<p>The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific</p><p>in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery.</p><p>In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases.</p> / <p>Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.</p>

Immunoglobulin-mediated inhibition of amyloid fibril formation in vivo is a promising strategy for the treatment of protein misfolding diseases such as the amyloidoses. Here we focus on transthyretin amyloidoses, a group of protein conformation diseases caused by the misfolding of the serum protein transthyretin into fibrillar structures that deposit in specific organs and tissues—often with serious pathological consequences. Using a structure-guided immunological approach, we report a novel antibody that selectively recognizes monomeric, misfolded conformations of transthyretin in vitro. Raised to an epitope normally buried in the native form of transthyretin, this antibody was found to suppress transthyretin fibrillogenesis at substoichiometric concentrations in vitro. Overall, the selectivity and inhibitory nature of the antibody signals the potential use of conformation specific antibodies in the diagnosis and treatment of transthyretin amyloidoses, conditions which remain difficult to treat and are widely under/misdiagnosed at the current time.

Immunoglobulin-mediated inhibition of amyloid fibril formation in vivo is a promising strategy for the treatment of protein misfolding diseases such as the amyloidoses. Here we focus on transthyretin amyloidoses, a group of protein conformation diseases caused by the misfolding of the serum protein transthyretin into fibrillar structures that deposit in specific organs and tissues—often with serious pathological consequences. Using a structure-guided immunological approach, we report a novel antibody that selectively recognizes monomeric, misfolded conformations of transthyretin in vitro. Raised to an epitope normally buried in the native form of transthyretin, this antibody was found to suppress transthyretin fibrillogenesis at substoichiometric concentrations in vitro. Overall, the selectivity and inhibitory nature of the antibody signals the potential use of conformation specific antibodies in the diagnosis and treatment of transthyretin amyloidoses, conditions which remain difficult to treat and are widely under/misdiagnosed at the current time.

Immunoglobulin (Ig) proteins, also known as antibodies, are important molecules with great variability in their amino acid sequences. Aberrantly overproduced monoclonal Ig light chain (LC) proteins may aggregate into a β-sheet featured structure, and deposit in the extracellular space; this pathologic process, called primary amyloidosis or Ig LC amyloidosis (AL) causes problems to multiple organs during the course of the disease. Post-translational modifications (PTMs), which remain to be explored, are likely an important factor affecting the formation of AL fibrils. In addition, therapeutic monoclonal antibodies (mAbs) are widely employed because of their high specificity and low side effects. Using plants as the expression platform is commercially attractive although this approach has been hampered by low protein expression yield and undesired glycosylation patterns. The investigations detailed in this dissertation focus on the analyses of Ig proteins derived from several human and plant sources. A method combining 2D SDS-PAGE separation and mass spectrometry (MS) analysis was used for de novo sequencing of Ig in a fat biopsy for which the corresponding DNA was unavailable, and for characterizing the LC proteins found in autopsied kidney, serum and urine samples from patients with AL amyloidosis whose LC-DNA was sequenced. The PTMs of each LC were extensively characterized with different enzymes and various tandem MS techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) and electron transfer dissociation (ECD). PTMs observed include truncations, mono-/di-chlorination of the tyrosine residues and a nitrile group formed from the primary amine on lysine side chains. All these may play critical roles in the fibrillogenesis and/or the disease pathogenesis. Experimental evidence supports the hypothesis that the proteolytic processing of amyloidogenic LCs occurs after deposition in the organ. Characterization of a plant-derived HSV8 mAb was accomplished using high-performance liquid chromatography separation and gel display followed by various MS methods. Three N-terminal and one C-terminal truncations were found. The N-glycan moiety attached to the heavy chain was also analyzed. The MS method established helps to elucidate important structural information on therapeutic mAbs in complex mixtures, potentially contributing to optimization of plant systems that may produce more stable mAbs.

Light chain (AL) amyloidosis is a disease that occurs due to the presence of a small plasma-cell clone, which produces amyloidogenic light chains. These chains can misfold and aggregate, leading to the deposition of amyloid fibrils in tissues. If left untreated or if treatment is ineffective, this can result in irreversible organ dysfunction and eventual death. Current therapeutic treatments generally target and remove the clonal plasma cell population responsible for secreting full-length light chains which is not always effective or safe, however, a different approach to halt pathological LC misfolding would be to inhibit the amyloidogenesis cascade at its starting point. Small molecules have been identified that have the ability to bind to highly conserved residues in the interface between heavy and light chains which can be used to potentially impede the process of amyloid fibril deposition before the native FL LC can misfold or undergo proteolysis to form amyloid fibrils. To test whether small-molecule kinetic stabilizers are effective in stabilizing light chains, we measured the ability of the small molecule to bind to LCs, and the ability of light chains to aggregate and unfold in the absence and presence of small-molecule. Our findings suggest that the binding of stabilizers to the interface between variable domains of the LC dimer can increase equilibrium stability and decrease the rate of aggregation, thereby delaying the onset of amyloid formation.

<p>Over 20 different plasma proteins have been shown to have the capacity to undergo conformational changes and self-assemble into highly stable and insoluble amyloid fibrils. </p><p>One, transthyretin (TTR), consists of 127 amino acid residues arranged in eight β-strands (named A to H) and is involved in two different clinical forms of amyloidosis. In familial amyloidotic polyneuropathy (FAP), mutated TTR is found in the amyloid deposits while in senile systemic amyloidosis (SSA) only wild type TTR is present in the amyloid deposits.</p><p>In this study, we have identified a novel form of amyloidosis that is caused by the deposition of an N-terminal fragment of apolipoprotein A-IV (apoA-IV). Interestingly, apoA-IV amyloid was found deposited in a patient that also suffered from SSA. Thus, this patient had two biochemically distinct and concurrent forms of amyloidosis that were derived from apoA-IV and TTR. </p><p>We have also discovered that two different morphological deposition patterns (identified as patterns A and B) exist in TTR-derived amyloidosis. Pattern A, observed in all SSA patients studied and in half of the FAP patients examined contained large homogenous deposits that were composed of short randomly oriented fibrils. In contrast, pattern B was observed in the remaining FAP patients and was represented by smaller-sized deposits that consisted of longer fibrils that were arranged in parallel bundles. The predominant TTR component deposited also differed between the two amyloid patterns. Amyloid pattern A contained mainly C-terminal TTR fragments while pattern B amyloid consisted of full-length TTR. Our findings suggest that two different mechanisms of fibril formation may exist in TTR-derived amyloidosis. </p><p>We have found two epitopes, corresponding to strand C and H that are surface-exposed in TTR-derived amyloid fibrils but hidden and part of the hydrophobic core in the native molecular structure. This indicates that TTR undergoes partial unfolding during fibril formation. </p>

Over 20 different plasma proteins have been shown to have the capacity to undergo conformational changes and self-assemble into highly stable and insoluble amyloid fibrils. One, transthyretin (TTR), consists of 127 amino acid residues arranged in eight β-strands (named A to H) and is involved in two different clinical forms of amyloidosis. In familial amyloidotic polyneuropathy (FAP), mutated TTR is found in the amyloid deposits while in senile systemic amyloidosis (SSA) only wild type TTR is present in the amyloid deposits. In this study, we have identified a novel form of amyloidosis that is caused by the deposition of an N-terminal fragment of apolipoprotein A-IV (apoA-IV). Interestingly, apoA-IV amyloid was found deposited in a patient that also suffered from SSA. Thus, this patient had two biochemically distinct and concurrent forms of amyloidosis that were derived from apoA-IV and TTR. We have also discovered that two different morphological deposition patterns (identified as patterns A and B) exist in TTR-derived amyloidosis. Pattern A, observed in all SSA patients studied and in half of the FAP patients examined contained large homogenous deposits that were composed of short randomly oriented fibrils. In contrast, pattern B was observed in the remaining FAP patients and was represented by smaller-sized deposits that consisted of longer fibrils that were arranged in parallel bundles. The predominant TTR component deposited also differed between the two amyloid patterns. Amyloid pattern A contained mainly C-terminal TTR fragments while pattern B amyloid consisted of full-length TTR. Our findings suggest that two different mechanisms of fibril formation may exist in TTR-derived amyloidosis. We have found two epitopes, corresponding to strand C and H that are surface-exposed in TTR-derived amyloid fibrils but hidden and part of the hydrophobic core in the native molecular structure. This indicates that TTR undergoes partial unfolding during fibril formation.

The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery. In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases. / Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.