Neuronal and microglial involvement in beta-amyloid protein ...

AmericanJournal ofPathology, Vol. 137, No. 2, August 1990 Copyright ©) American Association ofPathologists

Rapid Communication Neuronal and Microglial Involvement in f3-Amyloid Protein Deposition in Alzheimer's Disease Patrick Cras,* Mitsuru Kawai,* Sandra Siedlak,* Paul Mulvihill,* Pierluigi Gambetti,* David Lowery,t Patty Gonzalez-DeWhitt,t Barry Greenberg,t and George Perry* From the Institute ofPathology, *Case Westernt Reserve

University, Cleveland, Ohio; and The Upjohnt Kalamnazoo, Michigan

CompanyJ

This study was undertaken to localize amyloidprecursor protein (APP) and to determine bow APP might be released and proteolyzed to yield the f3amyloidprotein depositsfound in senileplaques in the brains of Alzbeimer's disease patients. We found that antibodies to recombinantly expressed APP labeled many normal neurons and neurites. In addition, dystrophic neurites in different types of senile plaques and degenerating neurons in the temporal cortex and hippocampus of Alzbeimer's disease patients were immunostained. We also detected small clusters of dystrophic APP immunoreactive neurites that were not associated with fl-amyloid protein deposits. Microglia was involved in different types of senile plaques and often were associated closely with APP immunoreactive neurites and neurons. The greatest concurrence of APP immunoreactivity and reactive microglia was seen in the subiculum and area CA1, regions with a high density of congophilic plaques and subject to intense Alzbeimer's pathology. Our findings suggest that neuronally derived APP is the sourcefor senileplaque 3-amyloid protein, while microglia may act as processing cells. (Am JPathol 1990, 13 7:241-246)

Abundant extracellular ,B-amyloid protein (,B-AP) deposition in senile plaques (SP) and the presence of neurofibrillary tangles (NFT) are diagnostic criteria for Alzheimer's disease (AD).1-3 /3-amyloid protein is a 4.2-kd polypeptide, derived from a larger membrane-spanning molecule called amyloid precursor protein (APP), a glycoprotein that is produced by cells throughout the body.4 Three forms of APP have been described and are referred to as APP695, APP751, and APP770, reflecting the number of

amino acids (AA) coded for by their respective cDNAs.5 6 The secreted form of APP751/770 is protease nexin 112 and contains a 56-AA insert that has structural homology to the Kunitz type of protease inhibitors.47 In situ hybridization studies with cDNA probes to APP have shown that in the brain, neurons are the principal producers of APP mRNA.89 If and how APP is produced in neurons, other brain cells10 or is derived from a systemic source is related to the ,B-AP protein deposited in SP remains controversial. It is generally accepted that in systemic amyloidosis, macrophages play an important role in amyloid formation.11 Because sequences spanning the whole length of the APP molecule occur extracellularly12 as well as in neurites,13 phagocytic cells indeed could be involved in further APP processing and releasing of the ,-AP, which then would aggregate to form amyloid fibrils. Microglial cells show phagocytic activity and, together with dystrophic neurites, are frequently found in association with amyloid deposits in AD. 14,22 The present study examines the presence and distribution of APP, ,B-AP, and microglia in brains of subjects with AD and controls to define the site of origin and processing of the amyloid deposits in AD.

Material and Methods Tissue, Fixation, and Pretreatment Hippocampus and temporal cortex obtained after death from eight cases of AD16 (age range, 77 to 83 years; postmortem interval, 3 to 10 hours) and four patients without history of dementia (age range, 54 to 77 years; postmortem interval, 4 to 16 hours) was fixed with one of the following fixatives: 4% paraformaldehyde, Bouin's fixative, periodate-lysine-paraformaldehyde (PLP), and methacarn. In preliminary studies, we found that immunostaining for APP was optimal after PLP or methacarn fixation Supported by a Fogarty International Fellowship to Drs. Cras and Kawai and by NIH grants K04-AG00415 and AG-007552, AG-08155. Accepted for publication June 5, 1990. Address reprint requests to Dr. Patnck Cras, Institute of Pathology, Case Western Reserve University, 2085 Adelbert Rd., Cleveland, OH 44106.

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followed by paraffin embedding. We found that pretreatment with 70% formic acid for 5 minutes or 2% periodic acid17 for 16 hours before immunostaining enhanced /AP immunoreactivity on formaldehyde, Bouin's, and PLPfixed tissue. Immunostaining after methacarn fixation was optimal without treatment.

CA)'9 were used to localize, respectively, N-, C-terminal, and 3-AP regions of APP.12,18 3) The monoclonal antibody TALl B5 (Dako, Santa Barbara, CA), which reacts with the HLA-DR a-chain,20 was used as a marker for HLA-DR-expressing microglia. 4) Antibodies to ubiquitin (42D8, courtesy of Dr. V. Fried), and related epitopes (Alz-50, gift of Dr. P. Davies; 5E2, gift of Dr. K. Kosik) and phosphorylated neurofilament heavy chains (SMI-34, Sternberger-Meyer, Inc., r,

Antibodies Four sets of antibodies were used in this study: 1) Antibodies to recombinant APP rabbit antisera to the protein expressed in Escherichia coli (antiColi695) and to the modified APP695 and APP770 expressed in Sf9 insect cells (respectively, antiBac695 and antiBac770) were used in these studies. APP695 was expressed in E. coli and purified in fully denatured form from total cell extracts by preparative electroelution from SDS-polyacrylamide gels. Modified APPs were expressed in Sf9 cells as baculovirus recombinants: the valine codon, two positions C terminal to the 42 AA amyloid core protein, was replaced by a termination codon. This alteration functionally deletes the C-terminal 56 AA to direct the secretion of the expressed protein, which was then purified from serum-free conditioned media. Details of this work and evidence for the proper folding of the protein generated in insect cells will be reported (D Lowery, P Gonzalez-DeWhitt, R Altman, B Greenberg, manuscript in preparation). The specificity of the antibodies made to recombinant APP (antiBac695 and antiBac770) was validated by their recognition of APP protein bands at 105 and 125 kd on a immunoblot of APP purified from cerebrospinal fluid (a gift of Drs. M. Palmert and S. Younkin).18 On an immunoblot prepared from AD hippocampus, similar bands were stained, while in addition, we found lower-molecularweight bands whose relation to APP is unestablished. On immunodot blots, antiBac695 and antiBac770 showed no recognition of synthetic 3-AP (APP695 sequence 597-637). 2) Previously described antibodies to synthetic peptides corresponding to APP695 sequences 45-62, 638658, and 597-637 (Peninsula Laboratories, Belmont,

Jarretsville, MD).

Immunostaining Immunostaining was performed according to the unlabeled antibody bridge technique.21 Double immunostaining was done by localizing the first immunolabeling with diaminobenzidine (brown), followed by treatment with formic acid or periodic acid before application and visualization of the second antiserum with 4-chloro-naphtol (blue). Controls were done to exclude cross-reactivity between antibodies. Immunostaining with rabbit preimmune sera was completely negative. Specific staining was completely abolished after incubation of antiBac770 with 50 gg/ml of combinant APP770.

re-

Quantification To quantify the relationship between amyloid deposits and APP immunoreactive neurites and microglia, we double immunostained adjacent tissue sections. We divided the amyloid deposits into diffuse plaques on the one hand and core-containing classical plaques on the other hand.15 For each of three AD cases, we studied one to three sections of the whole hippocampal region with the 20X objective of a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, FRG) and counted the number of amyloid deposits that had APP immunoreactive neurites within their confines. The number of amyloid deposits having APP immunoreactive neurites is expressed as the absolute number and as a percentage of the specific type of senile plaque. We did a similar enumeration for the association of

Figure 1. A: Small zeuritic clusters (arrouheads) are labelled with antitiac 770, as uell as coarse cjtoplasmic granules in a neuron (arrow). B: Numerous core containinig plaques shou' immunolabelling of surrounding dystrophic neurites and intercellular amyloid wisps uith antiBac 770. C: Intensely APP immunoreactii'e neuron, showing satellitosis by glial cells. D: Small APP positive tieulritic cluster (brown) in a sectioni double immunostainedfor f3-AP after formic acid treatment. There is nto /3-AP deposition, which would appear blue. E: LargerAPPpositive neuritic cluster (brown), with interspersed f-AP deposition (blue). Neurons in the dentate gyrus show granuilar cytoplasmic APP immunloreactivity (upper part ofphotograph). F: Senile plaque u'ith fl-AP-immunoreactive core (blue) and surrounding APP immunoreactive neurites (brown). G: HLA-DR-immunoreactive microglia (brown) in diffiise amyloid deposits (blue) demonstrated with /3-AP immunostain. H: APP-immunoreactive neurites (brown) in a diffuse amyloid deposit (blue). I: HLA-DR-immunoreactive microglia (browun) associated with APP containing neurites (blue). Immunoperoxidase staini using diaminobenzidine (browni) or 4-chloro-naphthol (blue) substrate solution. A-C: Single immunostain for APP' (antiBac 770). D-F: Double immuniostaini: AP'P (anti-Bac 770) was demonistrated with diaminobenzidine and fi-AP (anti-597-63 7) with 4-chloro-naphthol. G: Double immuniostaini: HLA-DR-diaminobenzidine, f3-AP-4-chloro-niaphthol. H: Double immunostain: Al'P-diaminobenzidinie, f-AP-4-chloro-ntaphthol. 1: Double immuniostain: HLA-DR-diaminobenzidine, APP-4-chloro-ntaphthol. Sectionis shown in B andC were counterstained with hematoxylin. Magnifications, A: 25OX; B andG: 125X; C-F andH-1, 50OX.

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the fl-amyloid protein deposits and HLA-DR-positive microglia. Microglia was considered to be associated if their cell body or processes were confined within the area of amyloid deposit. We also determined the number of APP immunoreactive neurite clusters associated with HLA-DRpositive microglia. For APP immunoreactive neurites, microglia were considered to be associated if their cell processes were intermingled with the neurites and/or their cell bodies were closely adjacent to the cluster.

Results Immunocytochemistry Antibodies directed to all forms of recombinant APP gave essentially similar results. In AD brain, these antibodies identified dystrophic neurites and neurons (Figures 1 A and B). Some neurofibrillary tangles and neurophil threads, as well as normal neurites, were immunostained (not shown). In the subiculum and area CAl of the hippocampus, we found many small clusters of intensely APP immunoreactive neurites that were apparently unrelated to core containing SP (Figures 1 A and D). As revealed by double staining for ,B-AP, some of these clusters were associated with scant amyloid deposits (Figure 1 E). There were also curly and thickened APP immunoreactive neurites in diffuse amyloid deposits (Figure 1 H). In addition to immunolabeled dystrophic neurites (Figure 1 F), small extracellular amyloid wisps in corecontaining senile plaques (Figure 1 B) were stained. Immunolabeling with antisera directed to C- and N-terminal domains of the APP molecule also resulted in predominantly neuritic staining (Figures 2A and B), along with some diffuse amyloid

deposits (not shown). Granular cytoplasmic APP immunoreactivity was noted in neurons (Figure 1 E). Although the identity of most of the granules was not established, the granules of granulovacuolar degeneration also were labeled. In perikarya containing neurofibrillary tangles, the inclusion was rarely stained but the adjoining cytoplasm often contained APPimmunoreactive granules. Pyramidal neurons, mostly localized in layers IlIl and V of the entorhinal cortex and the subiculum, showed especially intense immunostaining for APP (Figures 1 A and C). The nuclei of these neurons often were pyknotic and peripherally displaced, and satellitosis by glial cells (Figure 1C), some of which were HLA-DR positive, was often present. On rare occasions, HLA-DR-positive cells were involved in neuronophagia. Control brains showed weak, although definite, staining of normal neurons and neurites with antiBac770. The hippocampus of a 77-year-old mentally intact patient had ADtype changes and showed the same APP staining as described for the AD cases. HLA-DR-positive microglia were found throughout the cortex and white matter of AD brains. Microglial cells were often intertwined between dystrophic neurites and broad

Figure 2. A: Neurites (arrowheads) in senile plaque stained with an anitiserum to N-terminal domain ofAPP molecule. B: Comparable regiont stained with an antiserum to the C-terminal domain of the APP molecule. Both sectionis shown in A and B were counterstainied with hematoxylin. Magnifications, SOOX.

processes infiltrated the amyloid core of the SP. In many diffuse-type plaques, microglia were surrounded by #-AP deposit (Figure 1G) (compare with Itagaki et a122). Many

dystrophic neurites, whether part of different types of SP or occurring in small isolated clusters, were associated with HLA-DR-positive cells (Figure 11). When the same senile plaque was examined in a serial section, virtually all the APP immunoreactive neurites showed ubiquitin immunoreactivity. Most APP-positive neurites in the AD cases also showed T immunoreactivity, but there were many more neurites demonstrated with the r stain. Similar results to the T antibody were obtained with the antibody to

phosphorylated neurofilament heavy chain.

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Quantification Almost one half of diffuse type plaques had APP immunoreactive neurites within their confines (Figure 1 H; Table 1), some of which were thickened and curly. Some of these neurites showed positivity for markers of cytoskeletal alterations (not shown). Essentially all of the amyloid corecontaining plaques showed intensely APP immunoreactive nondystrophic and dystrophic neurites. More than one half of the diffuse plaques contained HLA-DR immunoreactive microglia, with a morphology consistent with that of the resting state (Table 2). Essentially all amyloid core-containing plaques presented HLADR-positive cells with broad cytoplasm and processes. Two hundred twenty-five (55%, 2 AD cases combined) of 409 APP immunoreactive neuritic clusters were associated with microglial cells.

Discussion In a previous study we found that sequences from all APP domains are represented in the SP and probably define its locus12 (see also Ishii et a123). Therefore the key feature of SP localization has shifted from solely the site of amyloid deposition to the factors responsible for APP accumulation. Studies of in situ hybridization using APP cDNA probes have demonstrated that neurons contain the largest amount of APP mRNA. APP mRNA is also present, however in astrocytes, endothelial cells, and microglia although, apparently at a lower level.6 Furthermore, because APP is expressed in virtually every tissue,24 it might enter the brain from the blood through breaches in the blood brain barrier. We observed that relatively large amounts of APP are present in neuronal perikarya and their processes and that many APP-immunostained neurites are dystrophic and associated with different amounts of j-AP deposits. Table 1. Associationi ofAmnyloid Precuirsor Protein Immunioreactive Neturites with Sentile Plaques

Case 1 Case 2 Case3 Total

Core-containing plaques

Diffuse plaques APP + APP 52 31 193 283 317 180 425 631

APP0 1 7 8

APP + 14 61 108 183

(40%)*

(4%)t

(96%)t

(60%)*

The presence of APP immunoreactive neurites was evaluated by studying sections double stained for ,3-AP and APP. Diffuse and corecontaining senile plaques were counted in the hippocampus of three AD cases; results are expressed as number of diffuse or amyloid core plaques with or without APP immunoreactive neurites. The difference in proportion between the two types of plaques is significant at P = 0.0001. * Expressed as percentage of diffuse plaques. t Expressed as percentage of core-containing plaques.

Table 2. Associationi of HLA-DR Expressinig Microglia with Senile IPlaques Core-containing plaque Diffuse plaque HLA-DR - HLA-DR + HLA-DR - HLA-DR + 2 58 153 187 Case 1 44 0 166 Case 2 176 87 1 55 124 Case 3 3 157 416 477 Total

(47%)*

(53%)*

(2%)t

(98%)t

The presence of HLA-DR-positive cells was evaluated by studying sections double stained for T-AP and HLA-DR. Diffuse and core-containing senile plaques were counted in the hippocampus of three AD cases; results are expressed as number of diffuse or amyloid core plaques with or without HLA-DR-positive microglia. The difference in proportion between the two types of plaques is significant at P = 0.0001. * Expressed as percentage of diffuse plaques. t Expressed as percentage of core-containing plaques.

Small neuritic clusters were found in the absence of any f-AP, suggesting that these may represent early stages of SP. Some of the APP immunoreactive dystrophic neurites occurring in these small clusters appeared to contain straight or paired helical filaments (data not shown). It may be postulated that neurites becoming dystrophic accumulate relatively large amounts of APP that is improperly processed and results in extracellular accumulation of 3AP. Because many neurons containing abundant APP show signs of degeneration, it is possible that they also contribute to the formation of f-AP deposits.25 In agreement with the neuronal origin of the extracellular APP, we found no evidence for the presence of large amounts of APP in astrocytes26 or microglia. Although the absence of APP immunoreactivity in these cells could be due to rapid secretion, it is unlikely that they play a role in the production of APP generating the amyloid in SP. Nevertheless the possibility that these cells play a role in the processing of APP is conceivable. This is especially attractive for microglia because of their role as macrophages. ' 1 Another possible source of f-AP deposits in the cerebral parenchyma, recently emphasized, is the vasculature.27 This appears unlikely, however, in view of our finding that SPs bear no topographic relationship with cerebral microvessels.2e Nevertheless it is possible that vascular amyloid deposits are derived from locally available APP.Y In conclusion, our findings suggest that the extracellular APP12 present in the parenchyma of AD brains originates from degenerating neurons and their processes, while microglia might play a role in f-AP formation and deposition as APP-processing cells.

References 1. Bondareff W, Mountjoy CQ, Roth M, Hauser DL: Neurofibrillary degeneration and neuronal loss in Alzheimer's disease. Neurobiol. Aging 1989, 10:709-715

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2. Oltersdorf T, Fritz LC, Schenk DB, Lieberburg I, JohnsonWood KL, Beattie EC, Ward PJ, Blacher RW, Dovey HF, Sinha S: The secreted form of the Alzheimer's amyloid precursor with the Kunitz domain is protease nexin-Il. Nature 1989, 341:144-147 3. Wisniewski HM, Wegiel J, Wang KC, Kujawa M, Lach B: Ultrastructural studies of the cells forming amyloid fibers in classical plaques. Can J Neurol Sci 1989, 16:535-542 4. Tanzi RE, Gusella JF, Watkins PC, Bruns, GAP, St GeorgeHyslop P, Van Keuren ML, Patterson D, Pagan S, Kurnit DM, Neve RL: Amyloid ,B protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science 1987 235:880-884 5. Kitaguchi N, Takahasi Y, Tokushima Y, Shiojiri S, Ito H: Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity. Nature 1988, 331:530-532 6. Schmechel DE, Goldgaber D, Burkhart DS, Gilbert JR, Gajdusek DC, Roses AD: Cellular localization of messenger RNA encoding amyloid-beta-protein in normal tissue and in Alzheimer disease. Alzheimer Dis Assoc Disord 1988, 2:96-111 7. Ponte P, Gonzalez-DeWhitt P, Schilling J, Miller J, Hsu D, Greenberg B, Davis K, Wallace W, Lieberburg I, Fuller F, Cordell B: A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature 1988, 331: 525-527 8. Bahmanyar S, Higgins GA, Goldgaber D, Lewis DA, Morrison JH, Wilson MC, Shankar SK, Gajdusek DC: Localization of amyloid f-protein messenger RNA in brains from patients with Alzheimer's disease. Science 1987, 237:77-80 9. Card JP, Meade RP, Davis LG: Immunocytochemical localization of the precursor protein for beta-amyloid in the rat central nervous system. Neuron 1988,1:835-846 10. Stern RA, Otvos L, Trojanowski JQ, Lee VM-Y: Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alzheimer's disease fl-protein recognize amyloid and diverse glial and neuronal cell types in the central nervous system. Am J Pathol 1989,134:973-978 11. Zucker-Franklin D, Fuks A: The role of the macrophage phagocytic system (MPS) in the development of secondary amyloidosis. In GG Glenner EF Osserman, EP Bendit, AS Cohen, D Zucker-Franklin, eds. Amyloidosis. New York, Plenum Press, 1986, pp 329-340 12. Perry G, Lipphardt S, Mulvihill P, Kancherla M, Mijares M, Gambetti P, Sharma S, Maggiora L, Cornette J, Lobl T, Greenberg B: Amyloid precursor protein in senile plaques of Alzheimer disease. Lancet 1988 Sept; ii:746 13. Koo EH, Sisodia SS, Archer DR, Martin LJ, Weidemann A, Beyreuther K, Fischer P, Masters CL, Price DL: Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport. Proc Natl Acad Sci USA 1990 87: 1561-1565 14. Cras P, Gheuens J, Lubke U, Boons J, Vandermeeren M, Heuverswijn HV, Martin J-J: A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells. J. Histochem Cytochem (In press) 15. Yamaguchi H, Hirai S, Morimatsu H, Shoji M, Ihara Y: A variety of cerebral amyloid deposits in the brain of Alzheimer type dementia demonstrated by beta-protein immunostaining. Acta Neuropathol 1988, 76:541-549

16. Khachaturian ZS: Diagnosis of Alzheimer's disease. Arch Neurol 1985 42:1097-1105. 17. Behrouz N, Defossez A, Delacourte A, Hublau P, Mazzucca M: Alzheimer's disease: Glycolytic pretreatment dramatically enhances immunolabelling of senile plaques and cerebrovascular amyloid substance. Lab Invest 1989, 61:579-583 18. Palmert MR, Siedlak S, Podlisny MB, Greenberg B, Shelton ER, Chan HW, Usiak M, Selkoe DJ, Perry G, Younkin SG: Soluble derivatives of the f amyloid protein precursor of Alzheimer's disease are labeled by antisera to the 3 amyloid protein. Biochem Biophys Res Commun 1989,165:182-188 19. Kang J, Lemaire H-G, Unterbeck A, Salbaum JM, Masters CL, Grzeschik K-H, Multhaup G, Beyreuther K, Muller-Hill B: The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 1987, 325:733-736 20. Adams TE, Bodmer JG, Bodmer WF: Production and characterization of monoclonal antibodies recognizing the achain of subunits of human la alloantigens. Immunology

1983, 50:613-624 21. Sternberger LA: Immunocytochemistry, 3rd edition. New York, Wiley, 1986 22. Itagaki S, McGeer PL, Akiyama H, Zhu S, Selkoe D: Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J Neuroimmunol 1989, 24:173-182 23. Ishii T, Kametani F, Haga S, Sato M: The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease. Neuropathol Appl Neurobiol 1989, 15:135-147 24. Selkoe DJ, Podlisny MB, Joachim CL, Vickers EA, Lee G, Fritz LC, Oltersdorf T: ,B-amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membraneassociated proteins in neural and nonneural tissues. Proc Natl Acad Sci USA 1988, 85:7341-7345 25. Allsop D, Haga S-I, Haga C, Ikeda S-I, Mann DMA, Ishii T: Early senile plaques in Down's syndrome brains show a close relationship with cell bodies of neurons. Neuropathol Appl Neurobiol 1989, 15:531-542 26. Siman R, Card JP, Nelson RB, Davis LG: Expression of betaamyloid precursor protein in reactive astrocytes following neuronal damage. Neuron 1989, 3:275-285 27. Selkoe DJ: Molecular pathology of amyloidogenic proteins and the role of vascular amyloidosis in Alzheimer's disease. Neurobiol Aging 1988,10:387-395 28. Kawai M, Harik Si, Kalaria RN, Perry G: Spatial relationship of amyloid plaques and capillaries in Alzheimer disease. Soc Neurosci Abst 1989,15:647 29. Kawai M, Gras P, Siedlak S, Lowery D, Gonzalez-DeWhitt P, Greenberg B, Skelton E, Gambetti P, Perry G: Role of vascular smooth muscle cells in amyloid deposition in cerebral amyloid angiopathy. J Neuropathol Exp Neurol 1990, 49:339

Acknowledgments The assistance of Rich Altman in generating recombinant expression constructs, of Che-Shen Tomich in expression work in E. Coli and of Satish Sharma, Jane Carter, and Jim Cornette in preparation of antisera to the recombinant proteins is gratefully

acknowledged.