Congo Red Staining Research Articles

Abstract 4138014: Incidence and Association of Amyloidosis Found at Carpal Tunnel Release to Development of Cardiac Involvement

Background: Carpal tunnel syndrome (CTS) associated with transthyretin cardiac amyloidosis (ATTR-CA) may manifest 5 to 10 years prior to cardiac diagnosis. Transthyretin amyloidosis (ATTR) identification in the tenosynovium isolated during carpal tunnel release (CTR) may facilitate earlier ATTR-CA diagnosis and treatment. Hypothesis: Amyloid discovered at CTR will be associated with, and/or serve as a precursor for, cardiac involvement. Aim: We aimed to determine the frequency and type of amyloid found at CTR as well as the incidence of cardiac involvement and/or treatment indication. Methods: A retrospective analysis was conducted on a convenience sample of adults undergoing CTR from 01/05/2021 to 03/24/2023. Biopsy specimens with confirmed amyloid via Congo red staining were analyzed using mass spectrometry (MS) for subtyping. Patients with amyloid in tenosynovial tissue were offered cardiac evaluation with history and physical, electrocardiogram, B-type natriuretic peptide, troponin-I HS, echocardiogram, and TcPYP nuclear scintigraphy. Patients with light-chain amyloidosis (AL) were referred to oncology, while patients with ATTR were offered genetic testing. Results: A total of 578 patients underwent CTR with tenosynovial sample excision. Amyloid involvement by Congo red staining was found in 43 patients (7.4%), with a definitive subtype confirmed by MS in 30 patients (5.2%). Samples were insufficient to define subtypes in 13 patients. Sole ATTR was found in 27 patients (4.7%), both AL and ATTR were found in 1 patient, and sole AL was found in 2 patients. Of the 28 ATTR patients (the patient with both ATTR and AL included), 24 pursued genetic testing, revealing all had wild-type ATTR. Of these 28 patients, 21 underwent cardiac evaluation, for which 2 had cardiac involvement on TcPYP and started tafamidis therapy. The patients with concomitant ATTR and AL or sole AL did not have evidence of cardiac involvement, however, chemotherapy was initiated as indicated. Unfortunately, 1 patient with AL on CTR did not pursue an evaluation. Conclusion: Incidental amyloid tissue found at CTR is common and associated with occult involvement warranting therapy in a minority of cases. Future cardiac involvement is unknown, and this cohort will be followed to determine future incidence. A startling number of patients were found to have occult systemic AL discovered at CTR. These incidental diagnoses may trigger lifesaving interventions, which may otherwise go undiagnosed.

Abstract 4112944: A diagnostic challenge overcome with persistent clinical suspicion in a case of cardiac AL amyloidosis

Case: A 62-year old male with a history of chronic kidney disease and paroxysmal atrial fibrillation presented with several months of fatigue, a progressive decline in functional status (NYHA IIIb-IV), weight loss, and intermittent lower extremity swelling. Blood pressure was 92/72 mmHg on presentation, and heart rate was 90 bpm. Physical examination revealed a jugular venous pressure of 12-14 cm H2O and 2+ bilateral lower extremity edema with mild wheezing. Methods/workup: Laboratories are shown in Table 1 and were notable for a serum free light chain ratio of 0.14. Transthoracic echocardiogram (figure 1) revealed a left ventricular (LV) ejection fraction of 33% with increased LV wall thickness and severe bi-atrial enlargement. Bone marrow biopsy revealed 2% lambda restricted plasma cells without evidence of amyloid deposits; metastatic bone survey was negative. A fat pad biopsy and endomyocardial biopsy stained negative for amyloid with Congo red. Cardiac MRI showed diffuse, circumferential subendocardial late gadolinium enhancement, most compatible with cardiac amyloidosis (figure 2). The patient also had a chest CT scan that demonstrated a few patchy nodular pulmonary infiltrates. Given his extensive negative workup, he was referred to pulmonary and endobronchial biopsy was positive for amyloid on Congo red staining (figure 3). Biopsy tissue typing revealed Lambda subtype AL amyloidosis. Management: The patient was started on Daratumumab, Cyclophosphamide, Bortezomib, and Dexamethasone by Hematology. Unfortunately, about a month after his diagnosis, he developed worsening heart failure despite treatment. He was admitted to an outside hospital with refractory cardiogenic shock and passed away during that admission. Discussion: The diagnosis of AL amyloidosis requires a high index of suspicion for prompt diagnosis and treatment. We present a case in which cardiac amyloidosis was suspected based on the patient’s clinical picture and cardiac imaging; however, multiple initial biopsies were negative. An expedited, multimodal and interdisciplinary diagnostic assessment is imperative as prognosis with chemotherapy is highly dependent on extent of AL disease, patient functional status, and presence of cardiac compromise. Elevated natriuretic peptides, troponins, autonomic dysfunction, and hypotension are negative prognostic features.

Abstract 4120573: Abrupt cardiac rupture of the patient with ATTR amyloidosis

A Japanese male in his 80s was admitted to our emergency department with chest pain. His prior history was syncope and ventricular tachycardia. Transthoracic echocardiography (TTE) revealed thickening of the basal part of the antero-septal wall and thinning of the basal part of the inferior wall. Coronary angiography (CAG), cardiac MRI and blood test revealed no specific abnormalities. The day before his emergency admission, he visited the cardiology outpatient department for a periodic examination of cardiac hypertrophy. ECG, chest X-ray, and TTE were unchanged from his prior examination. However, that night, he experienced chest pain and was transferred to our hospital. On arrival, his vital signs were critical, with a blood pressure of 70/59 mmHg. ECG indicated ST elevation in antero-lateral leads. TTE detected pericardial effusion, and contrast-enhanced CT identified two focal areas of non-enhancement in the myocardium and pericardial effusion without signs of aortic dissection. CAG revealed no significant stenosis or obstructive lesion. During coronary angiography, the patient deteriorated to asystole. Cardiopulmonary resuscitation and percutaneous pericardial drainage were initiated. Hemorrhagic pericardial effusion was aspirated. Immediate surgical hemostasis was undergone, revealing about 2 cm lacerations and hematoma in the anterior and inferior wall. After surgery, he complained of abdominal discomfort. Esophageal gastrointestinal endoscopy was undergone to investigate the abdominal discomfort, and he was diagnosed with early gastric cancer. Endoscopic submucosal dissection (ESD) was undergone, and histopathological examination revealed adenocarcinoma and eosinophilic amorphous materials deposited in the submucosal vessels. Congo red staining of the submucosal vessels revealed orange-red areas. A technetium-99m pyrophosphatase scintigraphy showed Grade 2 uptake in the heart. Transthyretin (TTR) staining was positive in the specimen from ESD, leading to a diagnosis of TTR amyloidosis. Genetic test for TTR was negative, which led to a diagnosis of wild-type TTR amyloidosis and tafamidis was started. In cases of acute pericardial effusion and/or cardiac rupture, it is imperative to consider cardiac amyloidosis as a potential etiology, in addition to myocardial infarction and aortic dissection, especially in patients with left ventricular wall thickening, neuropathy, or abnormal ECG, also known as “red flag”.

FAAH Inhibition Counteracts Neuroinflammation via Autophagy Recovery in AD Models

Endocannabinoids have attracted great interest for their ability to counteract the neuroinflammation underlying Alzheimer’s disease (AD). Our study aimed at evaluating whether this activity was also due to a rebalance of autophagic mechanisms in cellular and animal models of AD. We supplied URB597, an inhibitor of Fatty-Acid Amide Hydrolase (FAAH), the degradation enzyme of anandamide, to microglial cultures treated with Aβ25-35, and to Tg2576 transgenic mice, thus increasing the endocannabinoid tone. The addition of URB597 did not alter cell viability and induced microglia polarization toward an anti-inflammatory phenotype, as shown by the modulation of pro- and anti-inflammatory cytokines, as well as M1 and M2 markers; moreover microglia, after URB597 treatment released higher levels of Bdnf and Nrf2, confirming the protective role underlying endocannabinoids increase, as shown by RT-PCR and immunofluorescence experiments. We assessed the number and area of amyloid plaques in animals administered with URB597 compared to untreated animals and the expression of autophagy key markers in the hippocampus and prefrontal cortex from both groups of mice, via immunohistochemistry and ELISA. After URB597 supply, we detected a reduction in the number and areas of amyloid plaques, as detected by Congo Red staining and a reshaping of microglia activation as shown by M1 and M2 markers’ modulation. URB597 administration restored autophagy in Tg2576 mice via an increase in BECN1 (Beclin1), ATG7 (Autophagy Related 7), LC3 (light chain 3) and SQSTM1/p62 (sequestrome 1) as well as via the activation of the ULK1 (Unc-51 Like Autophagy Activating Kinase 1) signaling pathway, suggesting that it targets mTOR/ULK1-dependent autophagy pathway. The potential of endocannabinoids to rebalance autophagy machinery may be considered as a new perspective for therapeutic intervention in AD.

Adverse Effects of Aβ1-42 Oligomers: Impaired Contextual Memory and Altered Intrinsic Properties of CA1 Pyramidal Neurons

Aβ1-42 (amyloid beta) oligomers, the major neurotoxic culprits in Alzheimer’s disease, initiate early pathophysiological events, including neuronal hyperactivity, that underlie aberrant network activity and cognitive impairment. Although several synaptotoxic effects have been extensively studied, neuronal hyperexcitability, which may also contribute to cognitive deficits, is not fully understood. Here, we found several adverse effects of in vivo injection of Aβ1-42 oligomers on contextual memory and intrinsic properties of CA1 pyramidal neurons. Male rats underwent behavioral and electrophysiological studies 1 week after microinjections into the dorsal CA1 region, followed by histological analysis. After 1 week, Aβ1-42 oligomers impaired contextual learning without affecting basic physiological functions and triggered training-induced neuronal excitability. Furthermore, riluzole, a persistent sodium current (INaP) blocker, dose-dependently reduced Aβ1-42 oligomer-induced hyperexcitability. Congo red staining, which detects insoluble amyloid deposits, further identified labeling of CA1 pyramidal neurons while immunohistochemistry with lecanemab, which detects soluble Aβ oligomers, revealed immunoreactivity of both pyramidal and non-pyramidal cells in the target area. Therefore, our study suggests that a single injection of Aβ1-42 oligomers resulted in contextual memory deficits along with concomitant neuronal hyperexcitability and amyloid deposition in the CA1 region after 1 week.

Oral administration of butylated hydroxytoluene induces neuroprotection in a streptozotocin-induced rat Alzheimer’s disease model via inhibition of neuronal ferroptosis

BackgroundAlzheimer’s disease (AD) is the most common human neurodegenerative disorder worldwide. Owing to its chronic nature, our limited understanding of its pathophysiological mechanisms, and because of the lack of effective anti-AD drugs, AD represents a significant socio-economic challenge for all industrialized countries. Neuronal cell death is a key factor in AD pathogenesis and recent studies have suggested that neuronal ferroptosis may play a major patho-physiological role. Since ferroptosis involves free radical-mediated lipid peroxidation, we hypothesized that enteral administration of the radical scavenger butylated hydroxytoluene (BHT) might slow down or even prevent the development of AD-related symptoms in an in vivo animal AD model.Material and methodsTo test this hypothesis, we employed the rat model of streptozotocin-induced AD and administered butylated hydroxytoluene orally at a dose of 120 mg/kg body weight. Following BHT treatment, neuronal cell death was induced by bilateral stereotactic intraventricular injection of streptozotocin at a dose of 3.0 mg/kg body weight. Three weeks after surgery, we assessed the learning capabilities and the short-term memory of three experimental groups using the conventional y-maze test: (i) streptozotocin-treated rats (BHT pre-treatment), (ii) streptozotocin-treated rats (no BHT pre-treatment), (iii) sham-operated rats (BHT pre-treatment but no streptozotocin administration). After the y-maze test, the animals were sacrificed, hippocampal tissue was prepared and several biochemical (malonyl dialdehyde formation, glutathione homeostasis, gene expression patterns) and histochemical (Congo-red staining, Nissl staining, Perls staining) readout parameters were quantified.ResultsIntraventricular streptozotocin injection induced the development of AD-related symptoms, elevated the degree of lipid peroxidation and upregulated the expression of ferroptosis-related genes. Histochemical analysis indicated neuronal cell death and neuroinflammation, which were paralleled by aberrant intraneuronal iron deposition. The streptozotocin-induced alterations were significantly reduced and sometimes even abolished by oral BHT treatment.ConclusionOur data indicate that oral BHT treatment attenuated the development of AD-related symptoms in an in vivo rat model, most probably via inhibiting neuronal ferroptosis. These findings suggest that BHT might constitute a promising candidate as anti-AD drug. However, more work is needed to explore the potential applicability of BHT in other models of neurodegeneration and in additional ferroptosis-related disorders.

Higher detectability of amyloid with phenol Congo red compared with alkaline Congo red

IntroductionAmyloidosis is a heterogeneous group of conditions associated with tissue deposition of insoluble abnormal proteins that damage vital organs. Early diagnosis, when the deposits are minimal, determines the prognosis and requires histological confirmation. The commonly adopted gold standard technique is alkaline Congo red (ACR) staining, though its sensitivity is limited. There is a need for a simple and inexpensive screening method offering a better chance of detecting mini­mal amyloid deposits. The aim of this study was to compare amyloid detectability with ACR and phenol Congo red (PHCR) staining techniques for early detection of minimal deposits.Material and methodsWe assessed 452 tissue specimens (including adipose tissue, gastrointestinal mucosa, labial salivary gland, myocardium, and bone marrow) from 425 patients with clinically suspected systemic or local amyloidosis, which had been sent to the Pathology Laboratory of the National Institute of Geriatrics, Rheumatology and Rehabilitation in Warsaw. Adjacent sections from each specimen were stained with ACR and PHCR. If amyloid was detected, immunohistochemical typing was conducted. The consistency of the two staining methods was expressed as Cohen’s κ coefficient.ResultsA total of 169 tissue specimens (37%) yielded positive readings, with 93 cases ACR(+) and PHCR(+); 75 cases ACR(–) and PHCR(+), and 1 case ACR(+) and PHCR(–). The percentage agreement between the staining methods was 83%, with the Cohen’s κ coefficient value of 0.60 (95% CI: 0.52–0.69), which corresponds to moderate agreement according to Fleiss. Additional immunohistochemical amyloid typing, conducted in ACR(–) and PHCR(+) specimens, yielded conclusive results in 82% of cases.ConclusionsThe use of PHCR staining as a screening method in suspected amyloidosis improves amyloid (light chain, transthyretin, and amyloid A protein) detectability in various tissues. The PHCR staining specificity should be verified via electron microscopy and/or mass spectrometry.

Role of BIX01294 in the intracranial inhibition of H3K9 methylation lessens neuronal loss in vascular dementia model.

Dementia develops as a result of multiple factors, including cerebrovascular disease which is called vascular dementia (VD). Histone-3 lysine-9 dimethylation (H3K9me2) broadly increases during VD and inhibits neuroprotective gene expressions. So, we aimed to determine how H3K9me2 inhibitor (BIX01294) affects neuronal damage in VD. An in vivo model of VD was used followed by BIX01294 treatment. Behavioral tests, hematoxylin, and eosin (H&E), Congo red, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were carried out. Hippocampal phosphorylated cyclic-AMP responsive element binding protein (p-CREB), c-fos, brain-derived neurotrophic factor (BDNF), and H3K9me2, were detected by western blot analysis technique. Neurological deficit and anxiety-related behavior significantly reduced in the treatment group compared to the VD group (p < 0.05). BIX01294 improved spatial and passive avoidance memory (p < 0.01 and p < 0.05, respectively) compared to the VD group. Treatment with BIX01294 restored the level of p-CREB/CREB ratio (p < 0.05), cfos (p < 0.01), BDNF (p < 0.01), and suppressed H3K9me2 (p < 0.001) when compared to the VD group. BIX01294 microinjection reduced the apoptosis level in TUNEL staining (p < 0.05), and raised neural cell count in H&E staining (p < 0.01); amyloid beta accumulation significantly decreased in the treatment group (p < 0.05) compared to the VD group. In conclusion, long-term treatment with a low dose of BIX01294 can prevent the progression of neuronal loss in VD model by raising the expression of neurotrophic factors, and reducing the apoptosis level.

Down regulation of the c-Fos/MAP kinase signaling pathway during learning memory processes coincides with low GnRH levels in aluminum chloride-induced Alzheimer's male rats.

Aluminum chloride (Al) is associated with Alzheimer's disease (AD) and reproductive disorders. But the relationship between gonadotropin-releasing hormone (GnRH) and c-Fos levels, the end product of MAP-kinase signaling, in AD is unknown, so we aimed to investigate this relationship. We exposed rats to Al dissolved in drinking water (10 and 50mg/kg) for two and four weeks. The control group received only drinking water. At the end, the blood sample was collected under deep anesthesia and the brain was dissected on ice, and the testicular tissue was fixed in formalin. Amyloid beta (βA) plaques in brain regions and the number of CA1 neurons were evaluated by Congo red staining and cresyl violet staining. Activation of neuronal nitric oxide synthase (nNOS) was studied using NADPH-diaphorase. The levels of c-Fos and testosterone receptors in the target area were examined immunohistochemically. Brain GnRH levels were determined by blotting, and serum levels of gonadotropins and steroids were measured by enzyme-linked immunosorbent assay (ELISA). All data were analyzed using analysis of variance (ANOVA) at α = 0.05 level. The accumulation of βA plaque was observed along with a decrease in the number of CA1 pyramidal neurons and a significant decrease in the levels of c-Fos and GnRH in the brains of rats receiving Al, which was aligned with a significant decrease in serum levels of testosterone and LH. This study, for the first time, showed a link between dementia and a concomitant decrease in brain GnRH and c-Fos levels.

Tumefactive Pulmonary amyloidosis in an elderly male: A Diagnostic Dilemma

Abstract Introduction/Objective Tumefactive pulmonary amyloidosis is a rare variant of pulmonary amyloidosis characterized by the extracellular deposition of abnormal protein fibrils in the lung parenchyma, forming tumor-like masses called amyloidomas. Although most cases are clinically silent, it may present with findings that mimic a wide range of respiratory conditions (eg. primary or metastatic malignant nodules, granulomatous disease, multiple pulmonary hamartomas etc), thereby necessitating a comprehensive and broad-based approach to differential diagnosis. Methods/Case Report We present a case of an 88-year-old male with a history of smoking and multiple comorbidities. He presented with numerous bilateral pulmonary nodules identified on a CT scan, concerning for malignancy. A biopsy from a left upper lobe lesion revealed necrotic tissue and was non-diagnostic. A PET scan to assess for metastatic disease was unremarkable, and demonstrated mild hypermetabolic activity, confined to the lungs, with standardized uptake values (SUV) ranging from 4.3 to a maximum SUV of 8.9. A subsequent right lung biopsy was positive for amyloid, confirmed by Congo red staining. A Mass spectrometry analysis further identified the amyloid as AL type. Serum, and urine protein electrophoresis with immunofixation were negative for M protein and the serum free light chain assay was normal as well. Significantly, the bone marrow biopsy also yielded normal results, and the patient was diagnosed with a localized form of AL amyloidosis. Results (if a Case Study enter NA) NA Conclusion This case highlights how tumefactive pulmonary amyloidosis poses a diagnostic challenge due to its rarity and resemblance to malignancy on imaging. Careful histopathological examination, including immunohistochemical stains and amyloid typing, was pivotal in establishing the correct diagnosis and underscores the essential role of pathology and laboratory medicine in differentiating rare pulmonary conditions, along with the need for a multidisciplinary approach in complex diagnostic scenarios.

Secondary Amyloidosis Treated with Tocilizumab as a Complication of Temporal Arteritis.

Temporal arteritis is a large-vessel vasculitis mostly seen in the elderly. Amyloidosis may be secondary to a chronic inflammation of body organs. Here, we present the second case report of temporal arteritis complicated by amyloidosis that was successfully treated by tocilizumab. A 64-year-old female presented complaining of fatigue, fever, and diarrhea accompanied by abdominal pain. One year before presentation, she was diagnosed with temporal arteritis. She was treated with 15 mg/day oral prednisone for the last 6 months, with partial remission, but persistence of the fatigue and an elevated erythrocyte sedimentation rate (ESR, 56 mm/h). Physical examination showed tenderness of both temporal arteries and a soft abdomen. Colon tissue biopsy showed amyloid depositions in the vessels and stroma that were positive for Congo red staining. Tocilizumab was started with 8 mg/kg intravenous, the diarrhea resolved, and the arthralgia improved within 1 month, with a decrease in the ESR to 8 mm/h, and a C-reactive protein (CRP) level of 0.98 mg/dl. Monthly tocilizumab therapy remains efficacious 12 months later and was stopped due to lack of tocilizumab from the hospital. No side effects of tocilizumab were registered. Chronic inflammation may be complicated by amyloidosis in patients with rheumatic diseases and genetic predisposition. Therefore, it is important to screen for intestinal Amyloid A (AA) amyloidosis in individuals with gastrointestinal disorders complicated by rheumatic disorders. AA amyloidosis may be complicated by temporal arteritis and presented with gastrointestinal symptoms such as diarrhea. Amyloidosis is manifested by the deposition of insoluble protein aggregates in organs.Amyloid A (AA) amyloidosis occurs as a complication of chronic inflammation in patients with a genetic predisposition to rheumatic diseases.Temporal arteritis complicated with AA amyloidosis is extremely rare.

Identification and Growth Characteristics of Cellulase Producing Bacteria

This study was envisaged to from fresh cow dung using the Congo red staining method, and screened by measuring their endoglucanase activity (CMC enzyme activity), exoglucanase activity (Cex enzyme activity), and filter paper enzyme activity (FPA enzyme activity). Subsequently, the molecular, morphological characteristics, and growth characteristics were studied. Three strains showed clear transparent circles on the Congo red cellulose medium. LY3 exhibited strong enzyme production ability, with relatively higher enzyme activity than the other two strains. LY3 was found to be a dominant strain that required a temperature of 40℃and pH 7.0 for optimal growth. Based on the molecular and morphological characteristics, the three strains were preliminarily identified as Bacillus. Bangladesh J. Bot. 53(3): 849-856, 2024 (September) Special

7289 An interesting case of Diabetes Mellitus Type 1 induced Renal Tubal Acidosis Type 1

Abstract Disclosure: R. Ripa: None. A. Itani: None. S.K. Patel: None. Introduction: Renal Tubular Acidosis (RTA) are a group of disorders that involve dysfunctional transporters and channels in the renal system. These disorders can be acquired (idiopathic, medication side effects, and medical disorders) or inherited. The most common type worldwide is type 4 RTA, the main cause is diabetic nephropathy leading to hypoaldosteronism. The association of type 1 RTA with diabetes has rarely been reported in studies. Here, we report the presentation, diagnosis and management of a rare case of type 1 RTA in a patient with a history of diabetes mellitus type 1. Case: A male in his 50s with a 30-year history of uncontrolled type 1 diabetes mellitus on insulin pump and coronary artery disease presented to his cardiologist with worsening chest pain, fatigue, polyuria, weight loss of 15 lbs, and recent insulin pump change. Blood tests revealed serum bicarbonate of 10 mmol/L and he was sent to the hospital. On examination, he was hypotensive and bradycardic with dry mouth and decreased skin turgor. His blood tests were consistent with type 1 RTA, he was treated with intravenous fluids containing bicarbonate plus potassium and transitioned to oral potassium citrate on discharge. He underwent renal biopsy as work up was unrevealing for a cause of type 1 RTA. Biopsy revealed normal glomeruli with negative immunofluorescence and Congo red stain. The biopsy, however, did show mild patchy chronic interstitial fibrosis with lymphocytes and mild tubular atrophy. At one year follow up, he remained on oral potassium citrate with resulting serum bicarbonate of 26 mmol/L and a normal renal function. Discussion: Diabetes mellitus can lead to a variety of renal pathologies, specifically metabolic acidosis that can be either seen as diabetic ketoacidosis or hyporeninemic hypoaldosteronism (Type 4 RTA). In our patient, despite being a diabetic, he did not present with either elevated anion gap, ketonuria or hyperkalemic hyperchloremic acidosis typical of type 4 RTA. He was noted to have hypokalemic hyperchloremic acidosis, with a positive urine anion gap, and an alkaline urine, all characteristic of type 1 RTA. Acquired distal RTA occurs more commonly due to certain medications or autoimmune diseases as in Sjögren’s syndrome. The relationship between distal RTA and Sjögren’s syndrome has already been established, unlike the relationship with type 1 diabetes. There have been few case reports of distal RTA in association with type 1 diabetes mellitus, implying a possible autoimmune pathology. Prior case reports also not only had patients who were insulin dependent, but diabetes was present for more than a decade at least suggesting chronicity of disease is needed. Traditional management consists of alkaline therapy and treatment of the underlying disease, or removal of offending medication. In this case, we hope to highlight distal RTA in a diabetic who doesn’t meet criteria for DKA or type 4 RTA. Presentation: 6/1/2024

Development of keratin-based fibers fabricated by interfacial polyelectrolyte complexation for suture applications

Interfacial Polyelectrolyte Complexation (IPC) is a convenient way to produce composite, micro-scale fibers. In this paper, we report the successful development of novel keratin-based IPC fibers and also demonstrate the feasibility of using these fibers as sutures through a proof-of-concept in vivo study. Two composite fibers were produced: chitosan-keratin (CK) and keratin-keratin (KK). These fibers were evaluated for their physico-chemical, mechanical and biochemical properties. In the dry state, the CK fiber had a greater Young's modulus of about 2 GPa while the KK fiber registered a longer strain-at-break of about 100 % due to the strain-stiffening effect. Notably, the keratins were found to assemble into amyloids within the composite fibers based on Congo red staining and Wide-Angle X-Ray Scattering. Functionally, both fibers were malleable could be weaved, braided and knotted. When used as sutures to close incisional wounds in mice over 21 days, these fibers were found to elicit minimal host tissue response and were partially degraded over the duration. Interestingly, the KK fiber evoked a lower extent of immune cell response and fibrous capsule encapsulation that was comparable to commercial, non-absorbable Dafilon® sutures. This work demonstrated the possibility of producing keratin-based IPC fibers which may find practicality as medical sutures.

Luteolin alleviates airway remodeling in asthma by inhibiting the epithelial-mesenchymal transition via β-catenin regulation

BackgroundAsthma is a prevalent long-term inflammatory condition that causes airway inflammation and remodeling. Increasing evidence indicates that epithelial-mesenchymal transition (EMT) holds a prominent implication in airway reconstruction in patients with asthma. Flavonoids obtained from Chinese Materia Medica (CMM), such as Luteolin (Lut), exhibit various beneficial effects in various asthma models. Lut has been shown to mitigate various asthma symptoms, including airway inflammation, hyperresponsiveness, bronchoconstriction, excessive mucus production, pulmonary autophagy, and neutrophilic asthma. However, whether flavonoids can suppress EMT-associated airway remodeling in asthma and the fundamental mechanisms involved remain unclear, with no studies specifically addressing Lut in this context. PurposeTo evaluate the inhibition of airway remodeling in asthma by Lut and its potential mechanisms, while examining the significance of β-catenin in this process through cellular and animal studies. MethodsA BEAS-2B cell model stimulated by lipopolysaccharide (LPS) was established in vitro. Wound closure and Transwell assays were utilized to assess the cellular migratory ability. EMT- and fibrosis-related markers in LPS-stimulated cells were evaluated using RT-qPCR and western blotting. The status of the β-catenin/E-cadherin and β-catenin destruction complexes was evaluated using western blotting, immunofluorescence (IF) staining, and co-immunoprecipitation (Co-IP) analysis. The regulatory function of Lut in β-catenin-dependent EMT was further validated by β-catenin overexpression with adenovirus transduction and siRNA-mediated knockdown of β-catenin. Moreover, the counts of different types of bronchoalveolar lavage fluid (BALF) inflammatory cells from mice with asthma induced by ovalbumin (OVA) were evaluated in vivo using Congo red staining. Hematoxylin and eosin (H&E), Masson's trichrome, and periodic acid-Schiff (PAS) staining were used to evaluate collagen deposition, mucus production, and inflammation in murine lung tissues. Western blotting and immunohistochemistry (IHC) assays were used to assess EMT- and fibrosis-related markers in the lung tissues in vivo. ResultSix naturally derived flavonoids, including Lut, attenuated cell migration and prevented EMT in LPS-treated BEAS-2B cells. Moreover, Lut suppressed TGF-β1, MMP-9, fibronectin (FN), and α-smooth muscle actin (α-SMA) levels in LPS-stimulated BEAS-2B cells. Additionally, Lut downregulated the levels of β-catenin by modulating the β-catenin/E-cadherin and β-catenin destruction complexes, highlighting the pivotal role of β-catenin in EMT inhibition by Lut in LPS-stimulated BEAS-2B cells. Furthermore, Lut suppressed airway inflammation and attenuated EMT-associated airway remodeling through β-catenin blockade in OVA-induced asthmatic mice. The bronchial wall thickness notably reduced from 37.24 ± 4.00 μm in the asthmatic model group to 30.06 ± 4.40 μm in the Lut low-dose group and 24.69 ± 2.87 μm in the Lut high-dose group. ConclusionAccording to our current understanding, this research is the first to reveal that Lut diminishes airway remodeling in asthma by inhibiting EMT via β-catenin regulation, thereby filling a research gap concerning Lut and flavonoids. These results provide a theoretical basis for treating asthma with anti-asthmatic CMM, as well as a candidate and complementary therapeutic approach to treat asthma.

An in vitro cell model for exploring inflammatory and amyloidogenic events in alkaptonuria.

Alkaptonuria (AKU) is a progressive systemic inherited metabolic disorder primarily affecting the osteoarticular system, characterized by the degeneration of cartilage induced by ochronosis, ultimately leading to early osteoarthritis (OA). However, investigating AKU pathology in human chondrocytes, which is crucial for understanding the disease, encounters challenges due to limited availability and donor variability. To overcome this obstacle, an in vitro model has been established using homogentisic acid (HGA) to simulate AKU conditions. This model employed immortalized C20/A4 human chondrocytes and serves as a dependable platform for studying AKU pathogenesis. Significantly, the model demonstrates the accumulation of ochronotic pigment in HGA-treated cells, consistent with findings from previous studies. Furthermore, investigations into inflammatory processes during HGA exposure revealed notable oxidative stress, as indicated by elevated levels of reactive oxygen species and lipid peroxidation. Additionally, the model demonstrated HGA-induced inflammatory responses, evidenced by increased production of nitric oxide, overexpression of inducible nitric oxide synthase, and cyclooxygenase-2. These findings underscore the model's utility in studying inflammation associated with AKU. Moreover, analysis of serum amyloid A and serum amyloid P proteins revealed a potential interaction, corroborating evidence of amyloid fibril formation. This hypothesis was further supported by Congo red staining, which showed fibril formation exclusively in HGA-treated cells. Overall, the C20/A4 cell model provided valuable insights into AKU pathogenesis, emphasizing its potential for facilitating drug development and therapeutic interventions.

L’amylose – point de vue du pathologiste

Despite the advent of scintigraphic diagnosis of transthyretin amyloidosis, the role of pathologists remains central for the diagnosis and typing of amyloidosis. The anatomo-pathological diagnosis and typing of amyloidosis are hindered by pitfalls that should be known, linked to the Congo red staining (technical implementation and interpretation) and the difficulties of immunohistochemistry. The use of expert centers is recommended for difficult cases and when the type cannot be confirmed.

Virtual birefringence imaging and histological staining of amyloid deposits in label-free tissue using autofluorescence microscopy and deep learning

Systemic amyloidosis involves the deposition of misfolded proteins in organs/tissues, leading to progressive organ dysfunction and failure. Congo red is the gold-standard chemical stain for visualizing amyloid deposits in tissue, showing birefringence under polarization microscopy. However, Congo red staining is tedious and costly to perform, and prone to false diagnoses due to variations in amyloid amount, staining quality and manual examination of tissue under a polarization microscope. We report virtual birefringence imaging and virtual Congo red staining of label-free human tissue to show that a single neural network can transform autofluorescence images of label-free tissue into brightfield and polarized microscopy images, matching their histochemically stained versions. Blind testing with quantitative metrics and pathologist evaluations on cardiac tissue showed that our virtually stained polarization and brightfield images highlight amyloid patterns in a consistent manner, mitigating challenges due to variations in chemical staining quality and manual imaging processes in the clinical workflow.

Distinct prebiotic effects of polysaccharide fractions from Polygonatum kingianum on gut microbiota

This study investigated the physicochemical properties, digestive stability, and in vitro fermentation behavior of Polygonatum kingianum polysaccharide (PKP) fractions (PKP60, PKP70, PKP80) obtained through graded ethanol precipitation. High-performance gel permeation chromatography revealed significant molecular weight differences among the fractions, while reverse-phase high-performance liquid chromatography indicated consistent monosaccharide types with variations in their proportions. Uronic acid analysis confirmed that all polysaccharide fractions met the criteria for neutral polysaccharides. Congo red staining confirmed the presence of a triple-helix structure in all PKP fractions. Comprehensive analysis demonstrated that these fractions remained stable during in vitro digestion, as evidenced by consistent molecular weights and total carbohydrate content, with no significant production of free monosaccharides or reducing sugars. All PKP fractions were fermented by gut microbiota, resulting in the production of short-chain fatty acids. Beta diversity and structural analyses of gut microbiota revealed distinct modulatory effects associated with each PKP fraction. The PKP fractions promoted probiotic growth, especially PKP70, which significantly enhanced Bifidobacterium proliferation, indicating strong prebiotic potential. These findings underscore the importance of isolation and purification methods in determining the functionality and gut microbiota-modulating effects of plant-derived polysaccharides, emphasizing the need for in-depth research that extends beyond merely evaluating their source.

Amylum forms typical self-assembled amyloid fibrils.

Amyloid fibril formation is a central biochemical process in pathology and physiology. Over decades, substantial advances were made in elucidating the mechanisms of amyloidogenesis, its links to disease, and the production of functional supramolecular structures. While the term "amyloid" denotes starch-like features of these assemblies, no evidence of amyloidogenic behavior of polysaccharides has been so far reported. Here, we investigate the potential of amylum (starch) not only to self-assemble into hierarchical fibrillar structures but also to exhibit canonical amyloidogenic properties. Ordered amylum structures were formed through a sigmoidal growth process with characteristic amyloid features including typical nanofibril morphology, binding to indicative dyes, inherent luminescence, apple-green birefringence upon Congo red staining, and notable mechanical rigidity. These findings shed light on polysaccharide self-assembly and expand the generic amyloid phenomenon.