Research ArticlePrion Disease

Prion seeding activity and infectivity in skin samples from patients with sporadic Creutzfeldt-Jakob disease

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Science Translational Medicine  22 Nov 2017:
Vol. 9, Issue 417, eaam7785
DOI: 10.1126/scitranslmed.aam7785
  • Fig. 1. Western blot analysis of skin tissue from CJD and non-CJD patients.

    (A) Western blot analysis of proteinase K (PK)–resistant misfolded forms of the prion protein (PrPSc) in skin from the U.S. variant Creutzfeldt-Jakob disease (US vCJD) patient. The blot was probed with anti-C antibody against human PrP(220–231) with an exposure time of 5 or 50 min. The skin samples [PK-treated, PrPSc-concentrated fraction (P4) after enrichment by ultracentrifugation] were treated with PK (25 μg/ml) at 37°C for 1 hour, followed by addition of Roche complete protease inhibitor cocktail. A CJD brain homogenate (brain) was used as a positive control. The black arrow refers to the longer exposure time of the blot on the right corresponding to the right lane of the left panel (at normal exposure). (B) Western blot analysis of PrP from the US vCJD patient skin sample treated with PK and peptide N-glycosidase F (PNGase F) and probed with the 3F4 anti-PrP antibody. The CJD brain homogenate was used as a positive control. (C) Western blot analysis of PrP in skin or brain samples from the UK vCJD patient treated with PK or PK plus PNGase F and probed with the anti-PrP antibody 3F4. (D) Comparison of the amounts of PrPSc in the brain between the US vCJD and UK vCJD cases treated with PK. (E) Western blot analysis of PrPSc in skin samples from sCJD patient nos. 1 to 5, vCJD patient no. 6, and non-CJD patient nos. 8 to 17, treated with PK (top) or PK plus PNGase F (bottom). Skin samples from about 60 to 80 mg of skin tissues for each case (~6 mm × 6 mm in area) were used for PrPSc enrichment and loading onto the gel. The vCJD patient no. 6 skin sample was used as a positive control. The black arrows indicate the PK-resistant deglycosylated PrPSc. The results shown in (A) to (E) are representative of three independent experiments.

  • Fig. 2. RT-QuIC testing of sCJD and vCJD brain and skin samples.

    (A) Final tissue dilutions of 10−5 brain (green) and 10−3 skin tissue [first supernatant (S1) fraction in magenta and P4 fraction in black] from sporadic CJD (sCJD) patient nos. 1 to 7 were used to seed quadruplicate real-time quaking-induced conversion (RT-QuIC) reactions. Negative control reactions were seeded in quadruplicate with a 10−4 dilution of brain tissue from patients with Alzheimer’s disease (AD) or progressive supranuclear palsy (PSP), and a 10−3 dilution of S1 or P4 skin fractions from non-CJD patient nos. 8 to 17. A final SDS concentration of 0.001% in combination with 300 mM NaCl was used with recombinant bank vole PrP (rPrPsen) as the substrate. Similar results were seen in at least two independent experiments. Traces from representative RT-QuIC experiments are the average of percent thioflavin T (ThT) fluorescence readings from all four replicate wells, regardless of the intensity of their signal (y axis) plotted as a function of time (x axis). (B) Average of percent ThT fluorescence readings from four replicate reactions, skin samples from the lower back (LB; blue), skull apex (red), and an area near the left ear (ear; green) from patients with CJD and from those without CJD (non-CJD) as control. For visual clarity, SDs of the averages are only shown every 5 hours. (C) Dot plot of the final mean relative ThT fluorescence readings for each skin sample obtained from the lower back, skull apex, and ear of 16 additional sCJD patients, and 5 additional non-CJD patients were examined with the RT-QuIC assay. The horizontal and vertical black lines indicate the means and SD of 60 ± 37%, 62 ± 40%, and 75 ± 30% ThT fluorescence for sCJD patient skin samples from the lower back, apex, and ear, respectively. The dotted line in (B) and (C) indicates the 8.8% calculated ThT fluorescence threshold. All samples were tested in a blinded fashion.

  • Fig. 3. RT-QuIC testing of prion seeding activity in P4 skin fractions from sCJD and vCJD patients.

    P4 skin fractions from the sCJD and vCJD patients were analyzed using a second rPrPsen substrate, hamster rPrPsen (residues 23 to 231), which has been previously shown to help discriminate between these two human prion strains (21). Dilutions (10−3) of P4 skin fractions from four sCJD patients (nos. 1, 2, 4, and 5) and 1 vCJD patient (no. 6) were used to seed quadruplicate RT-QuIC reactions. Testing was performed using either bank vole rPrPsen in the presence of 300 mM NaCl and 0.001% SDS (A) or hamster rPrPsen in the presence of 300 mM NaCl and 0.002% SDS (B). Testing was performed twice with similar results. The data show the average fluorescence of replicate wells monitored over time.

  • Fig. 4. Prion infectivity of skin samples from sCJD patients.

    (A) Survival curves of TgNN6h or TgWV humanized Tg mice inoculated with skin homogenate from sCJD or non-CJD patients. Six- to 8-week-old TgNN6h (n = 5) or TgWV (n = 7) mice were intracerebrally inoculated with 30 μl of 5% skin homogenate from patients with sCJDMM2 or sCJDVV1. Five TgNN6h mice were intracerebrally inoculated with skin homogenate from a patient with sCJDMM2 (red diamonds), and seven TgWV mice were intracerebrally inoculated with skin homogenate from a patient with sCJDVV1 (blue squares). Seven TgWV mice were intracerebrally inoculated with skin homogenate from a non-CJD patient as a negative control (green line). (B) Western blot analysis of PrPSc in the brains of TgNN6h humanized Tg mice inoculated with skin homogenate from a sCJDMM2 patient. Lanes 1 and 2 show samples of uninoculated mouse brain (control); lanes 3 to 12 show brain samples from five mice inoculated with a skin homogenate from a sCJDMM2 patient; lanes 13 and 14 show brain samples from two mice inoculated with sCJD brain homogenate (brain) as a positive control; lanes 15 and 16 show brain homogenates from sCJD type 2 (T2) and type 1 (T1) patients treated with PK and PNGase F that were used as a reference for PrPSc banding patterns. Samples in lanes 1, 3, 5, 7, 9, and 11 were not PK treated, whereas samples in the other lanes were treated with PK (50 μg/ml) for 1 hour at 37°C. The immunoblot was probed with the anti-PrP antibody 3F4. (C) Western blot analysis of PrPSc in the brains of TgWV mice inoculated with skin homogenate from a patient with sCJDVV1. Lanes 1 to 10 show brain samples from five humanized Tg mice inoculated with skin homogenate from the sCJDVV1 patient; lanes 11 and 12 show brain samples from a mouse inoculated with brain homogenate from a sCJDVV2 patient (brain); lanes 13 and 14 show brain samples from a mouse inoculated with skin homogenate from a non-CJD patient as a negative control (non-CJD skin); lanes 15 and 16 show brain homogenates from patients with sCJD type 1 or type 2 as a positive control. Samples in lanes 1, 3, 5, 7, 9, 11, and 13 were not treated with PK, whereas samples in the other lanes were treated with PK (50 μg/ml) for 1 hour at 37°C. Because of the lower amounts of PrPSc in the brain samples of mice shown in lanes 2 and 4, more PK-treated mouse brain homogenate was loaded onto the gel for these two mice compared to brain homogenate for the other mice. This could have resulted in the slower migration of the PK-resistant PrPSc in mouse brain samples in lanes 2 and 4 compared to the other lanes. The blot was probed with the anti-PrP antibody 3F4. (D) Hematoxylin and eosin (H&E) staining and PrP immunohistochemistry of the cerebral cortex of TgNN6h humanized Tg mice before and after inoculation with sCJD patient brain homogenate. (a and e) Brain sections from uninoculated mice as a negative control; (b and f) brain sections from mice inoculated with sCJD brain homogenate as a positive control; (c, d, g, and h) brain sections from two mice inoculated with skin homogenate from a patient with sCJDMM2. (E) H&E staining and PrP immunohistochemistry using the anti-PrP 3F4 antibody of the cerebral cortex of TgWV Tg mice inoculated with sCJD or non-CJD patient skin or brain homogenate. (a and e) Brain sections from a Tg mouse inoculated with skin homogenate from a non-CJD patient as a negative control; (b and f) brain sections from a Tg mouse inoculated with brain homogenate from a sCJD patient as a positive control; (c, d, g, and h) brain sections from two mice inoculated with skin homogenate from a sCJDVV1 patient. Scale bars, 50 μm.

  • Table 1. Characteristics of sCJD and vCJD patients.

    sCJD, sporadic Creutzfeldt-Jakob disease; vCJD, variant CJD; MRI, magnetic resonance imaging; CSF, cerebrospinal fluid.

    Mean age, years (SD)59.4 (15.3)
    Mean duration, months (SD)10.7 (10.8)
    Male/female10:13
    (43.5%:56.5%)
    Race, n (%)
      White19 (82.6%)
      Black0 (0)
      Hispanic1 (4.3%)
      Mixed1 (4.3%)
      Unknown2 (8.7%)
    Cognitive symptoms, n (% with known symptoms)20 (100%)
    Myoclonus, n (% with known symptoms)10 (52.6%)
    Cerebellar symptoms, n (% with known symptoms)18 (94.7%)
    Visual symptoms, n (% with known symptoms)11 (57.9%)
    Pyramidal symptoms, n (% with known symptoms)8 (42.1%)
    Extrapyramidal symptoms, n (% with known
    symptoms)
    8 (42.1%)
    PSWCs on EEG, n (%)*0 (0)
    Brain MRI suggestive of prion disease, n (%)16 (88.9%)
    CSF 14-3-3 positive, n (%)13 (59.1%)
    Mean CSF τ concentration, pg/ml (SD)7136.8 (6237.0)
    Prion disease subtype, n (%)
      MM16 (26.1%)
      MM23 (13%)
      MV11 (4.3%)
      VV11 (4.3%)
      VV24 (17.4)
      MV1-23 (13%)
      VV1-21 (4.3)
      MM1-22 (8.7)
      vCJD2 (8.7)

    *Of 23 CJD patients, electroencephalography (EEG) was available for 13 cases. Four patients exhibited normal EEGs, and nine had abnormal EEGs but did not have periodic sharp wave complexes (PSWCs).

    †Study of Zerr et al. (16).

    ‡Study of Belay et al. (17).

    • Table 2. Sensitivity and specificity of RT-QuIC assay for CJD skin samples

      NA, not available.

      DiagnosisLower backApexArea near earOverall
      sCJDVV1NA+NA+
      sCJDMM1NA+NA+
      sCJDVV1-2NA+NA+
      sCJDMM2NA+NA+
      sCJDMV2NA+NA+
      sCJDMM1NA+++
      sCJDMM1+/−*+++
      sCJDMM1++++
      sCJDMM1+++
      sCJDMM1++/−++
      sCJDVV2NA+++
      sCJDVV2++++
      sCJDVV2NA+++
      sCJDVV2++++
      sCJDMM2+/−++
      sCJDMM2NA+++
      sCJDMM1-2++++
      sCJDMM1-2++
      sCJDMV1-2++++
      sCJDMV1-2++++
      sCJDMV1++++
      Sensitivity (%)928894100§
      Non-CJD
      Non-CJD
      Non-CJDNA
      Non-CJD
      Non-CJD
      Non-CJD
      Non-CJD
      Specificity (%)100100100100

      *Weak positive (+/−) means one of four or two of eight wells on two or more independent experiments.

      †Positive (+) RT-QuIC prion seeding activity.

      ‡Negative (−) RT-QuIC prion seeding activity.

      §RT-QuIC analysis of skin samples from the torso or forearm of the UK vCJD and US vCJD patients, respectively, was RT-QuIC–positive and was included in the overall sensitivity and confidence interval calculations.

      ¶RT-QuIC analysis of skin samples from the torso or forearm of eight non-CJD patients was RT-QuIC–negative and was included in the overall specificity and confidence interval calculations.

      • Table 3. End point RT-QuIC quantitation of seeding activity in brain and skin samples from CJD patients.
        Log SD50 per milligram of tissue*
        Patient no.DiagnosisBrainS1 skinP4 skin
        1sCJDVV1>9.76.94.8
        2sCJDMM1>8.5>6.84.9
        3sCJDVV1-28.05.1≤3.0
        4sCJDMM29.26.84.7
        5sCJDMV2>8.76.54.1
        6vCJDMM2>8.25.03.6
        7vCJDMM27.7≤3.0≤3.5

        *Log seeding dose 50 (SD50) was calculated, as described in Materials and Methods.

        Supplementary Materials

        • www.sciencetranslationalmedicine.org/cgi/content/full/9/417/eaam7785/DC1

          Methods

          Fig. S1. Western blotting and neurohistology of autopsy brain samples from sCJD and vCJD patients.

          Fig. S2. Representative immunohistochemistry of skin samples from two vCJD patients and a non-CJD patient.

          Fig. S3. Representative RT-QuIC end-point dilution analysis of brain and skin fractions from two CJD patients.

          Fig. S4. Western blot analysis of bank vole rPrPres RT-QuIC products from reactions seeded with brain or skin samples.

          Fig. S5. Western blot analysis of brain samples from TgNN6h or TgWV mice inoculated intracerebrally with sCJDMM2 or sCJDVV2 brain homogenate, respectively.

          Table S1. Summary of non-CJD cases.

        • Supplementary Material for:

          Prion seeding activity and infectivity in skin samples from patients with sporadic Creutzfeldt-Jakob disease

          Christina D. Orrú, Jue Yuan, Brian S. Appleby, Baiya Li, Yu Li, Dane Winner, Zerui Wang, Yi-An Zhan, Mark Rodgers, Jason Rarick, Robert E. Wyza, Tripti Joshi, Gong-Xian Wang, Mark L. Cohen, Shulin Zhang, Bradley R. Groveman, Robert B. Petersen, James W. Ironside, Miguel E. Quiñones-Mateu, Jiri G. Safar, Qingzhong Kong,* Byron Caughey,* Wen-Quan Zou*

          *Corresponding author. Email: wxz6{at}case.edu (W.-Q.Z.); bcaughey{at}nih.gov (B.C.); qingzhong.kong{at}case.edu (Q.K.)

          Published 22 November 2017, Sci. Transl. Med. 9, eaam7785 (2017)
          DOI: 10.1126/scitranslmed.aam7785

          This PDF file includes:

          • Methods
          • Fig. S1. Western blotting and neurohistology of autopsy brain samples from sCJD and vCJD patients.
          • Fig. S2. Representative immunohistochemistry of skin samples from two vCJD patients and a non-CJD patient.
          • Fig. S3. Representative RT-QuIC end-point dilution analysis of brain and skin fractions from two CJD patients.
          • Fig. S4. Western blot analysis of bank vole rPrPres RT-QuIC products from reactions seeded with brain or skin samples.
          • Fig. S5. Western blot analysis of brain samples from TgNN6h or TgWV mice inoculated intracerebrally with sCJDMM2 or sCJDVV2 brain homogenate, respectively.
          • Table S1. Summary of non-CJD cases.

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