Emerging Infections of International Public Health Importance

Objectives:

Have a working knowledge of the theory of how prions cause transmissible spongiform encephalopathy
Be familiar with the categories and clinical manifestations of the human transmissible spongiform encephalopathies

Definition of Transmissible spongiform encephalopathy (TSE)

Non-inflammatory diseases of humans and animals that share common pathologic features. They have long incubation periods and are caused by infectious proteins or prions.

Prions are a normal cellular protein which occurs in 2 forms, the normal form called PrPc for cellular or PrP-sen for sensitive because it is sensitive to a protease. The protein is highly conserved membrane glycoprotein which has 2 glycosylation sites: the C terminal where there are alpha helices (a folding form of the protein) and the N terminal region which has octapeptide repeats and 2 copper binding sites. The N terminal inserts into the membrane. This protein is present in numerous cells but is significantly represented in neurons.

In a paper by Collinge et al in Annual Reviews of Neuroscience 2001, they discuss the different models of these proteins with schematic models. In their models the alpha helices have 2 potential carbohydrate binding sites. This allows the protein to have both carbohydrates bound, only one bound or no binding. This is important in determining species differences or strain differences of the proteins.

The abnormal form of the prion has the same amino acid sequence as the normal form. The same gene encodes them; they are the same protein if you look at sequence. They differ by their folding. The abnormal form is called the PrPSc or PrP-resistant form. It is protease resistant because if you take this form and partially digest it with protease it survives. It has a different confirmation, a different 3 dimensional folding than the normal form. This is a novel concept that the folding of a protein determines whether this will be a normal protein or an abnormal form capable of causing disease. In the abnormal form there are more beta-pleated sheets instead of the alpha helices at the C-terminal.

The normal and abnormal forms are encoded with a gene, which is called the PRNP gene. On this gene there are a number of possible mutations. Different mutations at specific codons are associated with the different forms of transmissible spongiform encephalopathies.

GSS is associated with point mutations at codons 102, 117, 198, and 217. Familial CJD in Libyan Jews is associated with mutation at codon 200.

An important polymorphism occurs at codon 129. This codon encodes either methionine or valine. Because each person has 2 PRNP genes, people can be MM, VV or MV. In the general European population 38% are MM, 51% are MV and 11% are VV. Polymorphism at codon 129 determines whether codon 178 mutations lead to fatal familial insomnia or familial CJD. Homozygosity at codon 129 enhances susceptibility to CJD.

Studies done in mice have been able to demonstrate a distinct incubation period before neuropathology appears. The strain type is likely determined by the folding conformation of the protein and the sequence polymorphism. The copper and zinc binding sites may also play a role but this is still unclear.

Most common of the human TSEs. It has a worldwide distribution and affects men and women equally. Average age of onset is 60 years. It can affect 1 person per million annually. This is not a common disease. It has a characteristic clinical triad consisting of rapidly progressive dementia, myoclonus and a typical EEG. The clinic course usually begins with some type of prodrome such as anxiety, asthenia, insomnia, decreased appetite. The initial stages are demonstrated by sensory disturbances, confusion, and cerebellar ataxia. Late stages are manifested by more behavioral abnormalities, progressive dementia, visual problems, cerebellar, pyramidal, extrapyramidal and lower motor neuron signs. They also have stimulus sensitive myoclonus and can become akinetic and mute. The median survival time is 4.5 months.

This usually occurs by accidental inoculation by previously infected surgical instruments or human products. This disease has been associated with neurosurgery and contaminated dural grafts. It appears that the typical sterilization procedures for surgical instruments do not inactivate these abnormal prions. Peripheral inoculation of human growth hormone has also been shown to transmit this abnormal protein. Most of these patients present more with cerebellar ataxia and less cognitive impairment. The incubation time for iatrogenic CJD ranges from 2 years to 40 years or more.

Familial CJD is responsible for 5-15% of all CJD cases. The age of onset is earlier usually in late 40’s and the disease duration is longer, about 18 months.

Fatal Familial Insomnia is characterized by a progressive and intractable insomnia. Patients develop dysautonomia including labile blood pressure, heart rate and motor abnormalities. The thalamus tends to be an area particularly involved in this form. There appears to be an absence of PrPsc in the brain of those affected with this familial form.

This is an EEG from a patient with CJD. This shows triphasic waves associated with the myoclonus of the face. The EEG pattern is characteristic of this disease and is part of the clinical triad of sporadic CJD.

CSF 14-3-3 Immunoassay is a good test to rule in CJD, in a setting in which CJD is very likely. It should be used in persons that you feel have a high likelihood of CJD since if used indiscriminately there would be a large number of false positive results. Recent strokes or encephalitis are among the more common diseases that will give you a false positive test.

Neuroimaging has some but limited value. There may be increased signal in deep nuclei on T2 and FLAIR images. There may also be increased signal in cortex and basal ganglia on DWI.

The MRI shows patchy areas of increased signal in the cortex. Biopsy revealed CJD.

The definitive diagnostic study however is neuropathology. The gray matter is affected greater than the white matter. There is neuronal loss, spongiform changes, gliosis and no evidence of inflammation. Plaques can be variable in distribution and degree. With staining and ultra structurally they have the appearance of amyloid. These plaques can be seen in cerebellum of persons with Kuru, in 10% of CJD cases and in all those with GSS. You can stain the tissue with anti-PrP antibody.

The differential diagnosis for CJD is broad and should be thought of before a diagnosis of CJD is entertained. The most common differential would be the neurodegenerative disorders such as Alzheimer’s dementia. Even an unusual presentation of Alzheimer’s will be more common than sporadic CJD. Other etiologies to rule out include neurosyphilis, subacute sclerosing panencephalitis, Whipple’s disease, vasculitis, Hashimotos thyroiditis, intoxications with metals and drugs and subclinical status epilepticus.

In a study by Tschampa et al of 413 brain samples of people suspected of having CJD, 309 had sporadic CJD or familial TSE,104 did not and had other neurological conditions such as Alzheimer’s dementia or dementia with Lewy bodies.

Bovine Spongiform Encephalopathy (BSE) and variant Creutzfeld Jacob Disease (vCJD)

BSE was first identified in the United Kingdom in 1985 with only a few animals per herd affected. This increased to a peak incidence in 1992-1993. The disease likely entered the herds through meat and bone meal used a food supplement. These may have been contaminated with scrapie or sporadic BSE. This meal is produced by rendering offal and has been incorporated into animal feed since 1940. However, in the late 1970s the rendering process was changed and omitted the solvent extraction and extreme heat processes. This change happened because there was no longer a market for the tallow which was a product created by this process. It is thought that this change made it possible for this new infectious agent to persist in the meal produced. The process of feeding meat and bone meal to cattle has been banned since 1988 in the UK.

The classic signs of BSE include an animal becoming apprehensive, hyperesthetic (not wanting to be touched), uncoordinated with wide based stance and "mad". The incubation period is 4-5 years. Pathologic evaluation is similar in all animals affected. There was an estimated 750,000 infected animals that entered the human food chain.

Will et al described the first 35 cases of vCJD. They found that the median duration of illness was 14 (8-38) months; median age at death was 29 (18-53) years; psychiatric symptoms began early and ataxia occurred after a median of 6 months. The cognitive impairment and involuntary movements occurred later in the course of disease.

On diagnostic testing, there were no triphasic waves seen on EEG. The CSF 14-3-3 was positive in 13/35 (56%), there was bilateral pulvinar intensity on MRI in 20/35 (77%). Most interesting was that all the cases were methionine homozygotes at codon 129 of the PRNP gene.

Variant CJD is unique in a number of ways. One is that you can detect the abnormal prion protein outside of the central nervous system. This has raised the question as to whether there are simpler diagnostic tests that can be done such as tonsil biopsy. Some preliminary studies exploring this possibility showed the abnormal prion in persons known to have CJD in their tonsillar tissue. It has yet to be identified outside of this population.

A study by Andrews et al looked at the epidemiology of variant CJD in the UK. They looked at definite and probably cases of vCJD from 1994-1999. They found that the onset increased by 23% per year and deaths increased by 33% per year.

As of September 2001 there have been 107 deaths from vCJD in the UK. The true number of those infected is unknown and the incubation period is also unknown. The first vCJD cases began to appear approximately 10 years after the first BSE case in cattle. BSE is likely the cause for vCJD in human. Studies which inoculated BSE into the brains of macaques produces a TSE similar to that seen in vCJD. Nonhuman primates can be infected with BSE orally. Inoculation of BSE and vCJD into mice produces the same "signature" based on pathology and incubation period. The PrP-res glycoforms are the same for vCJD and BSE. Transgenic mice expressing bovine PrP show nearly identical incubation times, pathology and glycoform profiles after inoculation with BSE and vCJD.

The concern about possible blood transfusion and transfer of vCJD has led to a few studies. To date there has been no epidemiologic evidence of CJD transmission by blood transfusion. There have been no good studies with regard to variant CJD and blood transfusion so this is still an open area of study and debate.

Theoretically if you can interfere with the conversion of the normal form of the prion to the abnormal form you could destabilize the abnormal form. One method would be to have antibody-mediated clearance of PrPsc or abnormal form. This is work that Stanley Prusiner is doing.

A paper by Montrasio et al from Science 2000 showed that PrPsc first accumulates in lymphoid tissue in rodents infected with scrapie. This substance called lymphotoxin beta receptor immunoglobulin fusion protein (LTBR-Ig) causes loss of FDCs (follicular dendritic cells) from the spleen. Animals that were treated with LTBR-Ig a week before the scrapie inoculation showed no prion infectivity in their spleens at 3 weeks. If they waited a week after inoculation to start treatment with LTBR-Ig they had a decline in the infectivity of the spleen. This suggests that this immunoglobulin interferes with the propagation of the prion protein in the periphery. This study suggests that treating with the LTBR-Ig before inoculation can potentially prevent disease, given after inoculation can ameliorate the incubation time but not the disease process.

This holds some hope that an agent can be developed which will interfere with development of the FDCs could prevent or delay the progression of vCJD.

Quinacrine can get into brain tissue. Korth et al showed a reduction in the accumulation of the abnormal prion proteins in prion-infected cells treated with quinacrine. Given this data they are conducting a clinical trial based on this study.

In summary, prion disease is a new area of study and is clearly an emerging disease problem. Changes in the meat and bone meal production likely contributed to the appearance of BSE and subsequent development of vCJD in humans. The transmissibility of vCJD is still being explored and as yet no good definitive treatment is available. BSE surveillance systems are in place in most developed countries and as yet we have not seen a case of BSE or vCJD in the United States.

Study Questions:

Define prion.
What is PrP-res?
What are 3 categories of human transmissible spongiform encephalopathy?