Failure to Detect the Presence of Prions in the Uterine and Gestational Tissues from a Gravida with Creutzfeldt-Jakob Disease
2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.081045
ISSN1525-2191
AutoresXiangzhu Xiao, Leticia Miravalle, Jue Yuan, J.E. McGeehan, Zhiqian Dong, Robert E. Wyza, Gregory T. MacLennan, Alan M. Golichowski, G.G. Kneale, Nicholas B. King, Qingzhong Kong, Salvatore Spina, Rubén Vidal, Bernardino Ghetti, Karen Roos, Pierluigi Gambetti, Wen‐Quan Zou,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoThe vertical transmission of a prion disease from infected mothers to their offspring is believed to be one of the routes for the natural spread of animal prion diseases. Supporting this notion is the observation that prion infectivity occurs in the placenta of infected ewes. Furthermore, the prion protein (PrP), both in its cellular form (PrPC) and its pathological isoform (PrPSc), has been observed at the fetal-maternal interface of scrapie-infected sheep. However, whether these features of prion infectivity also hold true for human prion diseases is currently unknown. To begin to address such an important question, we examined PrP in the uterus as well as gestational tissues, including the placenta and amniotic fluid, in a pregnant woman with sporadic Creutzfeldt-Jakob disease (CJD). Although the proteinase K (PK)-resistant prion protein, PrP27-30, was present in the brain tissues of the mother, the PrP detected in the uterus, placenta, and amniotic fluid was sensitive to PK digestion. Unlike PrPC in the brain and adjacent cerebrospinal fluid, the predominant PrP species in the reproductive and gestational tissues were N-terminally truncated, similar to urine PrP. Our study did not detect abnormal PrP in the reproductive and gestational tissues in this case of CJD. Nevertheless, examination by a highly sensitive bioassay is ongoing to ascertain possible prion infectivity from CJD in the amniotic fluid. The vertical transmission of a prion disease from infected mothers to their offspring is believed to be one of the routes for the natural spread of animal prion diseases. Supporting this notion is the observation that prion infectivity occurs in the placenta of infected ewes. Furthermore, the prion protein (PrP), both in its cellular form (PrPC) and its pathological isoform (PrPSc), has been observed at the fetal-maternal interface of scrapie-infected sheep. However, whether these features of prion infectivity also hold true for human prion diseases is currently unknown. To begin to address such an important question, we examined PrP in the uterus as well as gestational tissues, including the placenta and amniotic fluid, in a pregnant woman with sporadic Creutzfeldt-Jakob disease (CJD). Although the proteinase K (PK)-resistant prion protein, PrP27-30, was present in the brain tissues of the mother, the PrP detected in the uterus, placenta, and amniotic fluid was sensitive to PK digestion. Unlike PrPC in the brain and adjacent cerebrospinal fluid, the predominant PrP species in the reproductive and gestational tissues were N-terminally truncated, similar to urine PrP. Our study did not detect abnormal PrP in the reproductive and gestational tissues in this case of CJD. Nevertheless, examination by a highly sensitive bioassay is ongoing to ascertain possible prion infectivity from CJD in the amniotic fluid. The transmissible prion diseases affecting both humans and animals are characterized by the accumulation of an infectious prion protein particle (PrPSc) mainly in the central nervous system (CNS) and occasionally in the peripheral tissues.1Prusiner SB Prions.Proc Natl Acad Sci USA. 1998; 95: 13363-13383Crossref PubMed Scopus (5161) Google Scholar, 2Prusiner SB Williams E Laplanche JL Shinagawa M Scrapie, chronic wasting disease, and transmissible mink encephalopathy.in: Prusiner SB Prion Biology and Diseases. Woodbury, New York2004: 545-594Google Scholar, 3Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 4Glatzel M Abela E Maissen M Aguzzi A Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease.N Engl J Med. 2003; 349: 1812-1820Crossref PubMed Scopus (293) Google Scholar Animal prion diseases such as scrapie in sheep and goats, chronic wasting disease in deer and elks, and bovine spongiform encephalopathy in cattle are believed to spread naturally by oral transmission, close contact between animals, and maternal transmission. Indeed, Western blot analysis and bioassays have demonstrated that PrPSc and prion infectivity are present not only in the CNS, but also in many peripheral tissues including the tonsils, spleen, lymph nodes, nasal mucosa, distal colon, ovaries, uterus, skeletal muscle, placenta, and amniotic fluid of affected animals.2Prusiner SB Williams E Laplanche JL Shinagawa M Scrapie, chronic wasting disease, and transmissible mink encephalopathy.in: Prusiner SB Prion Biology and Diseases. Woodbury, New York2004: 545-594Google Scholar In humans, the transmission of prion diseases has been observed in the acquired form of the disease including kuru, iatrogenic Creutzfeldt-Jakob disease (iCJD), and variant CJD (vCJD).5Will RG Acquired prion disease: iatrogenic CJD, variant CJD, kuru.Br Med Bull. 2003; 66: 255-265Crossref PubMed Scopus (196) Google Scholar Kuru is associated with cannibalistic rituals,6Gajdusek DC Zigas V Degenerative disease of the central nervous system in New Guinea: the endemic occurrence of "kuru" in the native population.N Engl J Med. 1957; 257: 974-978Crossref PubMed Scopus (381) Google Scholar, 7Alpers MP Gajdusek DC Changing patterns of kuru: epidemiological changes in the period of increasing contact of the Fore people with western civilization.Am J Trop Med Hyg. 1965; 14: 852-879PubMed Google Scholar whereas iCJD is caused by prion exposure in the course of medical or surgical procedures, and vCJD has been attributed to the consumption of prion-contaminated meat.8Brown P Brandel JP Preese M Sato T Iatrogenic Creutzfeldt–Jakob disease: the waning of an era.Neurology. 2006; 67: 389-393Crossref PubMed Scopus (175) Google Scholar, 9Will RG Ironside JW Zeidler M Cousens SN Estibeiro K Alperovitch A Poser S Pocchiari M Hofman A Smith PG A new variant of Creutzfeldt-Jakob disease in the UK.Lancet. 1996; 347: 921-925Abstract PubMed Scopus (1835) Google Scholar In addition to CNS, PrPSc has also been detected in the tonsils, spleen, lymph node, retina, optic nerve, rectum, adrenal gland, and thymus of vCJD and in the spleen and skeletal muscles of sporadic CJD (sCJD).3Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 4Glatzel M Abela E Maissen M Aguzzi A Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease.N Engl J Med. 2003; 349: 1812-1820Crossref PubMed Scopus (293) Google Scholar However, it remains unknown whether human prion diseases are vertically transmitted in pregnancy. For instance, none of four offspring born to four gravid women with CJD had reportedly developed the disease when they reached the respective ages of 22, 10, 7, and 3 years.10Berrebi A Cohen M Ayoubi JM [Creutzfeldt-Jakob disease and pregnancy.].J Gynecol Obstet Biol Reprod (Paris). 1997; 26: 755-759PubMed Google Scholar In addition, no reports are available concerning examination of PrP in the uterus and gestational tissues from prion-affected patients. We examined PrPSc distribution in CNS, uterus, and gestational tissues from a woman affected with prion disease, who had become pregnant and delivered a baby boy during the time she had the disease. Typical PK-resistant PrP core fragments and neuropathological changes, characteristic of sCJD (with PrPSc type 1 carrying a valine/valine polymorphism at codon 129 of PrP gene), were detected in the brain tissues obtained at either biopsy or autopsy. Although PrP was detectable in the uterus, placenta, and amniotic fluid, it was PK-sensitive. Moreover, neither conventional nor enrichment-based Western blot analysis revealed the presence of abnormal PrP species. In contrast to the PrP in the brain and cerebrospinal fluid (CSF), the PrP detected in the uterus and gestational tissues, including placenta and amniotic fluid, was N-terminally truncated, similar to that normally found in the urine. Although the presence of prion infectivity in tissue remains to be determined by highly sensitive bioassay, our current study suggests that abnormal PrP species, including both PK-resistant and PK-sensitive forms, are undetectable in the uterus and gestational tissues in sporadic CJD. Sodium phosphotungstic acid (NaPTA), proteinase K (PK), and phenylmethyl sulfonyl fluoride were purchased from Sigma Chemical Co. (St. Louis, MO). Peptide N-glycosidase F (PNGase F) was purchased from New England Biolabs (Beverly, MA) and used according to the manufacturer's protocol. Reagents for enhanced chemiluminescence (ECL Plus) were from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). Magnetic beads (Dynabeads M-280, tosyl-activated) were from Dynal Co. (Oslo, Norway). Anti-PrP antibodies, including rabbit anti-C-terminal antiserum immunoreactive to human PrP residues 220 to 231, mouse monoclonal antibody 3F4 against human PrP residues 109 to 112, mouse monoclonal antibody 1E4 against human PrP 97 to 108 (Cell Sciences, Canton, MA), and mouse monoclonal antibody 6H4 against human PrP 145 to 152 (Prionics AG, Zurich, Switzerland) were used.11Monari L Chen SG Brown P Parchi P Petersen RB Mikol J Gray F Cortelli P Montagna P Ghetti B Goldfarb LG Gajdusek DC Lugaresi E Gambetti P Autilio-Gambetti L Fatal familial insomnia and familial Creutzfeldt-Jakob disease: different prion proteins determined by a DNA polymorphism.Proc Natl Acad Sci USA. 1994; 91: 2839-2842Crossref PubMed Scopus (273) Google Scholar, 12Chen SG Teplow DB Parchi P Teller JK Gambetti P Autilio-Gambetti L Truncated forms of the human prion protein in normal brain and in prion diseases.J Biol Chem. 1995; 270: 19173-19180Crossref PubMed Scopus (453) Google Scholar, 13Kascsak RJ Rubenstein R Merz PA Tonna-DeMasi M Fersko R Carp RI Wisniewski HM Diringer H Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins.J Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar, 14Yuan J Xiao X McGeehan J Dong Z Cali I Fujioka H Kong Q Kneale G Gambetti P Zou WQ Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains.J Biol Chem. 2006; 281: 34848-34858Crossref PubMed Scopus (100) Google Scholar Consent to use autopsy and biopsy material for research purposes was obtained for all samples. The neuropathological examination of biopsy and autopsy specimens of the proband was performed at the Indiana Alzheimer Disease Center, Indianapolis, IN, and at the National Prion Disease Pathology Surveillance Center, Cleveland, OH (http://www.cjdsurveillance.com/). Biopsy brain tissues were immediately frozen in liquid nitrogen and stored at −80°C. The autopsy was performed within 20 hours of death. Clinical data and relevant hospital records were examined. Brains, CSF, and urine were from patients free of neurological disorders and PrP mutations indicated by neurohistology, immunohistochemistry, Western blotting, and genetic analysis at National Prion Disease Pathology Surveillance Center, Cleveland, OH. The normal uterine and placental tissues and amniotic fluid were provided by the Pathology Department Human Tissue Procurement Facility, Case Western Reserve University/University Hospital Case Medical Center, Cleveland, OH. The 10% (w/v) brain, uterus, and placenta homogenates were prepared in 9 vol of lysis buffer (10 mmol/L Tris, 150 mmol/L NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 5 mmol/L ethylenediaminetetraacetic acid, pH 7.4) with pestle on ice. When required, the tissue homogenates were centrifuged at 1000 × g for 10 minutes at 4°C to collect supernatants (S1). The recombinant g5p was isolated from Escherichia coli, transformed with an Ff gene 5-containing plasmid and purified using DNA cellulose affinity plus Sephadex G75 sizing columns as described.15Oliver AW Bogdarina I Schroeder E Taylor IA Kneale GG Preferential binding of fd gene 5 protein to tetraplex nucleic acid structures.J Mol Biol. 2000; 301: 575-584Crossref PubMed Scopus (43) Google Scholar The purity was >99% as determined by quantitation of Coomassie Blue-stained bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Genomic DNA was extracted from frozen brain tissue. The human prion protein gene (PRNP) coding region sequence and polymorphism at codon 129 were determined as previously described.16Goldfarb LG Petersen RB Tabaton M Brown P LeBlanc AC Montagna P Cortelli P Julien J Vital C Pendelbury WW Haltia M Willis PR Hauw JJ McKeever PE Monari L Schrank B Swergold GD Autilio-Gambetti L Gajdusek C Lugaresi E Gambetti P Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism.Science. 1992; 258: 806-808Crossref PubMed Scopus (602) Google Scholar, 17Parchi P Castellani R Capellari S Ghetti B Young K Chen SG Farlow M Dickson DW Sima AA Trojanowski JQ Petersen RB Gambetti P Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease.Ann Neurol. 1996; 39: 767-778Crossref PubMed Scopus (725) Google Scholar Histopathology and PrP immunohistochemistry were performed as described.18Pastore M Chin SS Bell KL Dong Z Yang Q Yang L Yuan J Chen SG Gambetti P Zou WQ Creutzfeldt-Jakob disease (CJD) with a mutation at codon 148 of prion protein gene: relationship with sporadic CJD.Am J Pathol. 2005; 167: 1729-1738Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar The sections were deparaffinized, rehydrated, and immersed in 98% formic acid for 1 hour at room temperature. Endogenous peroxidase was blocked by immersion in 8% H2O2 in methanol for 10 minutes. Sections were completely immersed in 1.5 mmol/L hydrochloric acid and microwaved for 10 minutes. After rinsing, they were incubated with 3F4 at 1:600, washed, and incubated with secondary antibody (goat anti-mouse, 1:50; Cappel, Durham, NC) followed by incubation with mouse PAP complex (1:250; Sternberger, Meyer Immunocytochemicals, Jarrettsville, MD). Diaminobenzidine tetrahydrochloride was used to visualize the immunoreactivity. The g5p molecule and 6H4 antibody (100 μg) was conjugated to 7 × 108 tosyl-activated magnetic beads in 1 ml of phosphate-buffered saline (PBS) at 37°C for 20 hours. The g5p- or 6H4 -conjugated beads were incubated with 0.1% bovine serum albumin in PBS to block nonspecific binding. The prepared g5p or 6H4 beads were stable for at least 3 months at 4°C. The specific capture of PrP by g5p or 6H4 was performed as described incubating S1 fractions from the total brain homogenates, CSF, or amniotic fluid and g5p- or 6H4-conjugated beads (10 μg mAb/6 × 107 beads) in 1 ml of binding buffer (3% Tween-20 and 3% Nonidet P-40 in PBS, pH 7.5).19Zou WQ Cashman NR Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form.J Biol Chem. 2002; 277: 43942-43947Crossref PubMed Scopus (80) Google Scholar, 20Zou WQ Zheng J Gray DM Gambetti P Chen SG Antibody to DNA detects scrapie but not normal prion protein.Proc Natl Acad Sci USA. 2004; 101: 1380-1385Crossref PubMed Scopus (96) Google Scholar After incubation with constant rotation for 3 hours at room temperature, the PrP-containing g5p or 6H4 beads were attracted to the sidewall of Eppendorf tubes by external magnetic force, allowing easy removal of all unbound molecules in the solution. After three washes in wash buffer (2% Tween-20 and 2% Nonidet P-40 in PBS, pH 7.5), the g5p and 6H4 beads were collected and heated at 95°C for 5 minutes in sodium dodecyl sulfate sample buffer (3% sodium dodecyl sulfate, 2 mmol/L ethylenediaminetetraacetic acid, 10% glycerol, 50 mmol/L Tris-HCl, pH 6.8). Precipitation of PrP aggregates by NaPTA was conducted as described with minor modification.3Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 14Yuan J Xiao X McGeehan J Dong Z Cali I Fujioka H Kong Q Kneale G Gambetti P Zou WQ Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains.J Biol Chem. 2006; 281: 34848-34858Crossref PubMed Scopus (100) Google Scholar Briefly, 10% (w/v) homogenates from brain, uterus, or placenta tissues were prepared in PBS lacking Ca2+ and Mg2+. The samples were centrifuged at 1000 × g for 10 minutes at 4°C. A 500-μl aliquot of supernatant was mixed with an equal volume of 4% (w/v) Sarcosyl prepared in PBS, pH 7.4, and incubated for 10 minutes at 37°C with constant agitation. Samples were adjusted to final concentrations of 50 U/ml Benzonase (Benzon nuclease; Merck & Co., Whitehouse Station, NJ) and 1 mmol/L MgCl2 and incubated for 30 minutes at 37°C with constant agitation. Subsequently, the samples were adjusted with 81.3 μl of a stock solution containing 4% (w/v) NaPTA and 170 mmol/L MgCl2 at a final concentration in the sample of 0.3% (w/v) NaPTA. Samples were incubated at 37°C for 30 minutes with constant agitation before centrifugation at 16,000 × g for 30 minutes. After careful isolation of the supernatant, the pellet was resuspended in 1× lysis buffer for Western blotting. Samples were resolved on 15% Tris-HCl Criterion precast gels (Bio-Rad, Hercules, CA) for one-dimensional gel electrophoresis. The proteins on the gels were transferred to Immobilon-P membrane (PVDF, Millipore, Bedford, MA) for 2 hours at 70 V. For probing the PrP molecule, the membranes were incubated for 2 hours at room temperature with either 3F4 (1:40,000), 6H4 (1:10,000), or anti-C-terminal antibody (1:4000) as primary antibody. After incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG or donkey anti-rabbit IgG at 1:3000, the PrP bands were visualized on Kodak film (Eastman-Kodak, Rochester, NY) by the ECL Plus (GE Healthcare, Fairfield, CT) as described by the manufacturer. The patient was a 41-year-old pregnant Caucasian woman who suffered from rapidly progressive behavioral regression and eventually failed to perform simple activities of daily living. Shortly after the onset of her symptoms she became pregnant. Concurrently, she had evidence of executive cognitive dysfunction followed by weight loss, insomnia, aggressive behavior, and inability to care for personal hygiene. The patient had not traveled outside of the United States nor had she had a blood transfusion, corneal transplant, or treatment with human growth hormone. There was no family history of dementia or ataxia. The history, clinical course, and MRI abnormalities were suggestive of CJD. As the disease progressed, the patient developed myoclonus and ataxia. The definitive diagnosis of sCJD was made by neuropathological, molecular genetic, and Western blot analyses performed at the Indiana Alzheimer Disease Center, Indianapolis, IN, and confirmed by the National Prion Disease Pathology Surveillance Center, Cleveland, OH. As the clinical neurological symptoms progressed rapidly to a state of severe dementia, a cesarean delivery was performed at 35 weeks of gestation, during which samples of the amniotic fluid, umbilical cord blood, placenta, a full thickness biopsy of the uterine wall, and a biopsy of the uterus in the location of the placental bed were collected for storage and research. Tissues from the uterus and placenta and ∼60 ml of the amniotic fluid were sent to the National Prion Disease Pathology Surveillance Center, Cleveland, OH, for PrP analyses (see below). After delivery of a vital baby weighing 2.61 kg and 45.5 cm in length, the patient was closely followed by her neurologists. Five months after the delivery, the patient was admitted to the hospital for dehydration. On examination, she was obtunded and had frequent myoclonic jerks. The patient died at age 42 years and 1 month, 6 months after the birth of her child. There were severe spongiform degeneration, astrogliosis, occasional ballooned neurons, and possible neuronal loss throughout the neocortex (Figure 1A). The hippocampal formation and the basal ganglia were also severely affected. The cerebellum showed minimal spongiform degeneration and no atrophy, and kuru plaques, while the brain stem was normal (Figure 1A). Immunostaining for PrP was weak and consistently showed a fine granular or synaptic pattern throughout the cerebral cortex (Figure 1B). The neuropathological changes observed in the brain biopsy samples were similar to those observed in the autopsy samples. To determine whether there was a mutation in the PRNP, DNA was extracted from brain tissues and assessed by genetic analysis. The polymorphism at codon 129 of PRNP was of valine/valine and no mutations were observed in the coding region of the PRNP in this patient. A piece of brain tissue (∼77 mg) obtained at biopsy was homogenized in lysis buffer. The samples treated with PK at 50 μg/ml were subjected to Western blot analysis with 3F4 antibody. Proteinase K (PK)-resistant PrP 27-30 (rPrPSc) was detected; the unglycosylated rPrPSc fragment migrated at ∼21 kDa, identifying the rPrPSc as type 1 (Figure 2A). The brain samples received at autopsy were also examined and the rPrPSc was confirmed to have a migration pattern similar to the rPrPSc detected in the biopsy tissue (Figure 2B). Western blot analysis showed that although PrPC was easily detectable in both amniotic fluid and uterus, rPrPSc was not observed in the CJD and non-CJD control samples using an antibody termed anti-C, raised against the carboxyl terminus of human PrP 220-231 (Figure 3A). Only faint PrP bands from both CJD and non-CJD placenta samples were observed in overexposed films and they were all PK-sensitive (Figure 3A). To determine whether there is a small amount of PrPSc in these tissues, a large amount of samples (6 ml of amniotic fluid) was subjected to a gene 5 protein (g5p)-based enrichment of PrPSc before Western blot analysis. G5p is capable of capturing both rPrPSc and PK-sensitive PrPSc (sPrPSc) species.14Yuan J Xiao X McGeehan J Dong Z Cali I Fujioka H Kong Q Kneale G Gambetti P Zou WQ Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains.J Biol Chem. 2006; 281: 34848-34858Crossref PubMed Scopus (100) Google Scholar, 20Zou WQ Zheng J Gray DM Gambetti P Chen SG Antibody to DNA detects scrapie but not normal prion protein.Proc Natl Acad Sci USA. 2004; 101: 1380-1385Crossref PubMed Scopus (96) Google Scholar, 21Gambetti P Dong Z Yuan J Xiao X Zheng M Alshekhlee A Castellani R Cohen M Barria MA Gonzalez-Romero D Belay ED Schonberger LB Marder K Harris C Burke JR Montine T Wisniewski T Dickson DW Soto C Hulette CM Mastrianni JA Kong Q Zou WQ A novel human disease with abnormal prion protein sensitive to protease.Ann Neurol. 2008; 63: 697-708Crossref PubMed Scopus (227) Google Scholar Although the g5p beads precipitated the abnormal PrPSc from 5 μl of CJD brain homogenate, PrP was not detectable in samples captured by g5p from 6 ml of amniotic fluid and 6 ml of uterine and placental homogenates each in both CJD and non-CJD controls (Figure 3B and data not shown). PrP was immunoprecipitable by 6H4-conjugated beads (Figure 3B). However, the PrP species precipitated from 12 ml of amniotic fluid of both CJD and non-CJD control was sensitive to PK digestion, suggesting that it is PrPC(Figure 3B). There were faint bands migrating between 20 kDa and 35 kDa in the PK-treated amniotic fluid from both non-CJD and CJD in the blots probed with1E4 against human PrP 97-108 and anti-C (Figure 3B and data not shown). These bands should not represent PrPSc because they were detected in both non-CJD and CJD samples (data not shown). Using sodium phosphotungstate (NaPTA), another reagent that has been widely used for precipitating abnormal PrP, a small amount of PrP was also precipitated from the uterus but not the placenta of both CJD and non-CJD (Figure 3, C and D). However, the PrP species precipitated by NaPTA from CJD and non-CJD uterus was sensitive to PK digestion (data not shown). We compared the electrophoretic mobility of PrP obtained from a variety of samples including amniotic fluid, uterus, placenta, brain, CSF, and urine. Using the anti-C to residues PrP 220 to 231, PrP was detected in all samples despite the very small amount in the placenta (Figure 4A). The mobility of most PrP species from the brain and CSF was between ∼38 kDa and ∼20 kDa. In contrast, the migration of most PrP species from the amniotic fluid, uterus, and placenta was between 35 kDa and 26 kDa. Moreover, using 3F4 antibody against PrP 109-112, PrP was only detected in the brain and CSF but not in the peripheral tissues (Figure 4B), indicating that predominant PrP species from the amniotic fluid, uterus, and placenta are N-terminally truncated, similar to the urine PrP. This is consistent with previous observations.22Tuo W Zhuang D Knowles DP Cheevers WP Sy MS O'Rourke KI Prp-c and Prp-Sc at the fetal-maternal interface.J Biol Chem. 2001; 276: 18229-18234Crossref PubMed Scopus (55) Google Scholar, 23Wong BS Green AJ Li R Xie Z Pan T Liu T Chen SG Gambetti P Sy MS Absence of protease-resistant prion protein in the cerebrospinal fluid of Creutzfeldt-Jakob disease.J Pathol. 2001; 194: 9-14Crossref PubMed Scopus (35) Google Scholar, 24Narang HK Dagdanova A Xie Z Yang Q Chen SG Sensitive detection of prion protein in human urine.Exp Biol Med (Maywood). 2005; 230: 343-349PubMed Google Scholar To further confirm that the dominant PrP species from the uterine and gestational tissues are N-terminally truncated, the samples were subjected to deglycosylation with PNGase F. After treatment with PNGase F, by using the anti-C antibody two major PrP fragments were detected in brain tissues and CSF migrating at ∼28 kDa and ∼18 kDa. In contrast, only one major PrP band was detected in the amniotic fluid, uterus, placenta, and urine migrating at ∼18 kDa (Figure 4C). As expected, the PrP band migrating at ∼28 kDa, corresponding to the full-length PrP, was only detected in brain tissue and CSF, but not in the uterine and gestational tissues and urine (Figure 4C). It is widely thought that vertical transmission constitutes one route for the natural spread of prion diseases in animals, including scrapie in sheep and goats and bovine spongiform encephalopathy in cattle. However, the precise mechanism underlying the transmission of prion infectivity from ewe to lamb remains poorly understood. Since it has been demonstrated that prion infectivity is harbored in the blood of sheep with scrapie,25Houston F McCutcheon S Goldmann W Chong A Foster J Sisó S González L Jeffrey M Hunter N Prion diseases are efficiently transmitted by blood transfusion in sheep.Blood. 2008; 112: 4739-4745Crossref PubMed Scopus (151) Google Scholar vertical transmission could simply result from the large accumulation of blood in the placenta. Indeed, PrPSc has been observed at the fetal-maternal interface of infected sheep.22Tuo W Zhuang D Knowles DP Cheevers WP Sy MS O'Rourke KI Prp-c and Prp-Sc at the fetal-maternal interface.J Biol Chem. 2001; 276: 18229-18234Crossref PubMed Scopus (55) Google Scholar, 26Race R Jenny A Sutton D Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis.J Infect Dis. 1998; 178: 949-953Crossref PubMed Scopus (172) Google Scholar As for human prion diseases, to date eight gravid women with CJD (including the current case) have been documented through the completion of pregnancy and delivery of their neonates.10Berrebi A Cohen M Ayoubi JM [Creutzfeldt-Jakob disease and pregnancy.].J Gynecol Obstet Biol Reprod (Paris). 1997; 26: 755-759PubMed Google Scholar, 27Bernoulli C Siegfried J Baumgartner G Regli F Rabinowicz T Gajdusek DC Gibbs Jr, CJ Danger of accidental person-to-person transmission of Creutzfeldt-Jakob disease by surgery.Lancet. 1977; 1: 478-479Abstract PubMed Scopus (443) Google Scholar, 28Tamai Y Kojima H Kitajima R Taguchi F Ohtani Y Kawaguchi T Miura S Sato M Ishihara Y Demonstration of the transmissible agent in tissue from a pregnant woman with Creutzfeldt-Jakob disease.N Engl J Med. 1992; 327: 649PubMed Google Scholar, 29Lane KL Brown P Howell DN Crain BJ Hulette CM Burger PC DeArmond SJ Creutzfeldt-Jakob disease in a pregnant woman with an implanted dura mater graft.Neurosurgery. 1994; 34: 737-740Crossref PubMed Scopus (68) Google Scholar In addition, an 11-month-old girl born to a mother with vCJD was suspected to have the disease, inasmuch as she presented with convulsions and stiff limbs and had difficulty swallowing.30Ashraf H The first case of mother-to child transmission of vCJD?.Lancet. 2000; 356: 1085Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar However, to our knowledge, the diagnosis of CJD in this case has never been established. In fact, no case of vCJD has ever been reported in a baby to date. The youngest patient (a 12-year-old girl) with vCJD had no maternal history of the disease.31Verity CM Nicoll A Will RG Devereux G Stellitano L Variant Creutzfeldt-Jakob disease in UK children: a national surveillance study.Lancet. 2000; 356: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Prion disease has been transmitted by inoculation to BALB/c mice with placenta and colostrum as well as leukocytes from umbilical cord blood samples, but not from amniotic fluid, from only one of the eight pregnant women.28Tamai Y Kojima H Kitajima R Taguchi F Ohtani Y Kawaguchi T Miura S Sato M Ishihara Y Demonstration of the transmissible agent in tissue from a pregnant woman with Creutzfeldt-Jakob disease.N Engl J Med. 1992; 327: 649PubMed Google Scholar The finding of human-to-human transmission of vCJD through blood transfusion32Llewelyn CA Hewitt PE Knight RS Amar K Cousens S Mackenzie J Will RG Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion.Lancet. 2004; 363: 417-421Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar further implies the possibility that transmission from infected gravid women to neonate could occur through the placenta, a part of fetoplacental blood reservoir. But, neither the presence nor the individual features of PrP have been reported in the reproductive and gestational tissues in CJD and nonprion disease cases. Genetic analysis revealed homozygosity valine at codon 129 and no mutation in the coding region of the PrP gene. Western blot of brain tissues obtained at biopsy and autopsy demonstrated the presence of rPrPSc type 1 (Figure 2), whereas neuropathological examination revealed severe spongiform degeneration, astrogliosis, and the presence of ballooned neurons associated with weak PrP immunostaining, displaying a fine granular pattern (Figure 1, Figure 2).33Gambetti P Kong Q Zou WQ Parchi P Chen SG Sporadic and familial CJD: classification and characterisation.Br Med Bull. 2003; 66: 213-239Crossref PubMed Scopus (423) Google Scholar Cumulatively, these characteristics unquestionably define this case as sCJDVV1.33Gambetti P Kong Q Zou WQ Parchi P Chen SG Sporadic and familial CJD: classification and characterisation.Br Med Bull. 2003; 66: 213-239Crossref PubMed Scopus (423) Google Scholar Our study indicated that most of the PrP in the uterus, placenta, and amniotic fluid is truncated at the N-terminal and shares gel mobility (∼18 kDa) with C1, a C-terminal PrP fragment generated during normal metabolism of PrPC in the brain.12Chen SG Teplow DB Parchi P Teller JK Gambetti P Autilio-Gambetti L Truncated forms of the human prion protein in normal brain and in prion diseases.J Biol Chem. 1995; 270: 19173-19180Crossref PubMed Scopus (453) Google Scholar It is noteworthy that the gel mobility of the PrP fragment present in these tissues is similar to that of the urine PrP, but different from that of CSF and brain, in agreement with earlier observations.22Tuo W Zhuang D Knowles DP Cheevers WP Sy MS O'Rourke KI Prp-c and Prp-Sc at the fetal-maternal interface.J Biol Chem. 2001; 276: 18229-18234Crossref PubMed Scopus (55) Google Scholar, 23Wong BS Green AJ Li R Xie Z Pan T Liu T Chen SG Gambetti P Sy MS Absence of protease-resistant prion protein in the cerebrospinal fluid of Creutzfeldt-Jakob disease.J Pathol. 2001; 194: 9-14Crossref PubMed Scopus (35) Google Scholar, 24Narang HK Dagdanova A Xie Z Yang Q Chen SG Sensitive detection of prion protein in human urine.Exp Biol Med (Maywood). 2005; 230: 343-349PubMed Google Scholar The difference in the PrP profile between CNS and peripheral organs suggests that PrP may be subject to distinct metabolic pathways in the brain and peripheral organs. But the molecular mechanism associated with the generation of different PrP profiles needs to be further investigated. No significant differences in the amount and profile of PrP from these peripheral tissues were observed between normal controls and this CJD case. The rPrPSc type 1 was detected in the brain from the patient but not in the uterus, placenta, and amniotic fluid even after using g5p enrichment procedure. Although a small amount of PrP was precipitated from the uterus of the present case by NaPTA, a similar amount of PrP was also precipitated from the control (Figure 3C). Thus the captured PrP by NaPTA is unlikely to be associated with CJD. PrPC and PrPSc in the reproductive and gestational tissues have been well characterized in normal and scrape-infected sheep using Western blot analysis and immunohistochemistry.26Race R Jenny A Sutton D Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis.J Infect Dis. 1998; 178: 949-953Crossref PubMed Scopus (172) Google Scholar, 22Tuo W Zhuang D Knowles DP Cheevers WP Sy MS O'Rourke KI Prp-c and Prp-Sc at the fetal-maternal interface.J Biol Chem. 2001; 276: 18229-18234Crossref PubMed Scopus (55) Google Scholar, 34Tuo W O'Rourke KI Zhuang D Cheevers WP Spraker TR Knowles DP Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep.Proc Natl Acad Sci USA. 2002; 99: 6310-6315Crossref PubMed Scopus (72) Google Scholar N-terminally truncated and glycosylated PrPC that migrates at ∼23 to 37 kDa, has been observed in cotyledonary chorioallantois, allantoic fluid, endometrium, myometrium, oviduct, and ovary of pregnant and nonpregnant uninfected ewes.22Tuo W Zhuang D Knowles DP Cheevers WP Sy MS O'Rourke KI Prp-c and Prp-Sc at the fetal-maternal interface.J Biol Chem. 2001; 276: 18229-18234Crossref PubMed Scopus (55) Google Scholar In addition, PrPC in low or undetectable amounts was observed in intercotyledonary chorioallantois, amnion, and amniotic fluid. When detectable, PrPC had an electrophoretic mobility of ∼18 to 22 kDa after deglycosylation. The rPrPSc fragments were detected in enriched tissue homogenates of uterine caruncular endometrium and placental cotyledonary chorioallantois but not in other parts of the uterus or placenta, nor in amniotic fluid from pregnant scrapie-infected ewes.22Tuo W Zhuang D Knowles DP Cheevers WP Sy MS O'Rourke KI Prp-c and Prp-Sc at the fetal-maternal interface.J Biol Chem. 2001; 276: 18229-18234Crossref PubMed Scopus (55) Google Scholar By immunohistochemistry, PrPSc staining was observed mainly in the uterine endometrial epithelium, trophoblast cells, and multinucleate cells in zone A of the maternal endometrial crypts of scrapie-infected ewes.34Tuo W O'Rourke KI Zhuang D Cheevers WP Spraker TR Knowles DP Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep.Proc Natl Acad Sci USA. 2002; 99: 6310-6315Crossref PubMed Scopus (72) Google Scholar Interestingly, the rPrPSc bands present in sheep placenta had lower molecular mass than did corresponding bands from the brain.26Race R Jenny A Sutton D Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis.J Infect Dis. 1998; 178: 949-953Crossref PubMed Scopus (172) Google Scholar It has been proposed that the differences in the size and glycosylation patterns of the PK-resistant PrP in the placenta and brain represent unique PrPSc processing in different tissues.26Race R Jenny A Sutton D Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis.J Infect Dis. 1998; 178: 949-953Crossref PubMed Scopus (172) Google Scholar, 35Caughey B Race RE Ernst D Buchmeier MJ Chesebro B Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells.J Virol. 1989; 63: 175-181Crossref PubMed Google Scholar Although our current study did not detect abnormal PrP species in the uterus and gestational tissues, including the placenta and amniotic fluid from this CJD case, it must be emphasized that only portions of the uterine and placental tissues were examined. Given localization of PrPSc only in certain areas of the uterus and placenta of the infected ewes,22Tuo W Zhuang D Knowles DP Cheevers WP Sy MS O'Rourke KI Prp-c and Prp-Sc at the fetal-maternal interface.J Biol Chem. 2001; 276: 18229-18234Crossref PubMed Scopus (55) Google Scholar, 34Tuo W O'Rourke KI Zhuang D Cheevers WP Spraker TR Knowles DP Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep.Proc Natl Acad Sci USA. 2002; 99: 6310-6315Crossref PubMed Scopus (72) Google Scholar additional studies on more areas of these organs from gravid women with CJD are needed. Moreover, whether these tissues are infectious remains to be further determined by bioassay. Because the amniotic fluid is continually being swallowed and inhaled by the fetus during pregnancy, it is important to determine whether or not amniotic fluid harbors prion infectivity. Although close long-term follow-up of the offspring of the eight pregnant women with CJD is necessary, a definitive negative result of the transmissibility studies by direct bioassay or by amplification-based detection of reproductive and gestational tissues including the amniotic fluid would greatly relieve these children and their families from discomfort and anguish. We thank Francine Epperson for autopsy coordination; Brenda Dupree and Rose Richardson for technical support in histological and immunohistochemical analyses of the brain biopsy and autopsy specimens at the Indiana Alzheimer Disease Center; Kay Edmonds for tissue coordinating; Diane Kofskey for technical support in studies of histology and immunohistochemistry; and Kai Jing for technical support in diagnostic Western blot analysis of the autopsy brain sample at the National Prion Disease Pathology Surveillance Center.
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