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In 2019, 2020 and 2022, the Japanese Government approved the use of tafamidis and two technetium-scintigraphies for transthyretin amyloid (ATTR) cardiomyopathy, and announced the patient criteria for tafamidis therapy. In 2018, we had started a nation-wide pathology consultation of amyloidosis. To reveal the impact of approval of tafamidis and technetium-scintigraphy on the diagnosis of ATTR cardiomyopathy. Ten institutes participated in this study on the pathology consultation of amyloidosis and shared rabbit polyclonal anti-κ116-133, anti-λ118-134, and anti-transthyretin115-124 antibodies. Proteomic analysis was performed when the typing diagnosis by immunohistochemistry was unavailable. Out of 5400 consultation cases received from April 2018 to July 2022, the type of amyloidosis by immunohistochemistry was determined in 4119 of the 4420 Congo-red positive cases. The incidences of AA, ALκ, ALλ, ATTR, Aβ2M and others were 3.2, 11.3, 28.3, 54.9, 0.6 and 1.8%, respectively. Out of 2208 cardiac biopsy cases received, 1503 cases were ATTR positive. There were 4.0 and 4.9 times more total cases and ATTR-positive cases, respectively, in the last 12 months as compared to the first 12 months. The approval of tafamidis and technetium-scintigraphy raised the awareness of ATTR cardiomyopathy, leading to an upsurge in ATTR-positive cardiac biopsy cases. [ABSTRACT FROM AUTHOR]

The molecular pathogenesis of human amyloidosis has been elucidated greatly during the last 20 years. Based on the understanding of the molecular mechanisms of amyloid fibril formation and deposition, various kinds of new drugs and therapeutics have been emerging to improve the prognosis of amyloidosis and even cure this disease. In this review article, we first summarize the pathogenesis and state‐of‐the‐art therapeutics of representative types of systemic human amyloidosis, that is, immunoglobulin light chain‐related, transthyretin‐related, amyloid A‐associated and β2‐microglobulin‐related amyloidosis. Next, we describe the essential roles of pathological diagnosis, especially the typing diagnosis of amyloidosis to appropriately guide type‐specific therapies of amyloidosis patients. Finally, we introduce the activities of the government‐funded group for surveys and research of amyloidosis in Japan, especially the nation‐wide pathology consultation system of amyloidosis, which started in April 2018. The nation‐wide improvement of the typing diagnosis of amyloidosis is essential for the appropriate treatment and care of amyloidosis patients in Japan. [ABSTRACT FROM AUTHOR]

Amyloidosis refers to a group of diseases with amyloid fibrils deposited in various organs and is classified into more than 30 diseases in humans based on the kind of amyloid protein. In order to elucidate the molecular pathogenesis of human amyloidosis, we studied the molecular mechanism of amyloid fibril formation in vitro. We first developed a novel fluorometric method to determine amyloid fibrils in vitro based on the unique characteristics of thioflavin T. We next proposed a nucleation-dependent polymerization model to explain the general mechanism of amyloid fibril formation in vitro. Based on this model, we characterized the biological molecular interactions that promote or inhibit amyloid fibril formation in vitro and developed models of pathological molecular environment for inducing human β2-microglobulin-related amyloidosis in long-term hemodialysis patients. We also proposed a novel and attractive cytotoxic mechanism of β2-microglobulin amyloid fibrils, that is, the disruption of endosomal/lysosomal membranes by endocytosed amyloid fibrils. These findings may be useful to elucidate the molecular pathogenesis of other kinds of human amyloidosis. [ABSTRACT FROM AUTHOR]

Diverse human diseases, including various neurodegenerative disorders and amyloidoses, are thought to result from the misfolding and aggregation of disease-causative proteins, and thus are collectively called protein misfolding diseases. Natively folded disease-causative proteins generally undergo a β-sheet conformational transition through an energetically unfavourable process, and further polymerize into amyloid fibrils. In the case of β2-microglobulin-related amyloidosis, an extracellular protein misfolding disease, many kinds of biological molecules including glycosaminoglycans, proteoglycans and lipids partially unfold β2-microglobulin and catalyse its subsequent nucleus formation. After amyloid fibrils are formed, these biological molecules stabilize the β2-microglobulin fibrils. In the polyglutamine neurodegenerative diseases, an intracellular protein misfolding disease, molecular chaperones as well as the ubiquitin–proteasome and autophagy–lysosome protein degradation systems, which are called the protein quality control systems, strictly regulate protein misfolding, aggregation and disease progression. A family of extracellular chaperones also binds to misfolded proteins and inhibit amyloid fibril formation in the extracellular space. Protein misfolding and aggregation may be an ideal therapeutic target for protein misfolding diseases in general. [ABSTRACT FROM PUBLISHER]

Abstract: Although the formation of an α-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact β2-microglobulin. Although K3 formed an α-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH ∼2), the helical content was not high, indicating a low preference to do so. The partly α-helical conformation was converted with time into a highly ordered β-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at ∼20 and ∼10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence. [Copyright &y& Elsevier]

To understand the initial stages in the formation of amyloid fibrils of β2-microglobulin, a protein responsible for dialysis-related amyloidosis, the effects of heat on the acid-unfolded monomer at pH 2.5 were studied. In the presence of a low concentration of seed fibrils, differential scanning calorimetric thermograms of acid-unfolded β2-microglobulin monomers showed a large decrease in heat capacity with a sigmoidal temperature-dependence, which was subsequently released at higher temperature. Measurements of circular dichroism, atomic force microscopy, ultracentrifugation, and repeated differential scanning calorimetry indicated that the exothermic sigmoidal transition is accompanied by the conversion of about 12% of the monomeric β2-microglobulin molecules into amyloid fibrils, which subsequently dissociate into monomers at high temperature. Interestingly, amyloid fibrils, formed partly after the sigmoidal transition, exhibited a heating rate-dependent, kinetically controlled thermal response, indicating that 12% of the total protein is enough to exhibit the unique thermal response. On the other hand, the salt-induced protofibrils did not show such a calorimetric response, indicating that the kinetic thermal response is unique to the particular structure of fibrils. Taken together, although the calorimetric behavior of amyloid fibrils remains elusive, it may be interpreted in terms of the effects of heat associated with the formation, the association, and the unfolding of fibrils, in which the interactions between specific β-sheet structures and water molecules play a crucial role and are sensitively reflected in the heat capacity change in protein solution. [ABSTRACT FROM AUTHOR]

High hydrostatic pressure reversibly transforms the amyloid fibrils of β2-microglobulin (β2-m) into a more tightly packed, reorganized structure, which has provided insight into the polymorphic properties of amyloid fibrils. Here, to further investigate the molecular mechanism that controls fibril structure, seed-dependent fibril growth from an acid-unfolded monomeric form under high pressure was studied. At all pressures up to 400MPa, the fibril growth could be approximated by a single-exponential kinetics, although pressure above 300MPa decreased the growth rate significantly. The fibrils formed at high pressure were similar to the reorganized fibrils formed initially at ambient pressure and then pressurized, suggesting that the reorganized fibrils were formed directly at high pressure. A systematic investigation of the extension rate under various pressures indicated that the activation free energies for the original and reorganized fibrils are significantly different, suggesting that different amino acid contacts are involved in these two types of fibrils. On the other hand, for the seed-dependent extension reactions of both types of fibrils, the activation volume was much smaller than the change in reaction volume, implying that only small numbers of side-chain interactions are achieved in the transition state. Importantly, we observed a marked acceleration of fibril growth, i.e., maturation, on repeated self-seeding above 300MPa, revealing the coexistence of another type of fibril with a similar structure but with an increased growth-rate under high pressure. [Copyright &y& Elsevier]

In ß2-microglobulin-related (Aß2M) amyloidosis, a serious complication in patients on long-term dialysis, partial unfolding of ß2-microglobulin (ß2-m) is believed to be prerequisite to its assembly into Aß2M amyloid fibrils. Many kinds of amyloid-associated molecules, (e.g., apolipoprotein E (apoE), glycosaminoglycans (GAGs), proteoglycans (PGs)) may contribute to the development of Aß2M amyloidosis. In 1990s, the formation of Aß2M amyloid fibrils in vitro was first observed at low pH (2.0–3.0). Very recently, low concentrations of 2,2,2-trifluoroethanol (TFE) and the sub-micellar concentration of sodium dodecyl sulfate, a model for anionic phospholipids, have been reported to cause the extension of Aß2M amyloid fibrils at a neutral pH, inducing partial unfolding of ß2-m and stabilization of the fibrils. Moreover, apoE, GAGs, and PGs were found to stabilize Aß2M amyloid fibrils at a neutral pH, forming a stable complex with the fibrils. Some GAGs, especially heparin, enhanced the fibril extension in the presence of TFE at a neutral pH. Some PGs, especially biglycan also induced the polymerization of acid-denatured ß2-m. These findings are consistent with the hypothesis that in vivo, specific molecules that affect the conformation and stability of ß2-m and amyloid fibrils will have significant effects on the deposition of Aß2M amyloid fibrils. [ABSTRACT FROM AUTHOR]

AbstractBackgroundMethodsResultsConclusionsA high incidence of valvular involvement of amyloid in the setting of aortic stenosis (AS) has been reported. Amyloid derived from ApoAI (AApoAI) can form local amyloid deposits in the aortic valve. Although a high prevalence of concomitant severe AS and cardiac transthyretin-type amyloidosis (ATTR) has been reported, the prevalence of valvular involvement by ATTR and AApoAI is unclear.Using immunostaining and mass spectrometry, we analysed amyloid proteins in 97 aortic valves removed for valve replacement due to AS at Kyoto Prefectural University of Medicine between 2014 and 2021. Clinical information was also reviewed.Amyloid deposits were found in 44 cases (45%), of which 30 cases (68%) involved ATTR and 33 cases (75%) AApoAI. Statistical analysis showed significantly lower age and E/e′ among amyloid-positive cases compared with amyloid-negative cases and significantly lower brain natriuretic peptide, higher fractional shortening, and higher left ventricular ejection fraction among ATTR-positive cases compared with ATTR-negative cases. Seven recent patients underwent bone scintigraphy and ATTR cardiomyopathy was observed in only one case.AS symptoms can manifest earlier in patients with amyloid or ATTR deposition in the aortic valve than in patients without such deposition, even though left ventricular function is preserved. [ABSTRACT FROM AUTHOR]

Provides information on an experiment which compares the mechanisms of apolipoprotein E- and antioxidant- (AO-) mediated inhibition of beta-amyloid fibril formation in vitro, based on a nucleation-dependent polymerization model. Experimental procedures; Results; Discussion.

Discusses the concentration-dependent inhibitory effects of apolipoprotein E (apoE) on Alzheimer's beta-amyloid fibril (betaAf) formation. Differential effects of apo E on betaAf formation; Description of the kinetic properties of the inhibitory effect of apoE on f(1-40) formation.

An 85‐year‐old man with a history of aortic dissection suddenly fainted, underwent cardiac heart arrest, and died. An autopsy was performed, but the cause of death was not grossly identified. Congo red staining detected amyloid deposits in systemic organs, including the heart, lungs, liver, and kidneys. Immunohistochemical (IHC) analysis revealed immunoglobulin (Ig) λ light chain (‐λ) in systemic blood vessels and transthyretin (TTR) in the heart and lungs. Ig‐λ was predominantly positive in the blood vessels of the lungs, while TTR was detected in the alveolar septum. In the heart, Ig‐λ was positive in the endocardium and blood vessels, and TTR was positive in nodular deposits between cardiomyocytes. The concurrent deposition of Ig‐λ and TTR in the heart was further substantiated by laser microdissection (LMD)‐liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) at each deposition site. Despite systemic deposition of Ig‐λ, bone marrow biopsy findings were not diagnostic for multiple myeloma. In summary, we present an autopsy case of concurrent Ig‐λ and TTR deposition as revealed by IHC and LC‐MS/MS. When Congo red staining and IHC results are indeterminate due to the deposition of multiple amyloid proteins, LMD‐LC‐MS/MS is useful for determining the precursor protein. [ABSTRACT FROM AUTHOR]

Clinical presentation of systemic amyloidosis differs among subtypes, and accurate subtype classification is important for choosing the treatment. Amyloid transthyretin (ATTR) amyloidosis was the predominant among the recently consulted amyloidosis cases in Japan. To reveal the latest subtype frequency of systemic amyloidosis among autopsy cases in Japan. We analyzed systemic amyloidosis cases autopsied from January 2017 to December 2018, that were listed in the Annuals of the Pathological Autopsy Cases in Japan, Volumes 60 and 61. When the subtype was unclear, we performed a questionnaire survey, immunohistochemistry with in‐house rabbit polyclonal anti‐κ116–133, anti‐λ118–134, and anti‐transthyretin115–124 antibodies, and proteomic analysis. Out of 481 systemic amyloidosis cases listed in the Annuals, 411 cases were available for analysis (85.4%). We classified 399 of these systemic amyloidosis cases. ATTR was the most common subtype (44.4%, n = 177), followed by amyloid immunoglobulin light chain (AL) (38.8%, n = 155). Amyloid A and amyloid β2‐microglobulin were 9.3% (n = 37) and 6.0% (n = 24), respectively. Double deposition of amyloid was identified in 1.6% (n = 6). In 168 cases (42.1%), systemic amyloidosis was the main cause of death. Of these cases, AL was the most common subtype (47.6%, n = 80), followed by ATTR (41.1%, n = 69). ATTR is the most predominant subtype among the current autopsy cases in Japan. [ABSTRACT FROM AUTHOR]

Podocyte expression of fibroblast specific protein 1 (FSP1) is observed in various types of human glomerulonephritis. Considering that FSP1 is secreted extracellularly and has been shown to have multiple biological effects on distant cells, we postulated that secreted FSP1 from podocytes might impact renal tubules. Our RNA microarray analysis in a tubular epithelial cell line (mProx) revealed that FSP1 induced the expression of heme oxygenase 1, sequestosome 1, solute carrier family 7, member 11, and cystathionine gamma-lyase, all of which are associated with nuclear factor erythroid 2-related factor (Nrf2) activation. Therefore, FSP1 is likely to exert cytoprotective effects through Nrf2-induced antioxidant activity. Moreover, in mProx, FSP1 facilitated Nrf2 translocation to the nucleus, increased levels of reduced glutathione, inhibited the production of reactive oxygen species (ROS), and reduced cisplatin-induced cell death. FSP1 also ameliorated acute tubular injury in mice with cisplatin nephrotoxicity, which is a representative model of ROS-mediated tissue injury. Similarly, in transgenic mice that express FSP1 specifically in podocytes, tubular injury associated with cisplatin nephrotoxicity was also mitigated. Extracellular FSP1 secreted from podocytes acts on downstream tubular cells, exerting renoprotective effects through Nrf2-mediated antioxidant activity. Consequently, podocytes and tubular epithelial cells have a remote communication network to limit injury. [ABSTRACT FROM AUTHOR]

Despite the dedicated research of artificial intelligence (AI) for pathological images, the construction of AI applicable to histopathological tissue subtypes, is limited by insufficient dataset collection owing to disease infrequency. Here, we present a solution involving the addition of supplemental tissue array (TA) images that are adjusted to the tonality of the main data using a cycle-consistent generative adversarial network (CycleGAN) to the training data for rare tissue types. F1 scores of rare tissue types that constitute < 1.2% of the training data were significantly increased by improving recall values after adding color-adjusted TA images constituting < 0.65% of total training patches. The detector also enabled the equivalent discrimination of clinical images from two distinct hospitals and the capability was more increased following color-correction of test data before AI identification (F1 score from 45.2 ± 27.1 to 77.1 ± 10.3, p < 0.01). These methods also classified intraoperative frozen sections, while excessive supplementation paradoxically decreased F1 scores. These results identify strategies for building an AI that preserves the imbalance between training data with large differences in actual disease frequencies, which is important for constructing AI for practical histopathological classification. [ABSTRACT FROM AUTHOR]

Although Sepsis-3 doesn't require evidence of bacteremia to diagnose sepsis, clinicians often want to identify the causative pathogen at autopsy. In principle, if the blood cultures are the same at ante- and postmortem, the cause of death is obvious. However, interpretations of postmortem blood cultures are often difficult due to discordance, negativity, mixed infection, and contamination, of pathogens occupying ≥ 50% of the tests. To increase specificity identifying agonal phase sepsis in the situations where blood cultures are discordant, multiple or negative at postmortem, we established a scoring system using blood cultures, procalcitonin (PCN) showing highest sensitivity and specificity for postmortem serum, and bone marrow polyhemophagocytosis (PHP). Histological sepsis showed significantly higher levels of culture score (2.3 ± 1.5 vs. 0.4 ± 0.5, p < 0.001), PHP score (2.5 ± 0.8 vs. 1.0 ± 1.1, p < 0.001), and PCN score (1.8 ± 0.8 vs. 0.8 ± 0.6, p < 0.01) than non-septic patients. Receiver operating characteristic curve analysis indicated that estimation of three scores was the most reliable indicator for recognizing agonal phase sepsis. These findings suggest that the combination of these three inspections enables to determine the pathological diagnoses of sepsis even it is not obvious by discordant, mixed or negative blood cultures. [ABSTRACT FROM AUTHOR]

Papillary carcinoma is the most common thyroid malignancy. As the cytological diagnosis of papillary carcinoma is not difficult in patients with the usual type of lesion, fine-needle aspiration ( FNA) cytology is an effective method for preoperative evaluation. However, this modality is often ineffective in identifying the follicular variant of papillary thyroid carcinoma ( FVPTC) due to its similarity to other follicular lesions and the incompleteness of typical nuclear features. Therefore, we investigated the expression of immunocytochemical markers of papillary carcinoma in cytological specimens of FVPTC and evaluated their utilities. The immunoreactivity of HBME-1 and CD15 was investigated using 50 imprint smear cytological specimens obtained from thyroid lesions, including 13 FVPTC. The sensitivity and specificity of HBME-1 for FVPTC were 92% and 89%, respectively, while those of CD15 were 23% and 100%, respectively. In conclusion, HBME-1 is a sensitive marker of papillary carcinoma, including both usual type and FVPTC, in cytological specimens. Therefore, using HBME-1 immunocytochemistry in FNA cytology will lead to reduction of the incidence of false-negative diagnoses of FVPTC. Although CD15 is apparently inferior in terms of sensitivity for FVPTC, its excellent specificity will support the definitive diagnosis of thyroid malignancies, including FVPTC, after screening with HBME-1. [ABSTRACT FROM AUTHOR]

Rocken et al. analyzed 29 cases of localized AL amyloidosis and diagnosed 22 of 29 (76%) cases as an AL with LBN, while the plasmacytic lymphocytes adjacent to the amyloid deposits were negative for CD10, in contrast to our case [[2]]. These findings suggest that the peritumoral AL amyloidosis formation in FL may require the in situ plasmacytic differentiation of FL cells. [Extracted from the article]

Spleen tyrosine kinase (Syk), a non-receptor-type tyrosine kinase, has a wide range of physiological functions. A possible role of Syk in Alzheimer's disease (AD) has been proposed. We evaluated the localization of Syk in the brains of patients with AD and control participants. Human neuroblastoma M1C cells harboring wild-type tau (4R0N) were used with the tetracycline off (TetOff) induction system. In this model of neuronal tauopathy, the effects of the Syk inhibitors—BAY 61–3606 and R406—on tau phosphorylation and oligomerization were explored using several phosphorylated tau-specific antibodies and an oligomeric tau antibody, and the effects of these Syk inhibitors on autophagy were examined using western blot analyses. Moreover, the effects of the Syk inhibitor R406 were evaluated in vivo using wild-type mice. In AD brains, Syk and phosphorylated tau colocalized in the cytosol. In M1C cells, Syk protein (72 kDa) was detected using western blot analysis. Syk inhibitors decreased the expression levels of several tau phosphoepitopes including PHF-1, CP13, AT180, and AT270. Syk inhibitors also decreased the levels of caspase-cleaved tau (TauC3), a pathological tau form. Syk inhibitors increased inactivated glycogen synthase kinase 3β expression and decreased active p38 mitogen-activated protein kinase expression and demethylated protein phosphatase 2 A levels, indicating that Syk inhibitors inactivate tau kinases and activate tau phosphatases. Syk inhibitors also activated autophagy, as indicated by increased LC3II and decreased p62 levels. In vivo , the Syk inhibitor R406 decreased phosphorylated tau levels in wild-type mice. These findings suggest that Syk inhibitors offer novel therapeutic strategies for tauopathies, including AD. • Spleen tyrosine kinase (Syk) colocalizes with phosphorylated tau in AD brains. • Syk inhibitors decrease in vitro and in vivo the levels of phosphorylated tau. • Syk inhibitors inactivate tau kinases and activate tau phosphatases and autophagy. • Syk inhibitors decrease the levels of caspase-cleaved tau. • Syk pathways offer novel therapeutic strategies for tauopathies, including AD. [ABSTRACT FROM AUTHOR]

The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, β2-microglobulin, a protein responsible for dialysis- related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2- trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-β core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-β structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure. [ABSTRACT FROM AUTHOR]

Abstract: Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of β2-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid β-peptides but also during the processes of the temperature-induced conversion of β2-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules. [Copyright &y& Elsevier]

Heat-triggered conversion of the salt-induced thin and flexible protofibrils into well-organized thick and straight mature amyloid fibrils was achieved with β2-microglobulin, a protein responsible for dialysis-related amyloidosis. First, protofibrils that formed spontaneously at pH 2.5 in the presence of 0.5 M NaCl were aggregated by agitating the solution. Second, the aggregated protofibrils were heated in a cell of a differential scanning calorimeter (DSC). The DSC thermogram showed an exothermic transition with sigmoidal temperature dependence, resulting in a remarkably large decrease in the heat capacity of the solution. Third, on the basis of electron microscopy together with circular dichroism spectroscopy, seeding experiments, and a thioflavin T binding assay, the sigmoidal transition was found to represent the conversion of protofibrils into mature amyloid fibrils. Furthermore, DSC thermograms obtained at various heating rates revealed that the transition curve depends on the heating rate, implying that the effects of heat associated with the conversion to the mature fibrils are kinetically controlled, precluding an interpretation in terms of equilibrium thermodynamics. Taken together, these results highlight the importance of the change in heat capacity in addressing the biological significance of interactions between solvent water and amyloid fibrils and, moreover, in detecting the formation of amyloid fibrils. [ABSTRACT FROM AUTHOR]

Abstract: Alzheimer''s disease (AD) is one of the most common dementing disorders and has profound medical and social consequences. The initiating molecular event is unknown, and its pathophysiology is highly complex. However, free radical injury appears to be a fundamental process contributing to the neuronal death seen in this disorder, and many studies using surrogate markers of oxidative damage have provided evidence supporting this hypothesis. Various compounds with antioxidant ability attenuated the oxidative stress induced by amyloid β-protein (Aβ) in studies done in vitro and in vivo. Moreover, various antioxidants have been reported to inhibit the formation and extension of β-amyloid fibrils (fAβ), as well as to destabilize preformed fAβ in vitro. In cell culture experiments, destabilized fAβ were suggested to be less toxic than intact fAβ. In transgenic mice model studies, some antioxidant coumpounds reduced plaque burden in vivo. In this article, we review the recent advances in the research on the antioxidants that inhibit the formation of fAβ, as well as destabilize preformed fAβ. Although the mechanisms by which these compounds inhibit fAβ formation from Aβ, and destabilize preformed fAβ are still unclear, they could be key molecules for the development of preventives and therapeutics for AD. [Copyright &y& Elsevier]

Abstract: Inhibition of the accumulation of amyloid β-peptide (Aβ) and the formation of β-amyloid fibrils (fAβ) from Aβ, as well as the destabilization of preformed fAβ in the central nervous system would be attractive therapeutic targets for the treatment of Alzheimer''s disease (AD). Many studies have demonstrated that oxidative damage plays a central role in AD pathogenesis, as well as Parkinson disease (PD). Among the antioxidant strategies proposed, increasing evidence points to the possibility of achieving neuroprotection by dopamine agonists, as well as monoamine oxidase B (MAO-B) inhibitors. Actually, the beneficial effect of selegiline, a MAO-B inhibitor, in AD has been noted in several clinical studies. On the reverse, antimuscarinic agents have been reported to accelerate β-amyloidosis and senile plaque formation in PD. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of anti-Parkinsonian agents, dopamine, levodopa, pergolide, bromocriptine, selegiline, and trihexyphenidyl on the formation, extension, and destabilization of fAβ(1-40) and fAβ(1-42) at pH 7.5 at 37°C in vitro. The anti-Parkinsonian agents other than trihexyphenidyl dose-dependently inhibited fAβ formation from Aβ(1-40) and Aβ(1-42), as well as their extension. Moreover, these agents dose-dependently destabilized preformed fAβs. The overall activity of the molecules examined was in the order of: dopamine>selegiline>levodopa=pergolide>bromocriptine. Although the exact mechanism of the anti-amyloidogenic activity of these agents is unclear, these and other structurally related compounds could be key molecules for the development of therapeutics for AD and other conformational diseases. [Copyright &y& Elsevier]

Abstract: The pathogenesis of Alzheimer''s disease (AD) is characterized by cerebral deposits of amyloid β-peptides (Aβ) and neurofibrillary tangles which are surrounded by inflammatory cells. Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of NSAIDs such as ibuprofen, aspirin, meclofenamic acid sodium salt, diclofenac sodium salt, ketoprofen, flurbiprofen, naproxen, sulindac sulfide and indomethacin on the formation, extension, and destabilization of β-amyloid fibrils (fAβ) at pH 7.5 at 37°C in vitro. All examined NSAIDs dose-dependently inhibited formation of fAβ from fresh Aβ(1–40) and Aβ(1–42), as well as their extension. Moreover, these NSAIDs dose-dependently destabilized preformed fAβs. The overall activity of the molecules examined was in the following order: ibuprofen ≈ sulindac sulfide ≥ meclofenamic acid sodium salt>aspirin ≈ ketoprofen ≥ flurbiprofen ≈ diclofenac sodium salt>naproxen ≈ indomethacin. Although the mechanisms by which these NSAIDs inhibit fAβ formation from Aβ, and destabilize preformed fAβ in vitro are still unclear, NSAIDs may be promising for the prevention and treatment of AD. [Copyright &y& Elsevier]

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on β2-microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded β2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native β2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mM sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of β2-m, producing fibrils adapted to the respective pH. [ABSTRACT FROM AUTHOR]

Abstract: Inhibition of the formation of β-amyloid fibrils (fAβ), as well as the destabilization of preformed fAβ in the CNS, would be attractive therapeutic targets for the treatment of Alzheimer’s disease (AD). We reported previously that nordihydroguaiaretic acid (NDGA) and wine-related polyphenol, myricetin (Myr), inhibit fAβ formation from Aβ and destabilize preformed fAβ in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of coenzyme Q10 (CoQ10) on the formation, extension, and destabilization of fAβ at pH 7.5 at 37°C in vitro. We next compared the anti-amyloidogenic activities of CoQ10 with NDGA and Myr. CoQ10 dose-dependently inhibited fAβ formation from amyloid β-peptide (Aβ), as well as their extension. Moreover, it destabilized preformed fAβs. The anti-amyloidogenic effects of CoQ10 were slightly weaker than those of NDGA and Myr. CoQ10 could be a key molecule for the development of therapeutics for AD. [Copyright &y& Elsevier]

Abstract: β2-Microglobulin (β2-m) is a major structural component of dialysis-related amyloid fibrils. Kozhukh et al. [J. Biol. Chem. 277 (2002) 1310] prepared a series of peptide fragments of β2-m by the protease digestion and examined their ability to form amyloid fibrils in citrate buffer at pH 2.5. Among various peptides, a 22-residue K3 peptide corresponding to Ser20-Lys41 spontaneously formed amyloid fibrils in aqueous solution. This peptide also formed amyloid protofibrils in 20% (v/v) 2,2,2-trifluoroethanol (TFE). To investigate the influence of solvent conditions on fibril formation, we studied their structures by atomic force microscopy. In aqueous solution, fibrils had a diameter of 4 or 8nm and tended to cluster each other. On the other hand, protofibrils in 20% (v/v) TFE had a diameter of 2nm with no tendency of clustering. Intriguingly, when the K3 protofibrils were transferred from 20% (v/v) TFE to aqueous solution, some of them associated to form thicker fibrils with a diameter of 4–15nm and a left-handed helical twist. TFE is a hydrophobic solvent, so that hydrophobic interactions between molecules may be weakened. The results suggest that the fibrils in aqueous conditions are formed by the cooperative association of protofibrils at the growing ends of the fibrils, in which hydrophobic interactions play a major role. [Copyright &y& Elsevier]

Inhibition of the accumulation of amyloid β-peptide (Aβ) and the formation of β-amyloid fibrils (fAβ) from Aβ, as well as the destabilization of preformed fAβ in the CNS would be attractive therapeutic targets for the treatment of Alzheimer''s disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAβ formation from Aβ(1–40) and Aβ(1–42) as well as destabilizing preformed fAβ(1–40) and fAβ(1–42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of polymeric polyphenol, tannic acid (TA) on the formation, extension, and destabilization of fAβ(1–40) and fAβ(1–42) at pH 7.5 at 37 °C in vitro. We next compared the anti-amyloidogenic activities of TA with myricetin, rifampicin, tetracycline, and NDGA. TA dose-dependently inhibited fAβ formation from Aβ(1–40) and Aβ(1–42), as well as their extension. Moreover, it dose-dependently destabilized preformed fAβs. The effective concentrations (EC50) of TA for the formation, extension and destabilization of fAβs were in the order of 0–0.1 μM. Although the mechanism by which TA inhibits fAβ formation from Aβ as well as destabilizes preformed fAβ in vitro is still unclear, it could be a key molecule for the development of therapeutics for AD. [Copyright &y& Elsevier]

β2-Microglobulin (β2m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length β2m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of β2m, Ser20-Lys41, obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length β2m. This suggests that once the rigid native-fold of β2m is unfolded and additional factors triggering the nucleation process are provided, full-length β2m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through CyseS. The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length β2m accompanied with a characteristic lag phase, suggesting that the internal structures are similar. [ABSTRACT FROM AUTHOR]

Increasing numbers of proteins have been found to aggregate into insoluble fibers, collectively referred to as amyloid fibrils. To address the conformational stability of amyloid fibrils, we studied the effects of dimethylsulfoxide (DMSO), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on β2-microglobulin amyloid fibrils by circular dichroism, thioflavin T fluorescence, light scattering, and electron microscopy. When measured by circular dichroism and thioflavin T fluorescence, HFIP, and TFE dissolved the fibrils, producing predominantly helical conformations. However, these alcohols did not dissolve the amyloid fibrils completely as monitored by light scattering and electron microscopy. On the other hand, DMSO completely dissolved the amyloid fibrils although a high concentration [i.e., 80% (v/v)] was required. These results are consistent with the important role of hydrogen bonds in stabilizing amyloid fibrils. [ABSTRACT FROM AUTHOR]

Novel therapies have recently been used for different amyloidoses including hereditary transthyretin (ATTR, ATTRv) amyloidosis, wild‐type ATTR (ATTRwt) amyloidosis, immunoglobulin light chain (AL)amyloidosis, and amyloid A (AA) amyloidosis. Early, accurate diagnosis is important for improved prognosis and cure. However, early intervention with early diagnosis is often difficult, because these diseases are frequently difficult to identify given their phenotypic heterogeneity. This study aimed to analyze characteristics of patients with amyloidosis that was diagnosed at the Amyloidosis Medical Practice Center, Kumamoto University Hospital, which, as an amyloidosis center in Japan, conducts histopathological, proteomic, and genetic analyses of amyloidosis. We analyzed diagnostic results and disease manifestations of patients diagnosed during April 2012 and March 2019. This study reviewed 1937 consecutive consultations that provided clinical information. The patients' average age was 65.9 (median 69) years. Of all patients, 66.6% were male. Diagnoses included ATTRv: 13.4%, ATTRwt: 14.3%, ATTR (no genetic analysis consultation requested): 4.6%, ALλ: 19.7%, ALκ: 8.8%, AA: 3.0%, β2‐microglobulin (dialysis‐related): 0.8%, and others: 1.9%, no TTR gene mutation (no histopathological analysis consultation requested): 23.2% (including duplicate consultations). ATTRv amyloidosis cases included V30M in endemic areas: 7.4%, V30M in non‐endemic areas: 51.2%, and non‐V30M: 41.4%. Sixty‐six cases required laser microdissection and liquid chromatography‐tandem mass spectrometry. Initial manifestations included polyneuropathy: 13.2%, carpal tunnel syndrome: 5.6%, autonomic dysfunction: 2.7%, heart failure: 28.4%, cardiac hypertrophy: 2.2%, arrhythmia: 5.0%, renal impairment: 11.1%, and stroke: 1.8%. Amyloidosis types and manifestations were diverse. For early diagnosis and appropriate interventions, improved diagnostic systems are needed. [ABSTRACT FROM AUTHOR]

Background Chronic hypoxia may play a pivotal role in the development of diabetic nephropathy (DN). However, the precise mechanisms underlying progressive hypoxia-induced glomerular injury remain unclear. Methods We housed db/db mice in a hypoxia chamber (12% O2) for up to 16 weeks beginning at 8 weeks of age. Various urine, serum and kidney abnormalities and glomerular messenger RNA (mRNA) expression were compared with those in age-matched db/db mice housed under normoxia. Results Levels of urinary albumin and podocalyxin (PCX) were significantly higher in hypoxic mice early during hypoxia. Ultracentrifugation of urine samples revealed that podocytes in the hypoxic mice shed PCX-positive microparticles into the urine. After 16 weeks of hypoxia, the mice also had higher hematocrits with lower serum glucose and various degrees of mesangiolytic glomerulosclerosis with microaneurysms and the infrequent occurrence of nodular lesions. Immunohistologically, hypoxic mice showed significantly decreased endothelial cell densities early during hypoxia and decreased podocyte densities later. In both hypoxic and normoxic mice, glomerular macrophage and transforming growth factor-β1 (TGF-β1) staining significantly increased with aging, without changes in vascular endothelial growth factor or endothelial nitric oxide synthase (eNOS). Glomerular mRNA expression of monocyte chemoattractant protein-1, eNOS and TGF-β1 was significantly enhanced in the hypoxic mice. Conclusions These results indicate that chronic hypoxia induces advanced glomerulosclerosis with accelerated albuminuria triggered by mesangiolysis and podocyte injury in a murine model of DN. [ABSTRACT FROM AUTHOR]

Polyphosphate (polyP), which is found in various microorganisms and human cells, is an anionic biopolymer consisting of inorganic phosphates linked by high-energy phosphate bonds. Previous studies revealed that polyPs strongly promoted the amyloid formation of several amyloidogenic proteins; however, the mechanism of polyP-induced amyloid formation remains unclear. In the present study using β2-microglobulin (β2m), a protein responsible for dialysisrelated amyloidosis, we investigated amyloid formation in the presence of various chain lengths of polyPs at different concentrations under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) conditions. We found that the amyloid formation of β2m at acidic pH was significantly accelerated by the addition of polyPs at an optimal polyP concentration, which decreased with an increase in chain length. The results obtained indicated that electrostatic interactions between positively charged β2m and negatively charged polyPs play a major role in amyloid formation. Under neutral pH conditions, long polyP with 60 to 70 phosphates induced the amyloid formation of β2m at several micromoles per liter, a similar concentration range to that in vivo. Since β2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with β2m electrostatically. PolyPs appear to dehydratewater molecules around β2m under the unfolded conformation, leading to the preferential stabilization of less water-exposed amyloid fibrils. These results not only revealed the pH-dependent mechanism of the amyloid formation of β2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis. [ABSTRACT FROM AUTHOR]

Cerebral amyloid angiopathy (CAA) is a degenerative disorder characterized by amyloid-β (Aβ) deposition in the brain microvessels. CAA is also known to contribute not only to cortical microbleeds but also lobar hemorrhages. This retrospective study examined CAA pathologically in patients who underwent direct surgeries for lobar hemorrhage. Thirty-three patients with lobar hemorrhage underwent open surgery with biopsy from 2007 to 2016 in our hospital. Cortical tissues over hematomas obtained surgically were pathologically examined using hematoxylin, eosin stain, and anti-Aβ antibody to diagnose CAA. We also investigated the advanced degree of CAA and clinical features of each patient with lobar hemorrhage. In the 33 patients, 4 yielded specimens that were insufficient to evaluate CAA pathologically. Twenty-four of the remaining 29 patients (82.8%) were pathologically diagnosed with CAA. The majority of CAA-positive patients had moderate or severe CAA based on a grading scale to estimate the advanced degree of CAA. About half of the CAA-positive patients had hypertension, and four took anticoagulant or antiplatelet agents. In five patients who were not pathologically diagnosed with CAA, one had severe liver function disorder, three had uncontrollable hypertension, and one had no obvious risk factor. Our pathological findings suggest that severe CAA with vasculopathic change markedly contributes to lobar hemorrhage. The coexistence of severe CAA and risk factors such as hypertension, anticoagulants or antiplatelets may readily induce lobar hemorrhage. [ABSTRACT FROM AUTHOR]

Purpose: Bronchopulmonary dysplasia (BPD) is a respiratory complication characterized by abnormal alveolar development in premature infants. Geranylgeranylacetone (GGA) can induce heat shock protein (HSP) 70, which has cytoprotective effects against various stressors. Here, we investigated whether GGA protected neonatal lungs from hyperoxic stress in a murine BPD model, and measured the serum HSP70 levels in preterm humans treated with oxygen. Methods: Newborn mice were exposed to >90% oxygen and administered GGA or vehicle alone orally on days 1, 2, and 3 of life. At 2 days of age, HSP70 expression in the lung was determined by western blotting. At 8 days of age, the lungs were processed for histological analysis. Radial alveolar count (RAC) and mean linear intercept (MLI) were measured as parameters of alveolarization. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and cleaved caspase-3 immunohistochemistry. Serum HSP70 levels in preterm humans treated with oxygen were measured by enzyme-linked immunosorbent assay. Results: GGA administration enhanced the HSP70 expression to two-fold compared with normoxia-exposed and vehicle-treated mice. Hyperoxia reduced HSP70 expression, whereas GGA abrogated the effects. Hyperoxia-exposed mice exhibited more apoptotic cells in lung parenchyma and a more simplified alveolar structure with less RAC and larger MLI than normoxia-exposed mice. GGA suppressed the increase in apoptotic cells and the structural changes of the lungs induced by hyperoxia. Serum HSP70 levels of preterm human infants gradually decreased with age. Conclusions: GGA may attenuate hyperoxic injury in neonatal lungs and thereby may prevent the development of BPD. [ABSTRACT FROM AUTHOR]

Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT’s amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay. [ABSTRACT FROM AUTHOR]

The article reports that native structures of peptides in proteins have been thought to represent the most stable among the possible conformations that a peptide can take. However, amyloid fibrils are now considered to be stable structures for peptide aggregates. The relationship between the secondary structural preferences of individual residue native proteins and amyloid fibrils has not been fully addressed because only fibril structures of short peptides have been examined so far. The secondary structures of component residues in the peptide were deduced from the shifted amide I frequencies of peptides specifically labeled with on the carbonyl group of the main chain.

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described. [ABSTRACT FROM AUTHOR]

Although cadaver dissections are important for skull base surgeons to acquire anatomical knowledge and techniques, their opportunities are limited in Japan. The Autopsy Imaging Center of the University of Fukui Hospital has both a CT scanner and an MR unit solely for deceased patients. The authors applied the postmortem imaging to cadaver dissections and evaluated its usefulness in surgical education. Ten sides of five formalin-fixed cadaver heads were dissected by ten neurosurgeons. Five neurosurgeons were young, three were moderately experienced, and two were experts in skull base surgery. They performed orbitozygomatic, anterior transpetrosal, posterior transpetrosal, and transcondylar approaches. CT bone images were taken before and after dissections, and MR images were taken before dissection to merge with the CT bone images. The usefulness of the images for each neurosurgeon and for each skull base approach was evaluated. The postmortem imaging system was useful for all neurosurgeons, especially in anterior transpetrosal, posterior transpetrosal, and transcondylar approaches. They could find the insufficiency or excessiveness of their drilling of specific bony structures with the images. Even the experts in skull base surgery could identify regions in which they could add drilling safely to widen the surgical field more. The postmortem imaging system was useful for skull base cadaver dissections. This system is expected to be utilized for education and research on surgical anatomy. [ABSTRACT FROM AUTHOR]

Although organ weight gives pathologists information about the pathogenesis of diseases at autopsy, the knowledge is rarely helpful in postmortem virtual autopsy by computed tomography ( CT). To investigate the feasibility of liver weight estimation based on liver volume estimated from three-dimensional CT images and the specific gravity of liver, thirty cadavers who died in the University of Fukui Hospital and whose family members agreed to postmortem CT and autopsy were prospectively enrolled. Mean specific gravity of liver was 1.054 ± 0.009 g/mL (95% confidence interval: 1.0507-1.0573 g/mL). The specific gravity was positively correlated to Hounsfield unit ( HU) values of less than 40 (cases with moderate to severe fatty deposition) and remained stable between 1.05 to 1.065 g/mL for HU values greater than 40 (cases with mild or no fatty change). The liver weight estimated by our formula corresponded well to the actual liver weight, and the correlation coefficient was 0.96 ( P < 1 × 10−13). The estimated liver weight calculated from estimated liver volume and the specific gravity of 1.055 g/mL was highly accurate, whereas the specific gravity should be reduced by 2%-3% in patients with an HU value less than 40 due to fatty deposition. [ABSTRACT FROM AUTHOR]

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with β2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding. [ABSTRACT FROM AUTHOR]

Abstract: Cerebral amyloid angiopathy is caused by deposition of the amyloid β-peptide which consists of mainly 39–40 residues to the cortical and leptomeningeal vessel walls. There are no definite in vitro systems to support the hypothesis that the vascular basement membrane may act as a scaffold of amyloid β-peptide carried by perivascular drainage flow and accelerate its amyloid fibril formation in vivo. We previously reported the critical roles of interfaces and agitation on the nucleation of amyloid fibrils at low concentrations of amyloid β-peptide monomers. Here, we reproduced the perivascular drainage flow in vitro by using N-hydroxysuccinimide-Sepharose 4 Fast flow beads as an inert stirrer in air-free wells rotated at 1rpm. We then reproduced the basement membranes in the media of cerebral arteries in vitro by conjugating Matrigel and other proteins on the surface of Sepharose beads. These beads were incubated with 5μM amyloid β(1–40) at 37°C without air, where amyloid β(1–40) alone does not form amyloid fibrils. Using the initiation time of fibril growth kinetics (i.e., the lag time of fibril growth during which nuclei, on-pathway oligomers and protofibrils are successively formed) as a parameter of the efficiency of biological molecules to induce amyloid fibril formation, we found that basement membrane components including Matrigel, laminin, fibronectin, collagen type IV and fibrinogen accelerate the initiation of amyloid β-peptide fibril growth in vitro. These data support the essential role of vascular basement membranes in the development of cerebral amyloid angiopathy. [Copyright &y& Elsevier]

Abstract: Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer''s disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be “activated” to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils. [Copyright &y& Elsevier]

Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately, β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins. [ABSTRACT FROM AUTHOR]

We report the case of a 38-year-old woman diagnosed with Gitelman syndrome. A kidney biopsy showed abundant floating cells in the Bowman's space of the mildly cystic glomeruli, moderate tubulointerstitial changes and apparent intimal thickening of small arteries. These floating cells were immunohistologically identified as podocytes, by the expression of podocalyxin, vimentin, Wilms' tumor 1, synaptopodin and nephrin with positivities of 100 %, 88.4 %, 80.4 %, 74.7 % and 22.6 %, respectively. In these phenotypes, nephrin expression was notably decreased in both detached and capillary-attached podocytes in comparison with normal control podocytes. Immunostaining of both detached and capillary-attached podocytes for Bax, Bcl-2, desmin, fibroblast-specific protein-1, α-smooth muscle actin and Ki-67 was negative, as were TUNEL assays. These results suggest that apoptosis and epithelial−mesenchymal transition were not the main cause of podocyte detachment in this patient. In addition, levels of urinary podocalyxin were not elevated, suggesting the detached podocytes were not excreted in the urine. To the best of our knowledge, this is the first report of severe intraglomerular non-apoptotic detachment of podocytes in Gitelman syndrome. This podocyte detachment may be associated with nephron obstruction and reduced nephrin expression. [ABSTRACT FROM AUTHOR]

Background: Allergen-specific immunotherapy has been anticipated to be a disease-modifying therapy for food allergies. We previously reported that CD8+ regulatory T cells may prevent antigen-sensitized mice from developing allergic diarrhea. Because oligomannose-coated liposomes ( OML) have been shown to induce MHC class I-restricted CD8+ T cell responses, we analyzed the adjuvant activities of OML for inducing regulatory CD8+ T cells and mucosal tolerogenic responses in allergen-sensitized mice. Methods: The BALB/c mice that were previously sensitized to ovalbumin ( OVA) were intranasally immunized with OVA-encased in OML ( OVA- OML) or OVA-encased in non-coated liposomes ( OVA- NL). We assessed allergic diarrhea induced by oral OVA administration, OVA-specific immunoglobulin production, and cytokine production in the intestines and mesenteric lymph nodes ( MLNs). Results: Intranasal immunization with OVA- OML, but not OVA- NL, suppressed the development of allergic diarrhea. This was associated with in vitro Ag-induced IL-10 production and the in vivo expansion of CD8+ CD28− and CD4+ CD25+ Foxp3+ T cell populations among mesenteric lymph node mononuclear cells, and was significantly ablated by anti- SIGNR1 or anti- CR3 m Abs. Up-regulation of serum OVA-specific Ig E was suppressed, whereas OVA-specific Ig G1, Ig G2a, and soluble Ig A production were enhanced by intranasal administration of OVA- OML. Adoptive transfer of CD8+ CD28− T cells but not CD28+ CD8+ T cells from the MLNs of OVA- OML-treated mice ameliorated the development of diarrhea. Conclusion: These results suggest that intranasal immunization with Ag-encased OML may be an effective immunotherapy for food allergies, as it induces a subset of regulatory CD8+ T cells as well as CD4+ CD25+ Foxp3+ T cell and modulates humoral immune responses in allergen-sensitized mice. [ABSTRACT FROM AUTHOR]