General Pathology

AmyloidosisFibrinoid, Hyalin

Institute of Pathology, 1st Faculty of Medicine, Charles University, Prague

Jaroslava Dušková

Jaroslava Duško

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Amyloidosis, fibrinoid and hyalin –

disturbances of protein metabolism - contents

Amyloid

definition and history

morphology of amyloid

Amyloidoses – classification

systemic amyloidoses and

their complications

localized amyloidoses and

their complications

Diagnosis of amyloid –

clinical and morphological

Reversibility & therapy of

amyloid

Fibrinoid change of collagen

definiton

morphology of fibrinoid

change of collagen

complications, clinical

importance

Hyalin

definition

morphology

complications, clinical

importance

Jaroslava Duško

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AmyloidosisDEF.:

disorder of protein

metabolism accompanied

with abnormal extracellular

deposition of proteinaceous

material - amyloid

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Amyloid = starch likemisfolded PROTEIN with abundant

proteoglycans and glycosaminoglycans

Karl Freiherr von Rokitansky (1804-1878)

Rudolf Ludwig Karl Virchow (1821-1902).

Amyloid - nature - history

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Karl Freiherr von

Rokitansky (1804-1878)

Austrian pathologist, born February 19, 1804, Königgratz, Böhmen, Austrian Empire (now Hradec Králové, East Bohemia, Czech Republic);

died July 23, 1878, Wien.

Handbuch der

pathologischen

Anatomie IInd Band,

Wien 1842

1st description of amyloid

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The Vienna School of Pathology

(& Czech Specialists)

Karl Rokitansky, Josef Škoda

-signatures from Prague´s Institute of Pathology memorial book

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German anatomist and

pathologist,

Rudolf Ludwig Karl

Virchow (1821-1902).

Virchow´s macroscopy

reactions for amyloidJarosla

va Dušková

Amyloidosis – morphology

Macroscopy:

small amounts – invisible

larger deposits – enlarged,

firm, waxy organs

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Amyloidosis renis

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Virchow I

JJK

Virchow II

H2SO4

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Ultrastructure &

Biochemistry of Amyloid

90-95% non branched fibrils diam. 10-12nm

5-10% p-component - glycoprotein + fibronectin, laminin, collagen 4

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Jaroslava Duško

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Amyloidosis

conformational disease(Carrell and Lomas, Lancet, 1997)

„…arises when a constituent protein

undergoes a change in size or

fluctuation in shape with resultant

self - association and tissue

deposition“ pleated β – sheet structure

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Conformational diseases (Carrell and Lomas, Lancet, 1997)

Amyloidosis

Prionoses - transmissible spongiform

encephalopathies (incl. CJD)

m. Alzheimeri

pleated β – sheet structure

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Amyloidosis

Classification:

according to the source protein

(2015 - 31 different identified)

according to the distribution

systemic (generalised)

localised

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Systemic Amyloidosis - I.

AL - imunocyte dyscrasia associated

light chains Ig (mostly )

„primary“

Distribution: tongue, heart, GIT, liver, spleen, kidney

Associated diseases: Monoclonal Gammapathy of Uncertain Significance (MGUS), plasma cell myeloma, B cell lymphoma, …

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Pathogenesis of Amyloid

Marin-Argany M et al: Mayo clinic, Rochester, USA

Mutations can cause light chains to be too stable or

too unstable to form amyloid fibrils.Protein Sci. 2015 Aug 24. doi: 10.1002/pro.2790. [Epub ahead of print]

Light chain (AL) amyloidosis is an incurable (???jd) human

disease, where the amyloid precursor is a misfolding-prone

immunoglobulin light-chain.

Certain mutations either decrease (H32Y and H70D) or

increase (R65S and Q96Y) the protein thermal stability.

Interestingly, the most and the least stable mutants, Q96Y

and H32Y, do not form amyloid fibrils under physiological

conditions.

Within a thermal stability range, the most stable protein in

this study is the most amyloidogenic protein.

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Pathogenesis of Amyloid

Ryšavá R. 1st Fac. of Medicine, Charles Univ. Prague

AL amyloidosis: advances in diagnostics and treatment.Nephrol Dial Transplant. 2018 Oct 8.

doi: 10.1093/ndt/gfy291. [Epub ahead of print]

AL amyloidosis is a systemic disease which still has unsatisfactory survival of patients.

The monoclonal light chains kappa (κ) or lambda (λ) deposit and accumulate in different tissues.

Renal involvement is very frequent with the development of nephrotic syndrome and renal failure

Histologic confirmation is based on Congo red & polarized light detection of amyloid deposits

AL amyloidosis must be distinguished from other systemic forms of amyloidoses with renal

involvement, AA amyloidosis, amyloidosis with heavy chain deposition, fibrinogen Aα or ALECT2 (leukocyte chemotactic

factor 2) deposition.

Immunofluorescence (IF) plays a key role here.

Formalin-fixed paraffin-embedded tissue after protease digestion, immunohistochemistry or laser

microdissection with mass spectrometry should complete the diagnosis in unclear cases.

Standard treatment with melphalan and prednisolone or with cyclophosphamide and

dexamethasone has been replaced with newer drugs used for the treatment of multiple

myeloma-bortezomib, carfilzomib and ixazomib or thalidomide, lenalidomide and

pomalidomide. High-dose melphalan supported by autologous stem cell transplantation

remains the therapeutic option for patients with low-risk status.

These new treatment options prolong survival from months to years and improve the

prognosis in a majority of patients.

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Systemic Amyloidosis - II.

AA - reactive systemic amyloidosis

SAA = Serum Amyloid Associated

protein „secondary“

Distribution: liver, kidney, spleen, GIT, lymph nodes, bowel, adipose tissue

Associated diseases: rheumatoid arthritis, chronic

infections (tb, leprosy, bronchiectasiae,

osteomyelitis, IBD, neoplasms MLH , RCC…)

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Amyloidosis interspecies transmission

Westermark GT, Westermark P.

Serum amyloid A and protein AA: molecular mechanisms

of a transmissible amyloidosis. FEBS Lett. 2009;583:2685-2690.

Amyloidosis is experimentally

transmissible in mice and inter species

(enhancing factor - extract from tissues with amyloid)

Amyloidosis can be induced by food

AA amyloid is a part of human food

foie gras experiment: mice fed for 5 days

with extract developped systemic amyloidosis

within 8 weeks

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Systemic Amyloidosis - III.

Wild –type (senile) systemic/cardiac ATTR

25% people over the age of 80 years (!)

-normal (wild-type) transthyretin TTR (prealbumin)

-mostly heart & vessels involvement

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Systemic Amyloidosis - IV.

A2 - hemodialysis associated

2 microglobulin

Hereditary

AA - Familial Mediterranean Fever

ATTR - Famil. polyneuropatia

transthyretin (mutated form)

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Systemic Amyloidosis - complications

diminished functions of some organs, esp.

KIDNEY FAILURE

IIIrd stage Amyloid nephrosis

severe proteinuria - coag. disorders – infections, hyperlipidemia, lipiduria, edemas

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Localised Amyloidosis - I.

Senile cardialATTR - transthyretin -

(structurally normal)

Senile cerebral

A - -amyloid protein

(a part of amyloid precursor protein-APP = transmembrane glycoprotein)

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Cardiac Amyloidosis – clinical manifestations

Dilated Cardiomyopathy (predominant systolic dysfunction)

Restrictive cardiomyopathy (predominant diastolic dysfunction)

Congestive heart failure

Rhytm abnormalities

Coronary insufficiency

Valvular dysfunction

Pericardial tamponade

Enhanced sensitivity to digitalis glycosides

Atrial thrombosis - embolisation

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Cardiac Amyloidosis

11 out of 31 known amyloid proteins can be found in the heart

only one – ANF limited to the heart

clinically important: ATTR, AL– AL therapy: chemoth, transplantation of stem cells and

heart

– ATTR therapy: heart and liver transplantation

– farmacotherapy

Different depositions : endocardium & valves, myocardium, vessels, pericardium

Typing: – immuno /peroxidase /fluorescence /electron microscopy

– mass spectrometry-based proteomics with laser dissection of amyloid deposits from paraffin blocks

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Localised Amyloidosis - I.

Senile cardialATTR - transthyretin -

(structurally normal)

Senile cerebral

A - -amyloid protein

(a part of amyloid precursor protein-APP = transmembrane glycoprotein)

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Pathogenesis of Amyloid

Penke B, Bogár F, Fülöp L. University of Szeged, Hungary

β-Amyloid and the Pathomechanisms of Alzheimer's Disease:

A Comprehensive View.

Molecules. 2017 Oct 10; 22(10). pii: E1692. doi: 10.3390/molecules22101692.

Protein dyshomeostasis is the common mechanism of neurodegenerative diseases such as

Alzheimer's disease (AD).

Aging is the key risk factor,

The extensive and complex network of proteostasis declines during aging and is not able to maintain

the balance between production and disposal of proteins.

Different cellular stress conditions result in the up-regulation of the neurotrophic, neuroprotective

amyloid precursor protein (APP).

Enzymatic processing of APP may result in formation of toxic Aβ aggregates (β-amyloids).

Chronic cerebral hypoperfusion causes dysfunction of the blood-brain barrier (BBB), and thus the Aβ-

clearance from brain-to-blood decreases.

Microglia-mediated clearance of Aβ also declines, Aβ accumulates in the brain and causes

neuroinflammation.

Protein folding is the basis of life and death.

Recognition of the above mentioned complex pathogenesis pathway resulted in novel drug targets in

AD research.

Jaroslava Duško

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Autophagy of Brain Amyloid

Uddin MS, Stachowiak A, Mamun AA, Tzvetkov NT, Takeda S, Atanasov AG,

Bergantin LB, Abdel-Daim MM, Stankiewicz AM. International team of authors.

Autophagy and Alzheimer’s Disease: From Molecular

Mechanisms to Therapeutic Implications.Front Aging Neurosci. 2018 Jan 30;10:04. doi: 10.3389/fnagi.2018.00004. eCollection 2018.

Conclusion

Despite much of the data presented in the review being acquired in studies performed on

animal models, we propose that:

Properly functioning autophagy is crucial for the normal aging of neurons.

Malfunction in neuronal autophagy is one of the key factors influencing the development

of neurodegenerative disorders, including AD.

The autophagy plays a key role in the metabolism of Aβ and tau protein, the mTOR

pathway, neuroinflammation, and in the endocannabinoid system, all of which may

mediate its effect on AD.

Accordingly, autophagy-targeted therapeutic approaches may lead to the development of

novel therapeutic strategies for the management of AD.

Jaroslava Duško

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Localised Amyloidosis - II.

Endocrine

ACal - ca medullare gl. thyreoideae

AIAPP - islets of Langerhans associated

AANF - isolated atrial amyloidosis

atrial natriuretic polypeptide

Nodular tumoriform amyloid deposits(tongue, lung,larynx, skin, urinary bladder, orbita)

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Clinical Symptoms of Amyloid

1. nephrotic proteinuria (edemas)

2. weekness, fatigue, loss of weight,

collapses, heart failure,

cardiomyopathy

3. hepatomegaly

4. idiopatic peripheral neuropathy

(parestesias)

5. diarrhoea, cachexia (GIT)

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Clinical Diagnosis of Amyloid

Scintigraphy (in vivo)using human serum amyloid component

marked with 123J

Echocardiography (atrial amyloid)

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Clinical Diagnosis of Amyloid

Biochemistry

sequening DNA -hered. forms

extraction of fibrils (from a biopsy

specimen)

spectrometry

sequening of the amyloid protein

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Amyloidosis – morphology

Macroscopy:

small amounts – invisible

larger deposits – enlarged,

firm, waxy organs

Jaroslava Duško

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Jaroslava Duško

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Jaroslava Duško

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Jaroslava Duško

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Morphological

Diagnosis of Amyloid

Macroscopy

–reaction Virchow I (sol. Lugolli)

Virchow II (H2SO4)Jarosla

va Dušková

Virchow I

JJK

Virchow II

H2SO4

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Morphological

Diagnosis of Amyloid

Microscopy:

– KONGO red (+ sirius red, saturn red, direct red)

+POLARISATION!

– thioflavine S,T

– crystal. violet (metachromasia)

– IMMUNOHISTOCHEMISTRY

(electron microscopy)

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Morphological Diagnosis of Amyloid

Materials:

– GIT (stomach, duodenum rectum, gingiva) biopsy

– kidney

– sural nerve & muscle

– fat aspiration biopsy – needle with an internal

diam. 0,7-1,2mm

Röcken Ch. Sletten K.: Amyloid in Surgical Pathology

Virchows Arch., 2003, 1-26

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Struma amyloidea

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Amyloidosis renis

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CONGO Red synthesized by young chemist at Bayer comp. 1883 as

the first of economically lucrative direct (not needing a

mordant) textile dyes

patented by AGFA 1885

(AktienGeselschaft Für Anilinfarbenfabrikation)

3 weeks after the conclusion of the

West Africa Conference

to Europeans in 1885, the word Congo evoked exotic

images of far-off central Africa known as The Dark

Continent

the Congo red stain was named „Congo“ for marketing

purposes by a German textile dyestuff company in 1885

Steensma DP: „Congo“ Red. Out of Africa? Arch. Pathol.Lab.Med.,2001, 125, 250-2

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Prionosis - CJD

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Reversibility of Amyloid

The deposits are NOT irreversible. e.g. Hrncic R. et al: Antibody mediated

resolution of light chain – associated amyloid deposits. Am.J. Pathol., 2000, 157,12369-46

Progression of generalised amyloidosis can be delayed or stopped by treatment of the underlying disease.

Röcken Ch. Shakespeare Ann: Pathology, diagnosis and pathogenesis of AA amyloidosis. Virchows Arch. , 2002, 440, 11-122

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Prevention & Therapy of Amyloid

Prevention & treatment of the underlying diseases

Vaccination against β am. protein in mice diminished senile plaque formation and improved memory.

Nature Medicine, 2001, 7, 18th Jan.

A β –based experimental therapies based on degrading enzymes.

Zlokovic et al.: Neurovascular Pathways and Alzheimer

Amyloid β-peptide. Brain Pathol. , 2005, 15, 78-83

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Solomon A., Murphy Ch.L., Westermark P.:

Unreliability of

Immunohistochemistry for Typing

Amyloid Deposits„Because the treatment as well as prognoses of patients with

amyloidosis is dependent on the amyloid type, it is crucial that

the nature of the fibrillar protein be established unequivocally

to avoid inappropriate and costly therapy that can have dire

and possible legal consequences.“

Archives of Pathology and Laboratory Medicine,2008, vol.

132, No. 1, pp. 14–14.

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Amyloid Diagnosis Immunohistochemistry

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Amyloid- DiagnosisImmunofluorescence most sensitive

fresh tissue sometimes needed

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Fibrinoid & Hyalin

disorders of protein metabolism

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Fibrinoid Change of

Collagen vessels and connective tissue damage

plasmorrhagia (leakage of plasma)

deposits of Ag-AB complexes

staining characteristics fibrin - likeJaroslava Duško

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Hyaline change

Definition (historical, descriptive):

intra- or extracellular change

of homogenous rose „ glassy“ appearance

in the H&E stained histological sections

(gr. hyalon = glass)

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Hyaline change

Extracellular:

corpus albicans, scars, hyalinoses of

serous membranes

Intracellular:

Crooke cells, Mallory hyaline,

Russell bodies

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Keloid

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Perisplenitis cartilaginea

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Perisplenitis

cartilaginea

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Fibrous pleural plaque – marker of possible asbestosis!

Hyalinosis pleurae

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Hyalinosis pleuraeMesothelioma

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Ca bronchogenesHyalinosis et metatases

carcinomatosae pleurae

parietalis

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Ultrastructure

Fibrinoid - collagen fibres

surrounded by plasma

proteins may be reversible

Hyalin – collagen fibres

increased in thickness,

changed architecture rather

stable

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H

y

a

li

n

o

s

i

s

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Hyalinosis

endocardii

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Fibroelastosis

endocardii

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Silicosis gr. II

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Hyaline change

Extracellular:

corpus albicans, scars, hyalinoses of

serous membranes, valves, …

Intracellular:

Crooke cells, Mallory hyaline,

Russell bodies

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Mallory (alcoholic)

hyalineJarosla

va Dušková

Mallory (alcoholic) hyaline

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Russel bodies

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Russel bodies

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Crooke´ cells

PAS –OG ACTH

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Significance of Fibrinoid

Change

diminished quality of the collagen ( firmness, permeability)

tendency to thrombosis in the

vessels, aneurysms formationJaroslava Duško

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Significance of Hyalin

Change

diminished quality of the

collagen ( elasticity)

ischemia in organs with

thickened arterial walls

intracellular - function, death

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