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In the Pipeline-Creutzfeldt-Jakob Disease
Seeding Prion Activity Identified in Skin Cells of Patients with Sporadic CJD

ARTICLE IN BRIEF

Researchers found prion seeding activity and infectivity in postmortem skin samples from patients with sporadic Creutzfeldt-Jakob disease. The findings suggest that some component of the skin could potentially seed, spread, and contaminate non-central nervous system surgical environments. But they said the chance of prion diseases being spread through casual contact is very low.

Low levels of the infectious form of the prion protein were detected in skin samples from patients who died with sporadic Creutzfeldt-Jakob disease (sCJD), according to a study published in the November 22 edition of Science Translational Medicine.

The findings suggest that some component of the skin could potentially seed, spread, and contaminate non-central nervous system surgical environments. And the skin biopsies may offer another method for diagnosing the fatal neurodegenerative condition, the study authors said.

The study authors say that the samples contain all layers of skin, including blood vessels and nerve endings, and there is little epidemiological evidence that prions are transmissible through the skin itself.

CJD is caused by the accumulation of a misfolded form of a bodily protein into an infectious, self-seeding form called prion protein scrapie associated (PrPSc). Sensitive assays can now detect prion disease in the cerebrospinal fluid, but a brain autopsy or biopsy remains the most accepted way of making a definitive diagnosis.

The major implication of the finding is that “skin prion-seeding activity could be a molecular marker for a postmortem (and potentially premortem) test for sCJD,” said the senior study author Wen-Quan Zou, MD, PhD, associate professor of pathology and neurology and director of the CJD Skin Project at Case Western University.

Currently, lumbar punctures are ordered to diagnose sCJD in suspected cases. Skin biopsy offers an alternative easier-to-access material, said Dr. Zou. In addition, in cultures that discourage brain autopsy, skin testing could be used to confirm a diagnosis at death, he said.

STUDY DESIGN, FINDINGS

For the current study, Dr. Zou sent autopsied or biopsied skins from 38 patients to Christina D. Orrú, PhD, a microbiologist at the National Institutes of Health's Rocky Mountain Laboratories in Montana: 21 had died from sCJD, two died from variant CJD (vCJD) associated with consumption of tainted meat from cows with mad cow disease, and 15 had died without any evidence of CJD. Skin samples were taken from the torso, limbs, top of the head, or next to the ear.

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DR. WEN-QUAN ZOU said the major implication of the finding is that “skin prion-seeding activity could be a molecular marker for a postmortem (and potentially premortem) test for sCJD.”

The scientists analyzed the samples for evidence of PrPSc with the traditional Western blotting and real-time quaking-induced conversion (RT-QuIC). RT-QuIC is a sensitive assay developed by scientists to detect and amplify prion-seeding activity; it uses recombinant prion protein to which potentially infectious tissue homogenate is added.

The research team also took skin tissue samples from two deceased sCJD patients and injected them into the brains of humanized transgenic mice. Western blotting detected PrPSc in one of five sCJD patients examined. The RT-QuIC detected prion seeding activity in at least one skin sample from all 23 CJD patients and did not find any activity in the non-CJD controls. The area near the ear had the highest and most consistent prion-seeding activity, in 94 percent of sCJD cases, the researchers reported.

“The seeding activity is weaker in the skin — about a thousand times lower than what we find in brain tissues from sCJD patients,” Dr. Zou said.

“It was surprising,” said Byron Caughey, PhD, a biochemist at the Rocky Mountain Laboratories. “The only data in the literature were from one patient with vCJD. As we develop more sensitive assays we can pick up smaller and smaller amounts of prion seeding activity.”

Dr. Orrú added that they could quantify the seeding, even after they diluted the sample several times. “We don't know the level of risk. But the chance of it being transmitted through skin should be very low.”

The scientists also wanted to see if skin samples from sCJD patients could infect animals. They intracerebrally inoculated 12 mice from two transgenic lines with skin homogenates from two sCJD patients. More than a year later, they observed that all the animals had some clinical signs, pathological evidence of disease, and detectable PrPSc. This was proof that the skin homogenates, which contain all layers of skin, blood vessels and nerve endings, could seed and infect their new host.

The scientists said that they have no clue yet where the prion seeds accumulate within the skin. Dr. Orrú said that it is probably seeding and spreading through the nerve endings, but they are exploring that in ongoing studies.

“We should emphasize that in no way does our study imply that prion transmission can occur via casual contact,” the scientists wrote in the paper. “The bioassays that we have performed involved inoculation of relatively large amounts of skin extracts directly into the brains of humanized transgenic mice, which is not likely to be recapitulated in humans in any sort of routine event.”

Dr. Caughey added: “Our data show that there are prions in the skin but they are at a very low level, and there is no evidence that these levels represent a transmission risk under practical clinical circumstances. We also don't know what part of the skin contains this prion seeding activity.”

The researchers are now looking to see whether they can detect this abnormal activity from skin samples in living people. They have analyzed samples but the study data are still blinded. “It's unlikely that we will ever find it on the skin surface,” Dr. Orrú said.

The research team will also be conducting studies in animals using contaminated medical instruments, mimicking general surgery that exposes the skin to the contaminated instruments. They are also designing studies with skin grafts to see whether specific parts of human skin from sCJD patients could trigger seeding in healthy animals. And they are now extending their studies to see whether skin can be validated as a useful biomarker for sCJD.

EXPERT COMMENTARY

Commenting on the study, Kurt Giles, PhD, associate professor of neurology at the Institute for Neurodegenerative Diseases at the University of California, San Francisco, said the findings were interesting and the research is “quite convincing that there is infectivity in the skin samples studied.”

“It does not mean that sCJD is transmissible through skin contact, though,” Dr. Giles said. “It is not clear what part of the skin sample contained misfolded prion protein. Not every sample was positive. We still need to understand how this happens. Certainly, it would be simpler to get a skin sample.”

Dr. Giles said the take-home message is that there is a potential new source of material to use in diagnosis of sCJD.

Graham S Jackson, PhD, a biochemist at the MRC Prion Unit at University College London and the UCL Institute of Prion Diseases in London, agreed that “it is not clear where the seeding and prions reside.”

“They didn't define which cell types they were derived from,” he explained. “You can't exclude the fact that the prions may have come from blood or nerve endings.”

He said several techniques are available for the diagnosis of sCJD, including detection using CSF samples and imaging of the brain by magnetic resonance imaging. His laboratory and others are looking for a pre-clinical diagnostic biomarker in blood.

“There are cases where epidemiology has spoken loudly,” said Sonia Vallabh, a PhD student at the Eli and Edythe L. Broad Institute of MIT and Harvard, referring to people who developed CJD decades after receiving growth hormone in childhood and/or people exposed to infectious prions through tainted meat. “But exposure through skin? That does not reach the level of direct epidemiological evidence,” the scientist said.

“Our ability to detect the presence of prions experimentally is getting much more sensitive, but we need to be cautious about how it speaks to the risk in the real world. They may be picking up sub-clinical amounts of prion material. It's important to balance understanding the risks with making sure that people don't become unnecessarily alarmed.”

THE BACK STORY: FINDING INFECTIVITY IN SKIN CELLS OF CJD

The current findings evolved from a body of work and experiments done collaboratively between Case Western Reserve University and the Rocky Mountain Laboratories. In 2013, Dr. Zou and his colleagues were making patient-derived induced pluripotent stem cells (iPSCs) and discovered misfolded prion protein in skin-derived fibroblasts from patients with sCJD and some asymptomatic PrP mutation carriers.

Shortly after the 2013 observation, Dr. Zou asked the scientists at the Rocky Mountain Laboratories to run the fibroblast samples through real-time quaking-induced conversion (RT-QuIC), which allows scientists to quantify seeding activity.

Byron Caughey, PhD, a biochemist at the lab, took CSF from animals and patients and added it to a 96-well assay plate. Any PrPSc seeds in the CSF were amplified by many orders of magnitude to levels that were detectable by a fluorescent dye.The RT-QuIC is almost 100 percent specific and 95 percent sensitive, and is now routinely ordered when there is a suspicion of CJD.

A test on a handful of samples suggested that RT-QuIC was sensitive enough to identify even weak signals of prion infectivity in skin.

LINK UP FOR MORE INFORMATION:

•. Orru CD, Yuan J, Appleby BS, et al. Prion seeding activity and infectivity in skin samples from patients with sporadic Creutzfeldt-Jakob disease stm.sciencemag.org/content/9/417/eaam7785. Sci Transl Med 2017; Epub 2017 Nov 22.