MMI 445 final

what is so unique about prion disease?

what is so unique about prion disease?

can develop spontaneously

can be inherited

can be acquired

UV resistant

no nucleic acid

what is the normal prion protein vs prion protein associated with disease?

normal: PrPsen or PrPC; 209 aa, can be digested by proteinase K, soluble in detergents, used in adhesion, metal binding

disease: PrPres; 209 aa, proteinase K can only digest the N terminus, forms insoluble aggregates, found in nervous system and lymphoid tissues

main difference is conformational

can both be di,mono, or unglycosylated

what is the conformation of PrPC?

3 alpha helices

2 small anti-parallel beta strands

sensitive to proteinase K

what is the conformation of PrPsc?

loses alpha content

increased beta sheets

resistant to proteinase K (cleaves at residue 90)

what 4 techniques are used to visualize prions?

1.) electron microscopy

2.) atomic force microscopy

3.) X-ray diffraction

4.) cryo-electron microscopy

*when PrPc becomes PrPsc, there tends to be a stacked conformation and unfolded paper clip appearance taken on with the infectious type

what are the 3 steps of the prion lifecycle?

1.) conversion: domino cascade

2.) accumulation

3.) spread: to other cells in brain

what is the end stage pathology of prion disease?

spongiform change

neuronal loss

astrocytosis

no immune response, as these are self antigens

what is the incidence of prion disease?

1-2 million cases per year

90% is sporadic CJD

10% genetic (familial CJD, FFI)

less than 1% acquired (kuru, variant CJD, iatrogenic)

what is sporadic CJD?

always fatal prion disease

causes ataxia, cortical visual symptoms, progressive dementia, myoclonus and akinetic mutism

average death after 4 months

definite diagnosis by neuropathological confirmation (after death)- typing of codon 129 (polymorphism of M or V)

mid 60’s average onset

what is the probable diagnostics of sporadic CJD?

rapid cognitive decline

at least 2 of the following: myoclonus, visual disturbances, ataxia, akinetic mutism

one of: EEG periodic discharges, MRI restricted diffusion, positive QuIC

what is real time quaking induced conversion?

incubate normal prion protein added to patient sample

if misfolded protein results, positive for prion disease

what are potential future targets for prion disease?

suppress PrPc

block conversion

improve clearance

contact with what region of a CJD patient would most likely cause transmission?

brain, spinal cord, dura mater, posterior eye segment, pituitary gland

what are the 2 common sources of iatrogenic CJD?

dura matter graft

growth hormone transplant

not transmitted sexually or vertically

no higher incidence in healthcare workers

what is varient CJD?

bovine to human transmission

-first round of cases had MM

-second round had MV

prions in brain and spinal cord

where are prions in chronic wasting disease found?

blood, saliva, urine, feces

why is it not likely for humans to acquire chronic wasting disease?

species barrier in prion proteins (shape)

potential for barrier to be crossed by adaptation through intermediate hosts, but hasn’t happend yet

lack of strain overlap

macaques cannot acquire the disease- more closely related to humans, squirrel monkeys can, mice recently have

what 2 human PRNP mutations may protect against CJD?

V127 and E219K

prevents misfolding into the infectious form

what are 3 types of genetic prion diseases?

1.) familial CJD

2.) PrP amyloidoses (GSS and peripheral amyloidosis)

3.) thalamic degeneration (FFI)

what is Gertmann-Straussler Scheinker?

heritable prion disease

early 50s onset

6 years

causes multicentric amyloid plaques

causes early ataxia, late cognitive decline, motor and parkinsonian features

what are the 4 different GSS clinical phenotypes?

1.) typical

2.) with areflexia ad paresthesia

3.) pure dementia

4.) CJD-like

what are the 4 different GSS neuropathological descriptions?

1.) parenchymal PrP amyloid and prominent spongiform changes

2.) parenchymal PrP amyloid and minimal/no spongiform changes

3.) parenchymal PrP amyloid coexisting with severe tau neurofibrillary pathology, and absent/minimal spongiform changes

4.) vascular PrP-cerebral amyloid angiopathy coexisting with severe tau neurofibrillary pathology

what is PrP amyloidoses?

can also cause peripheral syndromes or autonomic neuropathy

associated with nonsense mutations Q160X and Y163X

what is fatal familial insomnia?

heritable prion disease

progressive sleep distrubance, autonomic changes, tremor, dysarthia, ataxia, myoclonus, cognitive decline

1 year duration

atrophy of thalamus without spongiform change or amyloidosis

D178N in cis with M129

different disease with MM 129 vs MV 129

MRI and EEG can be normal

have to test for PRNP mutation

what is FFI disease presentation in MM 129 individuals?

shorter disease

sleep change, prominent myoclonus, autonomic dysfunction, spatial disorientation, hallucinations, weight loss

what is FFI disease presentation in MV 129 individuals?

longer disease

early visual symptoms and ataxia, later dysarthria, seizures, bulbar symptoms

what is sporadic fatal insomnia?

MM2T classification

more heterogeneity than FFI

only 29% have insomnia

major symptom is atypical parkinsonism

what is variably protease sensitive prionopathy?

small percentage of all sporadic prion diseases

transmissible to animals

majority are VV

can be equivalent of V180I familial CJD

older onset, longer duration

normal MRI and EEG

psychiatric, speech/language change, cognitive decline

causes 5-7 PrP bands on electrophloresis

what is a prion?

a protein that can be misfolded and self-template

aggregates can spread cell to cell

different conformations produce distinct phenotypes

misfolding leads to neurodegeneration

what are the 4 proteins in the prion family?

1.) amyloid beta (Abeta)

2.) Tau

3.) alpha-synuclein

4.) PrP

what prion proteins are associated with Alzheimers disease, Parkinsons, multiple systems atrophy, cortical basal degeneration?

AD: tau and Abeta

PD: alpha-synuclein

MSA: alpha-synuclein

CBD: Tau

where is MSA vs PD caused by alpha-synuclein commonly found?

MSA: glial

PD: neuronal

human MSA is transmissible when injected into mice, PD is not

why can prophylactic treatment to prion diseases not be administered?

would need to know the exact protein misfolded shape, there are too many different variations to do this

what are the 3 levels of host defense?

1.) barriers

2.) innate immunity

3.) adaptive immunity

what do TLR4, TLR7, TLR9 and RIG-1 recognize?

TLR3: dsRNA

TLR7: retroviral ssRNA

TLR9: unmethylated CpG dsDNA

RIG-1: abnormally terminated (PPP) on ssRNA

what does the IRF-3 vs the IFR-7 interferon system express?

IRF-3: IFN beta

IRF-7: IFN alpha and IFN beta

IFN beta is turned on first because IRF3 is always present but in its inactive form (needs to be phosphorylated)

IRF7 is an ISG product, IFN beta turns on ISG

what are features of orthomyxoviruses?

negative sense RNA

enveloped

helical nucleocapsid assembled in cytoplasm

influenza types A, B and C

respiratory transmission

infect upper and lower respiratory cells

what 3 obstacles must influenza pass to infect? How does it combat this?

1.) mucus

2.) cilia

3.) macrophages

use high viral load to overwhelm the immune system

what 2 surface glycoproteins are characteristic of influenza A?

1.) hemagglutinin: binds sialic acid sugars on surface of epithelial cells; 16 subtypes

2.) neuraminidase: detaches newly formed virions from host cell; 9 subtypes

what are 3 strategies to prevent interferon action?

1.) prevent IFN production (influenza)

2.) prevent IFN from reaching its target

3.) prevent action of anti-viral proteins

How does influenza avoid PRRs?

NS1 binds dsRNA to mask it from TLR3

NS1 also binds RIG-1 to prevent it from signaling a 5’ triphosphate

cap addition to 5’ end that releases the triphosphate

what is the innate immune response to influenza?

NS1 is good at suppressing signaling systems

full IFN response not seen until 2 days (8 rounds of replication occured before IFN response)

asymptomatic for 2 days but most contagious

what is the adaptive immune response to influenza?

1 week

all virus particles eliminated by 2 weeks

virus specific killer T cells

antibodies against glycoproteins lead to protective response

what is antigenic drift in influenza?

antigenic change by point mutation in HA or NA

due to no proofreading abilites

what is antigenic shift in influenza?

antigenic changes from re-assortment of gene segments

ex.) combining humans and animal subtypes

what are 3 requirements for a pandemic flu?

1.) new virus subtype

2.) infects humans and causes serious disease

3.) spreads easily and is sustainable

what are the 3 common antivirals used to treat influenza?

1.) amantadines

2.) oseltamivir

3.) zanamivir

what are 3 ways to diagnose respiratory viruses?

1.) rapid diagnotics using viral antigens

2.) cultures

3.) molecular methods (nucleic acid amplification tests)

what is the morphology of human papillomavirus?

small non-enveloped dsDNA

circular genome

100 different genotypes, differing in major capsid protein

13 are oncogenic

how is HPV transmitted?

sexually

what are HPV target cells?

attacks basal cells near basement membrane through cracks or tears of the skin

virions are shed from the top layer of mucosal surface

no capsid or viral particles are made in the basal cells

what is the role of HPV E7 and E6:

E6: binds P53 to prevent cell detecting abnormal replication, prevents apoptosis

E7: binds pRB and promotes DNA replication

how is the HPV genome organized?

early genes: produced in basal cells

late genes: capsid proteins, produced in maturing cells

how does HPV enter host?

L1 protein binds heparin sulfate on cell causing a conformational change

L1 then binds an unknown receptor which initiates endocytosis

L1/L2 undergo conformational changes decreasing affinity for primary receptors leading to membrane pitting and invagination

endocytosis independent of other proteins

what does HPV do once inside host cells?

capsid is partially removed (L1 gone, L2 stays)

genome replicated in host nucleus, all early genes

host polymerase synthesizes mRNA which remains episomal

L2 capsid formed first, then L1

how does transcription, translation and synthesis occur in DNA viruses?

genome is template for host polymerases to make + RNA

genome gets coated in protein capsid and virus leaves nucleus and exist cell

what are features of genital warts caused by HPV?

types 6 and 11

flat lesions, small cauliflower like bumps or stem like protrusions

rarely painful, but are itchy

what are features of common warts caused by HPV?

types 1 and 2

rough raised bumps on hands, fingers, elbows

may be painful

what are features of plantar warts caused by HPV?

hard, grainy growths on heels of balls of feet

may be uncomfortable

what are features of flat warts caused by HPV?

flat topped, sightly raised and darker than normal skin

children normally get them on the face, men in beard area, women on legs

what are 7 common regions HPV causes cancer?

1.) cervix

2.) oropharynx

3.) anus

4.) rectum

5.) vagina

6.) penis

7.) vulva

caused by types 16 and 18

what are the 2 classifications of cancer HPV causes?

infection of squamous cells: squamous cell carcinoma

infection of glands in cervix: adenocarcinomas

how are the majority of HPV infections cleared?

within a year

due to episomal DNA and continual basal cell proliferation, some daughter cells will receive all DNA, while others will receive none

viral genome eventually lost

immune system is very good at clearing infection (killer T cells are specific though)

how does HPV use a quiet lifecycle to avoid immune detection?

infects basal cells with minimal replication, maintains genome

increases replication after cell differentiation to exit from top layer and make immune system fight infection from the initial location, where it is no longer present

how does HPV resist detergents?

capsid

withstands detergents in condoms that can destroy HIV and HSV

how was an HPV vaccine developed?

L1 expression produced virus like particles which resemble HPV virions but hold no viral DNA

what are morphological features of toxoplasma gondii?

apicomplexan parasite

3 lineages

3 life stages

tachyzoites and bradyzoites and asexual

oocytes are sexual and produced in definitive host cat

humans are dead end hosts

colonization for life with tissue cysts

what is horizontal transmission of toxoplasma gondii?

intermediate host ingests sporulated oocysts from contaminated food or water which become tachyzoites which rapidly divide in macrophages

oocysts can remain active in soil for a year

cat ingests tissue cysts where oocysts form in the intestine, sporozoites develop in oocysts and get deposited in feces

what is vertical transmission of toxoplasma gondii?

mother to fetus by placental transfer of tachyzoites

causes congenital toxoplasmosis

what are 3 ways humans can get infected with toxoplasma gondii?

1.) ingest tissue cysts with bradyzoites in undercooked meat

2.) fecal oral ingestion of oocysts

3.) blood transfusions or organ transplants

what are the 3 risk factors associated with getting toxoplasma?

1.)sociodemographic

2.) biological

3.) lifestyle

what are 4 complications of toxoplasmosis?

1.) encephalitis

2.) myocarditis

3.) hepatitis

4.) pneumonia

what is the predominant symptom of toxoplasmosis in immunocompetent people?

cervical lymphadenopathy

short self-limiting infections

what is the symptoms of toxoplamosis in HIV patients?

increased chance of toxo encephalitis and eye infections

usually occurs from reactivation due to tissue cyst rupture in CNS

what is the symptoms of toxoplasmosis in solid organ transplant patients?

increase in CNS or pulmonary toxo

usually from reactivation

clinically similar to AIDS patients in brain, heart and lungs with fever presentation

3 months after transplant

rare

what is congenital toxoplasmosis?

acute maternal infection leads to tachyzoites in blood going to fetus

difficult to diagnose mom

transmission risk increases with increase gestation time

most severe effects in early gestation

neurological and intellectual sequelae can occur after birth with infection at late infection

what is the prenatal treatment for toxoplasmosis?

spiramycin (a parasitostatic)

pyrimethamine-sulfonamide (a parasitocidal that’s effective on tachyzoites)

what are the 3 direct detection methods for toxoplasmosis?

1.) detect tachyzoites or tissue cysts in body fluids or tissues

2.) enzyme immunoassay for toxoplasma antibodies

3.) detection of parasite DNA in body fluids or tissue samples

what are the 2 types of serological diagnosis methods for toxoplasmosis?

1.) acute infection IgM positive or serial specimans

2.) EIA sensitive and specific ( gold star)

what is the pathogenesis of toxoplasma?

invades all cell types

can form tissue cysts for protection from immune system

invasive at any cell cycle stage

if infection occurs in macrophage it will phagocytose into a phagosome which fuses with a lysosome and kill parasite

infection in other cells from entering a vacuole from plasma membrane invagination which doesn’t fuse with lysosome and doesn’t acidify

no flagella

attach via apical complex

what are 3 special structures in apicomplexans?

1.) micronemes

2.) rhoptries

3.) dense granules

what are micronemes?

made of many different proteins

50-100 per apical portion

function in cell-cell adhesion and motility

what are rhoptries?

club shaped transmembrane proteins

6-12 per parasite

secretion during host cell invasion

what are dense granules?

constitutive secretion

what are 4 changes in the host cell following infection of toxoplasma?

1.) facilitate parasite nutrient acquisition

2.) enhance host cell survival

3.) avoid strong immune response allowing infection to continue

4.) tissue cysts are a mechanism for parasite to avoid immune interference

what is the manipulation hypothesis of toxoplasmosis?

parasite changes organisms behaviour to benefit further survival and transmission of parasite

-increasing chances for entering cat

what are the host parasite interactions of toxoplasma?

obligate intracellular parasite

neutrophils have early role in killing parasite

inactive macrophages

infected cells resist apoptosis

need glucose, iron, arginine, ornithine, cholesterol, calcium

can sequester lysosome to acquire nutrients

calcium acquired from host cell cytosol

how do host cells deal with toxoplasma infection?

growth is dependent on tryptophan derived from arginine

NK or T cells produce IFN-gamma to stimulate fibroblasts to degrade arginine

also produce antibodies

MHC 1 critical

what vaccines are available for toxoplasma?

animal vaccination to reduce number of tissue cysts in sheep and goats

no human vaccination, but adult acquired toxo leads to lifelong immunity which prevents reactivation of bradyzoites and congenital transmission

what is the cause of malaria?

plasmodium

phylum apicomplexa

what are the 3 life stages of apicomplexa?

1.) shizogany (in human)

2.) gamogony (in human)

3.) sporogony (in vector)

what is the plasmodium lifecycle in humans?

sporozoites infect liver hepatocytes and undergo asexual reproduction

merozoites released from liver travel in blood and infect RBCs

become trophozoite and go back to merozoites to burst from RBC

some become gametocytes and get uptaken by vector

what is the plasmodium lifecycle in vector?

gametocytes form zygote and become ookinete

become sporozoite and travel to salivary glands to be transmitted to human

what is a hypnozoite?

dormant form of plasmodium in the liver

only in P. vivax and P. ovale

sporozoites that don’t immediately undergo shizogany

initiate reactivation after weeks or months

what are the symptoms of malaria?

non-specific prodromal symptoms

malaria febrile paroxysms: cold stage (30min to 1 hour), hot stage (2-6 hours), sweating stage (1-4 hours)

how does the time of febrile cycles differ between malaria species?

24 hours: falciparum

48 hours: vivax, ovale, knowlesi

72 hours: malariae

what are the 4 causes of anemia associated with malaria?

1.) RBC rupture

2.) hemolysis from autoantibody

3.) phagocytosis

4.) RBC production suspended in bone marrow

how do the age of RBCs infected differ by malaria species?

Falciparum: all RBC types

malariae: senescent RBCs (old)

vivax, ovale and knowlesi: reticulocyte (young)

what is hyperreactive malarial splenomegaly syndrome?

exaggerated immune response

excessive removal of infected RBCs from circulation in spleen causing inflammation

what are 3 complications of p. falicparum?

1.) cerebral malaria

2.) blackwater fever

3.) severe anemia