Physical And Chemical Characteristics Of Amyloidosis Notes

Amyloidosis

Amyloidosis is the term used for a group of diseases characterized by the extracellular deposition of a fibrillar insoluble proteinaceous substance called amyloid which has a common morphological appearance, staining properties, and physical structure but has variable protein (or biochemical) composition.

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Amyloid is a nineteenth-century term but the material has been more fully characterized and defined in the twentieth century. First described by Rokitansky in 1842, the substance was subsequently named by

• Virchow as ‘amyloid’ under the mistaken belief that the material was starch-like (amylin = starch). This property was demonstrable grossly on the cut surface of an organ containing amyloid which stained brown with iodine and turned violet on the addition of dilute sulfuric acid.
• By H and E staining under light microscopy, amyloid appears as an extracellular, homogeneous, structureless, and eosinophilic hyaline-like material.
• However, its confirmatory staining property is congophilia (positive with Congo red staining), followed by examination under polarising microscopy showing apple-green birefringence.
• The nomenclature of different forms of amyloid is done by putting the alphabet A (A for amyloid), followed by the suffix derived from the name of specific protein constituting amyloid of that type for example, AL (A for amyloid, L for light chain-derived), AA, ATTR, etc.

Physical And Chemical Characteristics:

Physical appearance by ultrastructural examination and chemical analysis for the study of its composition reveals the complex nature of amyloid.

It emerges that based on morphology and physical characteristics, all forms of amyloid are similar in appearance, but they are chemically heterogeneous. Based on these characteristics, amyloid is composed of 2 main types of complex proteins:

1. Fibril proteins comprise about 95% of amyloid.
2. Non-fibrillar components which include P-component predominantly; besides, there are several other different proteins which together constitute the remaining 5% of amyloid.

1.  Fibril Proteins:

By electron microscopy, it became apparent that the major component of all forms of amyloid (about 95%) consists of a meshwork of fibril proteins.

• These consist of delicate, randomly dispersed, nonbranching amyloid fibers having 4-6 fibrils, each measuring 7.5-10 nm in diameter and having an indefinite length.
• These fibrils are wound on each other and are separated by a clear space that contains regularly placed binding sites for Congo red dye.
• By X-ray crystallography and infrared spectroscopy, the fibrils are shown to have a cross-β-pleated sheet configuration which produces 1000 A° periodicity.
• These properties give amyloid its characteristic staining properties with Congo red dye and birefringence under polarising microscopy.

Based on these properties, Glenner gave amyloid an alternate name for β-fibrillosis.

Chemical analysis of fibril proteins of amyloid reveals the heterogeneous nature of amyloid.

Chemically, two major forms of amyloid fibril proteins were first identified in the 1970s:

1. AL (amyloid light chain) protein and
2. AA (amyloid associated) protein.

However, currently over 20 other biochemically different fibrillar proteins have been found in humans in different clinicopathologic settings:

AL Fibril Protein: Al amyloid fibril protein is derived commonly from the immunoglobulin light chain, which may be a complete light chain or may include an amino-terminal segment and part of the C region of the immunoglobulin light chain.

• AL fibril protein is more frequently derived from the lambda (λ) light chain (twice more common) than kappa (κ).
• However, in any given case, there is amino acid sequence homology. Rarely, a heavy chain of immunoglobulin may be associated with AL fibril protein.
• AL type of fibril protein is produced by immunoglobulin-secreting plasma cells.
• It is, therefore, seen in association with plasma cell dyscrasias and is included in primary systemic amyloidosis.

AA Fibril Protein: AA fibril protein is composed of protein with a molecular weight of 8.5- kD which is derived from a larger precursor protein in the serum called SAA (serum amyloid-associated protein) with a molecular weight of 12.5-kd.

• Unlike AL amyloid, the deposits of AA amyloid do not have sequence homology.
• In the plasma, SAA circulates in association with
• HDL3 (high-density lipoprotein). SAA is an acute phase reactant protein synthesized in the liver, in response to chronic inflammatory and traumatic conditions, and thus the level of SAA is high in these conditions.
• AA fibril protein is found in secondary systemic amyloidosis.
• Thus, it is seen secondary to several examples of chronic infections, autoimmune inflammatory diseases, and disseminated malignancies.

Other Fibril Proteins:

Apart from the two major forms of amyloid fibril proteins, a few other forms of proteins are found in different clinical settings:

Transthyretin (TTR):

It is a serum protein synthesized in the liver and normally transports thyroxine and retinol (trans-thy-retin).

• Single amino acid substitution mutations in the structure of TTR result in a variant form of protein that is responsible for this form of amyloidosis termed ATTR.
• ATTR is the most common form of heredofamilial amyloidosis seen in familial amyloid polyneuropathies.
• However, the deposit of ATTR in the elderly in the heart (senile cardiac amyloidosis) consists of normal TTR without any mutation.

Another interesting aspect of ATTR is that despite being inherited, the disease appears in middle age or elderly.

• Aβ2-microglobulin (Aβ2M): This form of amyloid is seen in cases of long-term hemodialysis (for 10-12 years). As the name suggests, β2M is a small protein that is a normal component of major histocompatibility complex (MHC) class I and has a β-pleated sheet structure.
• β2M is an 11.8 kDa protein that is not filtered by the hemodialysis membrane and thus there is a high serum concentration of β2M protein in these patients. Although the deposit due to Aβ2M may be systemic in distribution, it has a predilection for bones and joints.
•  Amyloid β-peptide (Aβ): Aβ is distinct from Aβ2M and is deposited in cerebral amyloid angiopathy and neurofibrillary tangles in Alzheimer’s disease. Aβ is derived from amyloid beta precursor protein (AβPP). The latter is a cell surface protein having a single transmembranous domain that functions as a receptor.
• The Aβ portion of this protein is seen extending into the extracellular region. Out of three intramembranous cleavage sites—secretase α, β, and γ, partial proteolysis of AβPP due to cleavage of β-secretase and γ-secretase sites generates Aβ i.e. amyloidogenic protein in Alzheimer’s disease.

 Endocrine amyloid from hormone precursor proteins:

It includes examples such as amyloid derived from pro-calcitonin (ACal), islet amyloid polypeptide (AIAPP, amylin), proinsulin (AIns), prolactin (APro), etc.

Amyloid of prion protein (APrP):

It is derived from precursor prion protein which is a plasma membrane glycoprotein. Prion proteins are proteinaceous infectious particles lacking in RNA or DNA. Amyloid in prionosis occurs due to an abnormally folded isoform of the PrP.

2. Non-fibrillar Components:

Non-fibrillar components comprise about 5% of the amyloid material.

These components include the following:

• Amyloid P (AP)-component: It is synthesized in the liver and is present in all types of amyloid. It is derived from circulating serum amyloid P-component, a glycoprotein resembling the normal serum α1-glycoprotein, and is PAS-positive.
• It is structurally related to C-reactive protein, an acute phase reactant, but is not similar to it. By electron microscopy, it has a pentagonal profile (P-component) or doughnut shape with an external diameter of 9 nm and an internal diameter of 4 nm.
• Apolipoprotein-E (apoE): It is a regulator of lipoprotein metabolism and is found in all types of amyloid. One allele, apoE4, increases the risk of Alzheimer’s precursor protein (APP) deposition in Alzheimer’s disease but not in all other types of amyloid deposits.
• Sulfated glycosaminoglycans (GAGs): These are constituents of matrix proteins; particularly associated is heparin sulfate in all types of tissue amyloid.

Other components:

Besides the above, other components such as α-1 anti-chymotrypsin, protein X, components of complement, proteases, and membrane constituents may be seen in the amyloid deposits.

Classification Of Amyloidosis:

Over the years, amyloidosis has been classified in several ways:

Based on the cause:  Into primary amyloidosis (due to plasma cell dyscrasias and the deposition occurring as a part of the disease itself) and secondary amyloidosis (as a complication of certain underlying known diseases).

• Based on the extent of amyloid deposition:  Into systemic (generalized) involving multiple organs and localized amyloidosis involving one or two organs or sites.
• Based on clinical location, into pattern 1 (involving the tongue, heart, bowel, skeletal and smooth muscle, skin, and nerves), pattern 2 (principally involving liver, spleen, kidney, and adrenals), and mixed pattern (involving sites of both pattern I and II).
• Based on precursor biochemical proteins:  Into specific type of serum amyloid proteins. With knowledge of the biochemical composition of various forms of amyloid and diverse clinical settings in which specific biochemical forms of amyloid are deposited, clinicopathologic classification of amyloidosis is widely accepted.
• According to this classification, amyloidosis is divided into 2 broad groups: systemic and localized, each having further subtypes depending upon clinical settings and the type of amyloid proteins. These types are discussed below.

Systemic Amyloidosis:

1. Primary Systemic (AL) Amyloidosis:

Primary amyloidosis consisting of AL fibril proteins is systemic or generalized in distribution.

• About 30% of cases of AL amyloid have some form of plasma cell dyscrasias, most commonly multiple myeloma (in about 15-20% cases), and less often other monoclonal gammopathies such as Waldenström’s macroglobulinemia, heavy chain disease, solitary plasmacytoma, and B cell lymphoma.
• The neoplastic plasma cells usually are a single clone and, therefore, produce the same type of immunoglobulin light chain or part of the light chain.
• Almost all cases of multiple myeloma have either λ or κ light chains in the serum and are excreted in the urine as BenceJones proteins.
• However, in contrast to normal or myeloma light chains, AL is twice more frequently derived from λ light chains.

The remaining 70% of cases of AL amyloid do not have evident B-cell proliferative disorder or any other associated diseases and are cases of true ‘primary’ (idiopathic) amyloidosis.

• However, by more sensitive methods, some plasma cell abnormalities are detected in virtually all patients with AL.
• The majority of these cases too have a single type of abnormal immunoglobulin in their serum (monoclonal).
• These patients also have some degree of plasmacytosis in the bone marrow, suggesting the origin of AL amyloid from precursor plasma cells.
• AL amyloid is a more prevalent type of systemic amyloidosis in North America and Europe and is seen in individuals past the age of 40 years. It is a rapidly progressive disease if not treated.
• Primary amyloidosis is often severe in the heart, kidney, bowel, skin, peripheral nerves, respiratory tract, skeletal muscle, and tongue (macroglossia).
• Treatment of AL amyloid is targeted at reducing the underlying clonal expansion of plasma cells. Median survival with no treatment after diagnosis is about one year.

2. Secondary/Reactive (AA) Systemic :

Amyloidosis The second form of systemic or generalized amyloidosis is reactive, inflammatory, or secondary, in which the fibril proteins contain AA amyloid.

• Secondary or reactive amyloidosis occurs typically as a complication of chronic infections (for example Tuberculosis, bronchiectasis, chronic osteomyelitis, chronic pyelonephritis, leprosy, and chronic skin infections).
• Non-infectious chronic inflammatory conditions associated with tissue destruction (for example.,  Autoimmune disorders such as rheumatoid arthritis, lupus, inflammatory bowel disease), some tumors (for example, Renal cell carcinoma, Hodgkin’s disease), and in familial Mediterranean fever, an inherited disorder (discussed below).
• Secondary amyloidosis is typically distributed in solid abdominal viscera like the kidney, liver, spleen, and adrenals.
• Secondary reactive amyloidosis is seen less frequently in developed countries due to containment of infections before they become chronic but this is the more prevalent type of amyloidosis in underdeveloped and developing countries of the world.

Classification of amyloidosis:

Secondary systemic amyloidosis can occur at any age and is the only form of amyloid that can also occur in children. Treatment lies in treating the underlying infectious or inflammatory disorder resulting in the lowering of SAA protein in the blood.

The contrasting features of primary and secondary systemic amyloidosis are.

3. Haemodialysis-Associated (Aβ2M) Amyloidosis:

Patients on long-term dialysis for more than 10 years for chronic renal failure may develop systemic amyloidosis derived from β2-microglobulin which is a normal component of MHC.

• The amyloid is deposited preferentially in the vessel walls at the synovium, joints, tendon sheaths, and subchondral bones.
• Carpal tunnel syndrome (median nerve compression presenting with numbness and tingling of the hand) is a common presentation.
• However, systemic distribution may also be observed in cases having bulky visceral deposits of amyloid.
• Cessation of dialysis after renal transplant causes symptomatic improvement.

4. Heredofamilial Amyloidosis:

A few rare examples of genetically determined amyloidosis having familial predisposition and seen in certain geographic regions have been described as under:

• Hereditary polyneuropathic (ATTR) amyloidosis:  This is an autosomal dominant disorder in which amyloid is deposited in the peripheral and autonomic nerves resulting in muscular weakness, pain, and paraesthesia, or may have cardiomyopathy.
• This type of amyloid is derived from transthyretin (ATTR) with single amino acid substitution in the structure of TTR; about 60 types of such mutations have been described. Though hereditary, the condition appears well past middle life.
• Amyloid in familial Mediterranean fever (AA): This is an autosomal recessive disease and is seen in the Mediterranean region (i.e. people residing in the countries surrounding the Mediterranean Sea for example, Sephardic Jews, Armenians, Arabs, and Turks).
• The condition is characterized by periodic attacks of fever and polyserositis i.e. inflammatory involvement of the pleura, peritoneum, and synovium causing pain in the chest, abdomen, and joints respectively.
• Amyloidosis occurring in these cases is AA type, suggesting a relationship to secondary amyloidosis due to chronic inflammation. The distribution of this form of heredofamilial amyloidosis is similar to that of secondary amyloidosis.
• Rare hereditary forms:  Heredofamilial mutations of several normal proteins have been reported for example, Apolipoprotein I (AApoAI), gelsolin (AGel), lysozyme (ALys), fibrinogen α-chain (AFib), cystatin C (ACys) and amyloid of familial dementia, etc. These types may also result in systemic amyloidosis.

Localized Amyloidosis:

1. Senile cardiac amyloidosis (ATTR):

Senile cardiac amyloidosis is seen in 50% of people above the age of 70 years. The deposits are seen in the heart and aorta. The type of amyloid in these cases is ATTR but without any change in the protein structure of TTR.

2. Senile cerebral amyloidosis (Aβ, APrP):

A senile cerebral amyloidosis is a heterogeneous group of amyloid deposition of varying etiologies that includes sporadic, familial, hereditary, and infectious.

Some of the important diseases associated with cerebral amyloidosis and the corresponding amyloid proteins are:

• Alzheimer’s disease (Aβ),  Down’s syndrome (Aβ), and transmissible spongiform encephalopathies (APrP) such as Creutzfeldt-Jakob disease, fatal familial insomnia, mad cow disease, kuru.
• In Alzheimer’s disease, a deposit of amyloid is seen as Congophilic angiopathy (amyloid material in the walls of cerebral blood vessels), neurofibrillary tangles, and in senile plaques.

3. Endocrine amyloidosis (Hormone precursors):

Some endocrine lesions are associated with microscopic deposits of amyloid.

The examples are as follows:

• Medullary carcinoma of the thyroid (from procalcitonin i.e. ACal).
• Islet cell tumor of the pancreas (from islet amyloid polypeptide i.e. AIAPP or amylin).
• Type 2 diabetes mellitus (from pro-insulin, i.e. AIns).

Contrasting features of primary and secondary amyloidosis:

• Pituitary amyloid (from prolactin i.e. APro).
• Isolated atrial amyloid deposits (from atrial natriuretic factor i.e. AANF).
• Familial corneal amyloidosis (from lactoferrin i.e. ALac).

4. Localised tumor-forming amyloid (AL):

Sometimes, isolated tumor-like formations of amyloid deposits are seen for example, Lungs, Larynx, Skin, Urinary Bladder, Tongue, Eye, and isolated atrial amyloid. In most of these cases, the amyloid type is AL.

Pathogenesis Of Amyloidosis:

The earliest observation that amyloidosis developed in experimental animals who were injected repeatedly with antigens to raise antisera for human use led to the concept that amyloidogenesis was the result of immunologic mechanisms.

Therefore, the AL variety of amyloid protein was isolated first. Although now it is known that amyloidosis is multifactorial, its exact pathogenesis is still controversial. However, in general, amyloid pathogenesis can be explained by the following sequence:

1. Pool Of Excess Precursor Protein:

In response to stimuli in distinct clinical settings, all forms of amyloid have increased serum levels of specific precursor proteins. However, high serum levels of precursor protein alone do not lead to a specific type of amyloid.

Common precursor proteins elevated in blood in different types of amyloidosis are as under:

• Precursor protein for AL amyloid: The stimulus for deposition of AL amyloid is excessive production of either intact immunoglobulin or its component ( for example, λ light chain, or κ light chain; rarely heavy chains).
  • These are synthesized by plasma cells, B lymphocytes, or their precursors in various monoclonal gammopathies for example, multiple myeloma, B cell lymphoma, and other plasma cell dyscrasias.
  • Light chains may be excreted in the urine as Bence-Jones proteins.
• Precursor protein for AA amyloid In AA amyloid: There is a high level of SAA protein which is a high-density lipoprotein (HDL₃) that is synthesized by the liver in response to longstanding tissue destruction for example, In chronic infections, chronic autoimmune inflammatory diseases, disseminated cancers.
  • Hepatic synthesis of SAA occurs by cytokines, notably IL-1 and IL-6, released from activated macrophages in these clinical settings.
• Other precursor proteins for different amyloid types:   Similarly, in different types of amyloid listed in Table, there is an elevated serum level of specific amyloidogenic precursor protein for example
  • In ATTR amyloid seen in heredofamilial amyloid and senile cardiac amyloid, there is a raised level of TTR protein in the blood.
  • In Aβ2M amyloid seen in patients on long-term hemodialysis, there is a high level of β2- microglobulin.
  • In Aβ amyloid seen in Alzheimer’s disease, there is a rise in β-peptide precursor protein in the blood.
  • In amyloid of prionosis seen in transmissible spongiform encephalopathies, there is a rise in prion proteins in the blood.
  • In endocrine amyloid, there is a rise in corresponding hormone precursor protein in serum ( for example, Procalcitonin in medullary carcinoma thyroid, proinsulin in type diabetes mellitus).

2. Reversible Misfolding And Aggregation:

The basic concept of the amyloid hypothesis as per present understanding is the misfolding and aggregation of precursor proteins. In

In other words, amyloidosis is a protein misfolding disorder:

• Normally:  The corresponding precursor proteins are soluble and are normally folded. If certain protein fractions are improperly folded, they are degraded by proteolysis by proteases in the cell or are destroyed by macrophages.
• In amyloidosis: There is either an excess of normal precursor proteins or a mutant form of abnormal precursor proteins.
• In either case, excessive or mutated forms of precursor proteins undergo limited proteolysis initiated by activated macrophages and lead to improper folding or misfolding of precursor proteins.

The misfolded proteins form oligomers which further form larger fibrils. Aggregation of oligomers and fibrils form the β-sheeted configuration of amyloid which binds to the basement membranes at the target tissues.

3. Non-Fibrillar Components:

• As stated earlier, all forms of amyloid contain nonfibrillar P components; others are apoE, glycosaminoglycans, etc.
• These substances are integrated into fibrillar proteins and cause irreversibility of protein aggregation, providing stability and protection to fibril proteins from solubilization or further degradation.

Amyloidosis: Characteristics Classification, Pathogenesis:

Amyloidosis is an extracellular deposition of fibrillar insoluble proteinaceous substance amyloid which has a common morphological appearance, staining properties, and physical structure but has variable protein (or biochemical) composition.

• By H and E staining under light microscopy, all forms of amyloid appear as extracellular, homogeneous, eosinophilic hyaline-like material; it stains positive with Congo red staining and shows apple-green birefringence on polarising microscopy.
• Biochemically, fibril proteins comprise about 95% of amyloid while non-fibrillar components constitute the remaining 5% of amyloid.
• Fibril proteins are predominantly of two types: AL (primary amyloid in association with plasma cell and B cell proliferative disorders) and AA (secondary amyloid seen in chronic infections, chronic inflammatory diseases, and advanced cancers)
• Others are transthyretin or ATTR (in heredofamilial forms), Aβ2-microglobulin or Aβ2M (in patients on long-term hemodialysis), amyloid β-peptide or Aβ (in Alzheimer’s disease), endocrine amyloid from hormone precursor proteins (in type 2 diabetes, medullary carcinoma thyroid, etc.) and amyloid of prion protein or APrP.
• Non-fibrillar components consist mainly of the P components seen in all forms of amyloid; others are apolipoprotein-E, sulfated glycosaminoglycans, etc.
• Pathogenesis of amyloid involves a rise in the level of a precursor of fibrillary protein (AL in primary and SAA in secondary form) followed by its limited proteolysis in the cell or in reticuloendothelial cells.
• This forms misfolded aggregates of fibrillary proteins which are prevented from further degradation by integration of non-fibrillary components.