The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway.

INTRODUCTION: Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations. METHODS: HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1ß (IL-1ß) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively. RESULTS: TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1ß, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1ß stimulation. These in vitro results correlated with patients' data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1ß induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation. CONCLUSIONS: The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1ß stimulation.

The intracellular quality control system down-regulates the secretion of amyloidogenic apolipoprotein A-I variants: a possible impact on the natural history of the disease.

Hereditary systemic amyloidosis caused by apolipoprotein A-I variants is a dominantly inherited disease characterised by fibrillar deposits mainly localized in the kidneys, liver, testis and heart. We have previously shown that the apolipoprotein A-I variant circulates in plasma at lower levels than the wild-type form (Mangione et al., 2001; Obici et al., 2004) thus raising the possibility that the amyloid deposits could sequester the circulating amyloidogenic chain or that the intracellular quality control can catch and capture the misfolded amyloidogenic chain before the secretion. In this study we have measured plasma levels of the wild-type and the variant Leu75Pro apolipoprotein A-I in two young heterozygous carriers in which tissue amyloid deposition was still absent. In both cases, the mutant was present at significantly lower levels than the wild-type form, thus indicating that the low plasma concentration of the apolipoprotein A-I variant is not a consequence of the protein entrapment in the amyloid deposits. In order to explore the cell secretion of amyloidogenic apolipoprotein A-I variants, we have studied COS-7 cells expressing either wild-type apolipoprotein A-I or two amyloidogenic mutants: Leu75Pro and Leu174Ser. Quantification of intracellular and extracellular apolipoprotein A-I alongside the intra-cytoplasmatic localization indicates that, unlike the wild-type protein, both variants are retained within the cells and mainly accumulate in the endoplasmic reticulum. The low plasma concentration of amyloidogenic apolipoprotein A-I may therefore be ascribed to the activity of the intracellular quality control that represents a first line of defence against the secretion of pathogenic variants.

Embryonic stem and haematopoietic progenitor cells resist to Aß oligomer toxicity and maintain the differentiation potency in culture.

Regenerative medicine deals with the possible use of stem cells to repair tissues damaged by aging and related diseases, including amyloidoses. In the latter case, the toxicity of the amyloid deposits can, in principle, question the possibility to graft specific tissues by undifferentiated cells. To assess whether stem cells are vulnerable to amyloid toxicity, we exposed, in culture, murine embryonic stem (ES) cells and haematopoietic progenitor (HP) cells to oligomers of the amyloidogenic peptide Aß42 at concentrations previously shown to be cytotoxic to several other cell types. These stem cells did not display any sign of apoptosis and their survival, proliferation and differentiation were not affected by the oligomers although the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay revealed that ES, but not HP, cells displayed some impaired ability to reduce the tetrazole salts possibly as a result of transient oxidative stress. Our results support a remarkable resistance of the investigated stem cells against amyloids and hence their potential use in cell therapy of Alzheimer's disease and, possibly, other amyloid diseases.

Dysfunctional inflammasome in Schnitzler's syndrome.

OBJECTIVE: IL-1beta plays a key role in the pathogenesis of Schnitzler's syndrome (SS). We have investigated inflammasome activity in peripheral blood mononuclear cells (PBMCs) from a patient affected by a variant type of SS. METHODS: PBMCs were purified by Ficoll and examined for ability to secrete IL-1beta and -18, expression and function of the P2X(7) receptor and expression of apoptosis-associated speck-like protein containing a caspase recruitment domaine (ASC) and NOD-like receptor protein 3 (NLRP3) before and after the therapy with steroid. Furthermore, extracellular adenosine 5'-triphosphate (ATP) blood levels were determined by luciferase assay. Expression of inflammasome components was measured by real time PCR and western blotting. RESULTS: PBMCs of patient with SS showed a high, spontaneous and lipopolysaccharide-stimulated, IL-1beta release but low response to stimulation with the P2X(7) agonist benzoyl ATP. P2X(7) expression was several fold increased, whereas ASC expression was dramatically decreased compared with PBMCs from healthy controls. NLRP3 expression was unchanged. Prednisone treatment induced remission of clinical symptoms and normalized IL-1beta secretion and P2X(7) and ASC expression. CONCLUSION: These findings reveal the presence of an overall derangement of the inflammasome and IL-1beta processing and release in SS.

Clinical, radiological, and biochemical features of a bilateral buttock amyloidoma emerging after 27 years of hemodialysis.

Deposition of amyloid in the buttock is a rare complication of dialysis related amyloidosis (DRA), but this localization is even rarer in other types of amyloidoses. We report here the clinical, radiological, and biochemical features of a patient who incurred into this complication after 27 years of hemodialysis. Imaging of the amyloid deposition by magnetic resonance imaging (MRI) documents the amyloid infiltration in the muscles of the buttock region and highlights a peculiar feature of amyloid fibrils deposition in the subcutaneous fat. The amyloid deposition is confirmed by biochemical and microscopic analysis of fibrils extracted from a biopsy specimen. Review of literature and the features of this case lead to speculation that the peculiar involvement of the buttock region including muscles and subcutaneous fat in DRA might derive from the propagation of amyloid initially deposited in the hip joint.

Identification of amyloidogenic light chains requires the combination of serum-free light chain assay with immunofixation of serum and urine.

BACKGROUND: The diagnosis of systemic immunoglobulin light-chain (AL) amyloidosis requires demonstration of amyloid deposits in a tissue biopsy and amyloidogenic monoclonal light chains. The optimal strategy to identify the amyloidogenic clone has not been established. We prospectively assessed the diagnostic sensitivity of the serum free light chain (FLC) kappa/lambda ratio, a commercial serum and urine agarose gel electrophoresis immunofixation (IFE), and the high-resolution agarose gel electrophoresis immunofixation (HR-IFE) developed at our referral center in patients with AL amyloidosis, in whom the amyloidogenic light chain was unequivocally identified in the amyloid deposits. METHODS: The amyloidogenic light chain was identified in 121 consecutive patients with AL amyloidosis by immunoelectron microscopy analysis of abdominal fat aspirates and/or organ biopsies. We characterized the monoclonal light chain by using IFE and HR-IFE in serum and urine and the FLC kappa/lambda ratio in serum. We then compared the diagnostic sensitivities of the 3 assays. RESULTS: The HR-IFE of serum and urine identified the amyloidogenic light chain in all 115 patients with a monoclonal gammopathy. Six patients with a biclonal gammopathy were omitted from the statistical analysis. The diagnostic sensitivity of commercial serum and urine IFE was greater than that of the FLC kappa/lambda ratio (96% vs 76%). The combination of serum IFE and the FLC assay detected the amyloidogenic light chain in 96% of patients. The combination of IFE of both serum and urine with the FLC kappa/lambda ratio had a 100% sensitivity. CONCLUSIONS: The identification of amyloidogenic light chains cannot rely on a single test and requires the combination of a commercially available FLC assay with immunofixation of both serum and urine.

Renal apolipoprotein A-I amyloidosis: a rare and usually ignored cause of hereditary tubulointerstitial nephritis.

Apolipoprotein A-I amyloidosis is a rare, late-onset, autosomal dominant condition characterized by systemic deposition of amyloid in tissues, the major clinical problems being related to renal, hepatic, and cardiac involvement. Described is the clinical and histologic picture of renal involvement as a result of apolipoprotein A-I amyloidosis in five families of Italian ancestry. In all of the affected family members, the disease was caused by the Leu75Pro heterozygous mutation in exon 4 of apolipoprotein A-I gene, as demonstrated by direct sequencing and RFLP analysis. Immunohistochemistry confirmed that amyloid deposits were specifically stained with an anti-apolipoprotein A-I antibody. The clinical phenotype was mainly characterized by a variable combination of kidney and liver disturbance. The occurrence of renal involvement seemed to be almost universal, although its severity varied greatly ranging from subclinical organ damage to overt, slowly progressive renal dysfunction. The renal presentation was consistent with a tubulointerstitial disease, as suggested by the findings of defective urine-concentrating capacity, moderate polyuria, negative urinalysis, and mild tubular proteinuria. Histology confirmed tubulointerstitial nephritis. Surprising, amyloid was restricted to nonglomerular regions and limited to the renal medulla. This location of apolipoprotein A-I amyloid differs sharply from other systemic amyloidoses that are mainly characterized by glomerular and vascular deposits. The tubulointerstitial nephritis as a result of hereditary apolipoprotein A-I amyloidosis is a rare disease and a challenging diagnosis to recognize. Patients who present with familial tubulointerstitial nephritis associated with liver disease require a high index of suspicion for apolipoprotein A-I amyloidosis.

A novel AbetaPP mutation exclusively associated with cerebral amyloid angiopathy.

Mutations in AbetaPP cause deposition of Abeta amyloid fibrils in brain parenchyma and cerebral vessels, resulting in Alzheimer's disease (AD) and/or cerebral amyloid angiopathy (CAA). We report a novel mutation (L705V) within the Abeta sequence of AbetaPP in a family with autosomal dominant, recurrent intracerebral hemorrhages. Pathological examination disclosed severe CAA, without parenchymal amyloid plaques or neurofibrillary tangles. This variant highlights the vascular tropism of mutated Abeta, resulting in CAA instead of the pathological hallmarks of AD.

Liver biopsy discloses a new apolipoprotein A-I hereditary amyloidosis in several unrelated Italian families.

BACKGROUND & AIMS: Hereditary systemic amyloidoses are autosomal dominant, late-onset disorders caused by mutations in the genes for a group of plasma proteins including transthyretin, lysozyme, fibrinogen Aalpha chain, gelsolin, apolipoprotein A-I, and apolipoprotein A-II. We investigated both phenotypic and genotypic aspects of apolipoprotein A-I amyloidosis unexpectedly disclosed by liver biopsy in 13 unrelated individuals with asymptomatic, persistent elevation of alkaline phosphatase and gamma-glutamyltransferase levels. METHODS: Immunoelectron microscopy was used for in situ characterization of amyloid deposits on liver biopsy specimens. Mutation analysis was performed by sequencing of the apolipoprotein A-I gene in all patients. Wild-type/variant apolipoprotein A-I ratio in plasma high-density lipoproteins was assessed by a peptide mass fingerprinting approach after purification of total apolipoprotein A-I of 2 patients. RESULTS: Family history was informative in 5 cases. Renal failure developed in 9 cases. Hypogonadism due to testicular involvement was observed. Amyloid fibrils specifically stained with anti-apolipoprotein A-I antibody. A novel (Leu75Pro) heterozygous mutation in the apolipoprotein A-I gene was present in affected individuals but not in controls. Variant apolipoprotein A-I was about 10% of the total protein in high-density lipoproteins. CONCLUSIONS: The high number of individuals with apparently sporadic disease might reflect widespread occurrence of this mutation in the population and a milder phenotype of this variant compared with other apolipoprotein A-I amyloidogenic mutants. These findings suggest that specific staining for amyloid should be performed on liver biopsy of individuals with asymptomatic chronic elevation of alkaline phosphatase and gamma-glutamyltransferase levels.