative predictive value, whereas colonic bi- This correlation may be related to increased exposure to parasites, opsy was 50% sensitive and 57% specific, with a 25% positive infectious agents, or injury over time, thereby predisposing older predictive value and an 80% negative predictive value. animals to reactive amyloidosis. In this survey, amyloid status Viral status. Of the 30 experimental animals, 10 were PCR- varied considerably among animals of the same age, housed in positive for SRV-2; 2 of the 10 SRV-2-positive animals were amy- similar environments. The effect of age on amyloid deposition loid-positive, and the remaining 8 were amyloid-negative. Both was shown to be independent of parasites and gastritis, suggest- SRV-2-positive, amyloid-positive animals had single-organ (ad- ing that genetics may also play a role in amyloid status. renal) amyloid deposits; 1 also was positive for STLV. This survey did not demonstrate an association between sys- Serology. Signifi cant (P < 0.05) differences in GGT, total choles- temic or GI amyloidosis and parasitism, as has been previously terol, albumin, total protein, ALP, and SAA were noted in amy- described.2,5 Previous studies17,43 describe an association between loidotic animals; the mean serum concentration of each analyte amyloidosis and viral infection, specifi cally retroviral infection, was signifi cantly (P < 0.05) elevated (GGT, TC, ALP, and SAA) or that was not demonstrated in this survey. Approximately 30% of decreased (albumin and total protein) compared with that from the animals examined were positive for SRV-2 by PCR. Of these animals without amyloid deposition. Serologic data are summa- animals, 20% had adrenal amyloidosis, whereas the majority rized in Tables 3, 4, and 5. Mean serum concentrations of glucose, (80%) were free of amyloid deposits. No SRV-2-positive animals bilirubin, ALT, ESR, BUN, AFP, CRP, and AST did not differ sig- had systemic, hepatic, or GI amyloidosis. nifi cantly between amyloid-positive and -negative animals. Ultrasonography was not diagnostic for amyloidosis. It is un- Immunoassays. The Anogen immunoassay failed to demon- known whether the echogenicity of amyloid fi brils is similar to strate serum amyloid A in macaque blood, whereas the TriDelta that of hepatic parenchyma or whether equipment quality or op- assay demonstrated signifi cantly (P < 0.05) elevated SAA with all erator skill (or both) was insuffi cient to detect subtle differences. forms of amyloid deposition (data are summarized in Tables 3, 4, Radiology for the detection of GI amyloid was not performed and 5). AFP was not demonstrated in macaque blood by immu- because the radiologic manifestations of abdominal amyloidosis noassay. ET-1 was demonstrated in macaque blood by ELISA, but are diverse and nonspecifi c.27 Hepatic radiology was predictive concentrations did not differ signifi cantly between amyloid-posi- of amyloidosis; hepatomegally (measured by C/B length and tive and -negative animals. sternal extension) may be attributed to amyloid fi bril deposition. 123 Vol 56, No 2 Comparative Medicine April 2006

Table 3. Statistically signifi cant serologic fi ndings between amyloid-positive and -negative animals Amyloid status N Minimum Maximum Mean Standard deviation Pa GGT + 13 15 136 89 32 (U/dl)b – 16 49 145 65 24 0.006

Albumin + 13 1.5 3.5 2.7 0.7 (g/dl)c – 16 2.4 3.6 3.2 0.4 0.04

SAA + 9 0 105 40 36 (μg/ml) – 14 0 83 14 22 0.04

Bilirubin + 13 0.2 1.0 0.54 0.24 0.60 (mg/dl)c – 16 0.2 0.8 0.56 0.17

ET-1 + 5 0.01 0.1 0.08 0.01 0.11 (pg/ml)d – 9 0.01 20 6.4 7.9

Mean age + 14 3.1 20.5 13.2 4.9 (y) – 16 4.7 15.6 9.3 3.5 0.02 a2-tailed Wilcoxon signed rank test. bWaNPRC colony normal, 0 to 49 U/dl. cWaNPRC colony normal, 3.5 to 5.2 μg/ml. dWaNPRC colony normal, 0.1 to 1.0 mg/dl.

Table 4. Statistically signifi cant serologic fi ndings between hepatic amyloid-positive and hepatic amyloid-negative animals Hepatic amyloid status N Minimum Maximum Mean Standard deviation Pa + GGT 9 15 136 95 37 b (U/dl) – 20 49 145 67 22 0.008

+ Cholesterol 9 122 170 143 19 c (mg/dl) – 20 44 162 114 25 0.004

+ SAA 6 0 105 44 36 μ ( g/ml) – 1708317250.049

+ Bilirubin 9 0.3 1.0 0.59 0.26 d 0.76 (mg/dl) – 20 0.2 0.8 0.53 0.17

+ Mean age 9 3.1 19.6 13.5 5.0 (y) – 21 4.7 20.5 10.1 4.1 0.04 aTwo-tailed Wilcoxon signed rank test. bWaNPRC colony normal, 0 to 49 U/dl. cWaNPRC colony normal, < 200 mg/dl. dWaNPRC colony normal, 0.1 to 1.0 mg/dl.

In moderate to severe cases of hepatic amyloidosis, radiologi- confi rming previous reports.14,23 Gamma-glutamyl transpeptidase cally detectable hepatomegaly (sternal extension of the liver) was is an enzyme associated with the microvilli of hepatocytes, biliary detectable by palpation. In 2 animals, abdominal radiographs epithelial cells, and renal tubular epithelium. Elevation in serum ruled out amyloidosis as the etiology of palpable hepatic sternal GGT is a sensitive indicator of hepatobiliary dysfunction.46 extension due other radiologically evident causes (hepatic dis- Animals with hepatic and GI amyloid demonstrated elevated placement). serum total cholesterol. Hypercholesterolemia may be related Our fi ndings suggest that endoscopic biopsy may be used to to hepatic or GI amyloid, as cholestasis and enteritis have both rule out GI amyloid (negative predictive value: 83% gastric, 80% been shown to elevate serum cholesterol.46 In addition, SAA is colonic). Gastric and colonic biopsies are moderately invasive transported in the blood by high-density lipoproteins. Elevations procedures that create excoriations and ulcerations. Because of in SAA are mirrored by elevations in high-density lipoprotein and associated risks of bowel perforation, internal bleeding, and infec- therefore total cholesterol.11 Ratios of high-density to low-density tion, a small (4 mm) biopsy tool was used for collection. The small lipoprotein were not considered in this survey. biopsy tool produced prominent edge crush artifact, compromis- Consistent with previous reports,10,14 ALP was elevated with ing structural morphology. Alternative methods of collection pro- GI amyloidosis. With the exception of 1 animal, the mean age ducing larger biopsy specimens or greater sample numbers may of hepatic amyloid-positive animals was 14.8 y (past the age for increase sensitivity but also risk of injury. pronounced bone growth), therefore the bone ALP isoenzyme Hepatic amyloid deposits accumulate within the sinusoidal did not markedly contribute to the total ALP concentration. The space of Disse, altering acinar morphology, compressing hepa- tissue most likely responsible for elevations in ALP is the liver; tocytes, and partially obstructing canaliculi. Therefore, a serum the intestinal isoenzyme represents a small fraction of total ALP.46 chemistry panel may be useful in diagnosing amyloid when eval- Intrahepatic cholestasis, due to the physical obstruction of bile uated in the context of functional cholestasis. This survey demon- (in this case, by amyloid fi brils), increases production of hepatic strated elevated GGT levels with all forms of amyloid deposition, ALP by hepatocytes and biliary epithelium.46 In this survey, 1 124 Detection of amyloidosis in pig-tailed macaques

Table 5. Statistically signifi cant serologic fi ndings between gastrointestinal amyloid-positive and gastrointestinal amyloid-negative animals

GI amyloid status N Minimum Maximum Mean Standard deviation Pa + GGT 7 75 127 101 21 0.003 (U/dl)b – 22 15 145 68 28 + Cholesterol 9 100 170 137 25 0.03 (mg/dl)c – 20 44 162 117 25 + Albumin 9 1.5 3.5 2.2 0.8 0.03 (g/dl)d – 20 2.4 3.6 3.1 0.4 + ALP 8 87 635 223 142 0.05 (U/l)e – 21 32 440 122 91 + Total protein 9 4.9 7.4 6.2 1.0 0.01 (g/dl)f – 20 5.4 7.9 7.1 0.6 + SAA 4 14 105 54 38 0.03 (μg/ml) –100831825 + Bilirubin 9 0.3 0.9 0.53 0.2 0.69 (mg/dl)g – 20 0.2 1.0 0.56 0.2 + Mean age 9 3.1 19.6 13.2 5 0.02 (y) – 21 4.7 20.5 10.2 4.2 aValues for cholesterol and albumin were obtained using 1-tailed Wilcoxon tests; all others were obtained using 2-tailed Wilcoxon tests. bWaNPRC colony normal, 0 to 49 U/dl. cWaNPRC colony normal, < 200 mg/dl. dWaNPRC colony normal, 3.5 to 5.2 g/dl. eWaNPRC colony normal, 36 to 122 U/l. fWaNPRC colony normal, 6.0 to 8.2 g/dl. gWaNPRC colony normal, 0.1 to 1.0 mg/dl. juvenile macaque (age, 3.1 y) was positive for amyloid and had The diagnostic value of hepatic biopsy was not evaluated in this an elevated ALP. Because ALP is physiologically elevated during study but has been shown to be of value in humans.53 periods of bone growth, the elevated ALP in this animal may be Elevations in AFP were not demonstrated in amyloidotic ma- related to bone growth instead of amyloid; therefore, this serum caque blood tested at the University of Washington Hospital Lab- value (1081 U/l) was excluded from ALP statistical analysis. oratory or in samples tested by ELISA. In nonpregnant women, Taken together, elevations in ALP, GGT, and cholesterol indicate serum AFP is used in diagnosis of hepatocellular carcinoma, cir- a cholestatic effect of hepatic amyloidosis; however, mean serum rhosis, and hepatocellular toxic injury.41,54,55 It was not possible to concentrations of total bilirubin were not signifi cantly elevated determine whether AFP is an appropriate test for hepatic amyloid in amyloidotic animals. Because amyloidosis reduces functional screening in macaques, because appropriate controls (samples hepatic mass, decreased bilirubin uptake, conjugation, and excre- from animals with neoplastic or cirrhotic livers) were not avail- tion may explain normal bilirubin levels in amyloidotic animals. able for the assays used. As such, suffi cient cross-reactivity with Biliary hyperplasia and bile pigment within Kupfer cells were not macaque AFP was not demonstrated for either assay. noted histologically. Similar to AFP ELISA, the Anogen SAA ELISA did not demon- Consistent with previous studies,10,14,39 amyloidotic animals strate serum elevations in amyloidotic macaques. Because appro- had low serum albumin. Hypoalbuminemia is related to GI loss priate controls were available, the lack of test sera reactivity may to a greater extent than to decreased hepatic production, because be attributed to insuffi cient assay cross-reactivity. The TriDelta animals with GI amyloid have lower albumin and total protein ELISA provided suffi cient cross-reactivity and sensitivity. Eleva- values than do animals with hepatic amyloid. It was not pos- tions in SAA were demonstrated in animals with infl ammatory sible to evaluate the potential for renal losses in all cases because conditions including infections (hepatic abscessation, peritonitis, kidneys were not uniformly available; however, renal amyloid encephalitis, chimeric simian–human immunodefi ciency virus, did not correlate with decreased total protein or albumin. A large or SRV-2), female reproductive conditions (parturition, abortion, number of aging pig-tailed macaques display renal disease8,19 and endometriosis), and neoplasia (pancreatic adenocarcinoma). unrelated to amyloid deposition. If such conditions can be ruled out through clinical evaluation, ALT, glucose, ESR, BUN, AFP, CRP, AST, and AST:ALT ratios an elevated SAA level may be an important indicator of amyloid were nondiagnostic for amyloidosis. These fi ndings contradict deposition. those of Hubbard and others23 who documented elevations in A close relationship and correlation between CRP and SAA has BUN and AST in amyloid positive animals. Such discrepancies are been established in man.30,35 Elevations in CRP have been dem- not surprising; serum analytes are nonspecifi c, indicating general onstrated in human cases of renal amyloidosis;30 this correlation organ dysfunction. Fluctuation in serum analytes may occur over was not demonstrated in macaques. The CRP assay used was a time and in individual animals, complicating interpretation. Ami- human assay that did not demonstrate suffi cient cross-reactivity notransferase (AST and ALT) values tend to fl uctuate over time; with amyloidotic or control (peritonitis, acute CNS infection, and as many as 60% of biopsy-confi rmed cases of hepatitis in humans abortion) macaque samples. A monkey (M. mulatta, M. iris, and are associated with occasional normal ALT values.15,38 Hepatic bi- M. fuscata)-specifi c assay to measure CRP has been developed26 opsy may be indicated in animals with fl uctuating analyte levels. and is reported to be more specifi c than human assays. Without

125 Vol 56, No 2 Comparative Medicine April 2006

Table 6. Summary of radiographic data Hepatic amyloidosis present Hepatic amyloidosis absent Lumbar extensiona Sternal extensionb C/B lengthc Lumbar extension Sternal extensiond C/B lengthe Minimum 0 0.6 4.2 0 0 2.1 Maximum 6.5 7.6 16 4 4.2 7.7 Mean 2.4 2.9 7.7 0.8 0.8 4.0 Standard deviation 2.2 2.4 1.5 1.4 1.4 0.4 N 7 7 7 16 16 17 aThe number of vertebrae to which the liver shadow extended on a ventrodorsal view. bExtension of the liver shadow below the sternum, as measured in intercostal spaces on a right lateral view. cThe length (cm) of the liver shadow from crux to base measured to the right of midline, perpendicular to the crux on a ventrodorsal view. dSignifi cantly different (P = 0.009, 2-tailed Wilcoxon test) versus hepatic amyloid-positive group. eSignifi cantly different (P = 0.004, 2-tailed Wilcoxon test) versus hepatic amyloid-positive group. access to a species-specifi c assay, it is not possible to conclude that References macaque CRP does not correlate with amyloidosis and SAA. 1. AVMA Panel on Euthanasia. 2001. 2000 Report of the AVMA Panel ET-1 is released upon deposition of cerebral amyloid.13 We hy- on Euthanasia. J Am Vet Med Assoc 218:669–696. pothesized that deposition of visceral amyloid deposits might 2. Bacciarini LN, Gottstein B, Pagan O, Rehmann P, Grone A. 2004. similarly increase ET-1 in macaques. Samples from macaques Hepatic alveolar echinococcosis in cynomolgus monkeys (Macaca with elevated ET-1 were not available for use as controls; how- fascicularis). Vet Pathol 41:229–234. 3. Baker HF, Ridley RM, Duchen LW, Crow TJ, Bruton CJ. 1994. In- ever, manufacturer controls and experimental macaque samples duction of beta (A4)-amyloid in primates by injection of Alzheimer’s demonstrated cross-reactivity with the human diagnostic ELISA. disease brain homogenate: comparison with transmission of spon- The lack of elevated ET-1 in amyloidotic animals may indicate giform encephalopathy. Mol Neurobiol 8:25–39. that amyloid-induced ET-1 is a central nervous system-specifi c 4. Banks KL, Bullock BC. 1967. Naturally occurring secondary amy- response. If so, it is likely that the animals examined were too loidosis of a squirrel monkey (Saimiri sciureus). J Am Vet Med Assoc young (overall mean age, 11.2 y) to develop cerebral plaques and 151:839–842. elevated ET-1. As with man, the incidence of cerebral plaques in 5. Benditt EP, Eriksen N. 1972. Chemical characteristics of the typical macaques increases with age. Uno and others48 have reported amyloidosis in monkeys. Acta Pathol 233:103–108. 6. Blanchard JL, Baskin GB, Watson EA. 1986. Generalized amyloidosis cerebral amyloid in 20% to 100% of animals ranging from 20 to in rhesus monkeys. Vet Pathol 23:425–430. 39 y of age. Animals younger than 20 y did not display cerebral 7. Bons N, Mestre N, Ritchie K, Petter A, Podlisny M, Selkoe D. plaques. Although cerebral tissue was not examined in the pres- 1994. Identifi cation of amyloid beta protein in the brain of the small, ent study, the mean animal age would indicate a low incidence of short-lived lemurian primate Microcebus murinus. Neurobiol Aging cerebral amyloidosis. 15:215–220. In conclusion, amyloidosis remains an antemortem diagnosis 8. Boyce JT, Giddens WE Jr, Seifert R. 1981. Spontaneous mesangio- by exclusion. Although the histologic presence of amyloid allows proliferative glomerulonephritis in pigtailed macaques (Macaca for a positive defi nitive diagnosis, the absence of amyloid does nemestrina). Vet Pathol 18(Suppl 6):82–88. 9. Brack M. 1997. Eikenella corrodens-caused botryomycosis-type pneu- not allow for a defi nitive negative diagnosis because of sample monia in a barbary ape (Macaca sylvanus). APMIS 105:457–462. size bias and nonuniform tissue distribution. Because antemortem 10. Casey HW, Kirk JH, Splitter GA. 1972. Generalized amyloidosis in diagnosis of amyloidosis would aid in interpreting clinical and re- a rhesus monkey. Lab Anim Sci 22:587–593. search abnormalities, a series of diagnostic tests were evaluated 11. Chait A, Han CY, Oram JF, Heinecke JW. 2005. Thematic review to increase the probability of a defi nitive diagnosis. Serology is series: the immune system and atherogenesis. Lipoprotein-associ- the most effi cient method of screening large numbers of animals. ated infl ammatory proteins: markers or mediators of cardiovascular Those with elevated GGT, ALP, and cholesterol, in combination disease? J Lipid Res 46:389–403. with normal bilirubin and decreased albumin and total protein, 12. Chapman WL Jr, Crowell WA. 1977. Amyloidosis in rhesus monkeys with rheumatoid arthritis and enterocolitis. J Am Vet Med Assoc should be evaluated further, because this combination may be 171:855–858. considered an amyloidotic profi le. Abdominal radiographs are 13. Deane R, Du Yan S, Submamaryan RK, LaRue B, Jovanovic S, Hogg diagnostic for hepatomegaly, which in the face of an amyloidotic E, Welch D, Manness L, Lin C, Yu J, Zhu H, Ghiso J, Frangione serologic profi le, may indicate amyloidosis. In this colony survey, B, Stern A, Schmidt AM, Armstrong DL, Arnold B, Liliensiek B, an elevated SAA level with an amyloidotic serologic profi le, nor- Nawroth P, Hofman F, Kindy M, Stern D, Zlokovic B. 2003. RAGE mal clinical exam, negative viral status, and radiographic hepato- mediates amyloid-beta peptide transport across the blood-brain megaly was diagnostic for amyloidosis. barrier and accumulation in brain. Nat Med 9:907–913. 14. Doepel FM, Ringlee DH, Petkus AR. 1984. Secondary amyloidosis in a rhesus macaque with chronic indwelling venous catheters. Lab Anim Sci 34:494–496. Acknowledgements 15. Dufour DR, Lott JA, Nolte FS, Gretch DR, Kofff RS, Seeff LB. 2000. This study was funded by National Institutes of Health grants RR07019- Diagnosis and monitoring of hepatic injury. II. Recommendations 23 “Biomedical Research Training for Veterinary Scientists” and RR00166 for use of laboratory tests in screening, diagnosis, and monitoring. “Washington National Primate Research Center.” The authors gratefully Clin Chem 46:2050–2068. acknowledge the assistance of the veterinary and research support staff 16. Ellsworth L, Farley S, DiGiacomo RF, Tsai CC. 1992. Factors as- of the WaNPRC. We thank the Department of Biostatistics (Center for sociated with intestinal amyloidosis in pigtailed macaques (Macaca Statistical Consulting at the University of Washington) for guidance in nemestrina). Lab Anim Sci 42:352–355. statistical analysis.

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