Advanced glycation end product-modified beta2-microglobulin is a component of amyloid fibrils of primary localized cutaneous nodular amyloidosis

Primary localized cutaneous nodular amyloidosis is a rare form of cutaneous amyloidosis. Amyloid fibrils in primary localized cutaneous nodular amyloidosis have been reported to be originated from immunoglobulin light chains. Immunohistochemical studies on the lesional skins of four patients with primary localized cutaneous nodular amyloidosis demonstrated that amyloid deposits of all cases showed a positive reaction with the antibodies for beta2-microglobulin and advanced glycation end products as well as immunoglobulin light chain (kappa or lambda). No beta2-microglobulin and advanced glycation end product immunoreactivity was found in the amyloid deposits of other primary localized cutaneous amyloidosis (lichen amyloidosis and macular amyloidosis). Double immunofluorescence study of the lesional skin of primary localized cutaneous nodular amyloidosis showed that anti-kappa light chain, anti-beta2-microglobulin and anti-advanced glycation end product antibodies mostly reacted with the same area of amyloid deposit. Amyloid proteins were sequentially extracted with distilled water from one case of primary localized cutaneous nodular amyloidosis and recovered in the five water-soluble fractions (fractions I-V). Immunoblot assay of amyloid fibril proteins demonstrated that immunoreactive polypeptides with anti-kappa light chain antibody (29 kDa) and with anti-beta2-microglobulin antibody (12 kDa) were detected in fractions I-V, whereas immunoreactive polypeptide with anti-advanced glycation end product antibody (12 kDa) was detected exclusively in fractions III-V but not in fractions I and II. Two-dimensional polyacrylamide gel electrophoresis revealed that 12 kDa polypeptide in fractions I and II was electrophoretically identical with authentic beta2-microglobulin and that beta2-microglobulin in fractions III-V was advanced glycation end product-modified beta2-microglobulin with more acidic pI value. These results indicate that beta2-microglobulin is another major component of amyloid fibrils in primary localized cutaneous nodular amyloidosis and that beta2-microglobulin in primary localized cutaneous nodular amyloidosis is partly subjected to the modification of advanced glycation end product.     

Galectin‐7 and actin are components of amyloid deposit of localized cutaneous amyloidosis

The precursor protein of localized cutaneous amyloidosis (LCA) is believed to be cytokeratins on the basis of previous immunohistochemical studies. To identify the candidate amyloid protein biochemically, amyloid proteins were extracted with distilled water from lesional skin of LCA associated with Bowen's disease. The proteins were resolved on one‐ or two‐dimensional polyacrylamide gel electrophoresis followed by characterization with immunoblot analysis. The proteins with multiple molecular weights of 50–67 kDa and two proteins with 25 and 35 kDa were identified as keratins, serum amyloid P component and apolipoprotein E, respectively. The unknown 14‐kDa (pI = 7.0) and 42‐kDa (pI = 5.4) proteins reacted with the antibody against galectin‐7 and actin, respectively. The protein with the molecular weight of 14 kDa was identified as galectin‐7 by MALDI‐TOF mass spectrometer. Their electrophoretic mobilities were identical with normal counterparts extracted from cultured normal human keratinocytes. Galectin‐7 and actin were detected by immunoblot assay in the water‐soluble fractions prepared from the lesional skins of two patients with primary LCA. Immunohistochemical studies of tumor‐associated (n = 9) and primary (n = 10) LCA revealed various degrees of positive immunoreactivities with the antibodies for galectin‐7 and F‐actin. Galectin‐7 and actin, which contain considerable amount of β‐sheet structure, may be candidate amyloidogenic proteins of primary and secondary LCA.     

The molecular skin pathology of familial primary localized cutaneous amyloidosis

Please cite this paper as: The molecular skin pathology of familial primary localized cutaneous amyloidosis. Experimental Dermatology 2010; 19: 416–423. Abstract: Familial primary localized cutaneous amyloidosis (FPLCA) is an autosomal dominant disorder associated with chronic itching and skin lichenification. In lesional skin, there are apoptotic basal keratinocytes and deposits of amyloid material on degenerate keratin filaments in the upper dermis. The genetic basis of FPLCA involves mutations in the OSMR and IL31RA genes but the disease pathophysiology is not fully understood. In this study, we identified new pathogenic heterozygous missense mutations in the OSMR gene (p.Val631Leu and p.Asp647Tyr) in two Dutch FPLCA families. We then compared gene expression profiles between FPLCA lesional skin (n = 4) and site‐matched control skin (n = 6). There was twofold or greater upregulation of 34 genes and downregulation of 43 genes. Most changes in gene expression (verified by quantitative RT‐PCR) reflected alterations in epidermal differentiation and proliferation consistent with lichenification, but we also noted a reduction in several interfollicular keratinocyte stem cell markers in FPLCA skin. Differences in gene expression were also noted for proteins involved in apoptosis and nerve conduction. Collectively, this study expands the molecular basis of FPLCA and provides new insight into the skin pathology of this condition.     

Coexistence of beta2 microglobulin and lambda light chain in amyloid fibrils of dialysis-unrelated plasma cell dyscrasia-associated systemic amyloidosis

BACKGROUND: Systemic amyloidosis occurs as a result of amyloid deposition in various tissues. The amyloid fibrils in systemic amyloidosis have been reported to originate from immunoglobulin light chains. OBJECTIVE: We studied the composition of amyloid fibrils from two patients with plasma cell-associated systemic amyloidosis (PASA). METHODS: A double immunofluorescence study of the lesional skin of PASA was undertaken. Amyloid proteins were extracted with distilled water from one case of PASA. RESULTS: The double immunofluorescence study showed that anti-lambda light chain and anti-beta2 microglobulin antibodies mostly reacted with the same area of amyloid deposit. Amyloid deposits from two patients with PASA who had never undergone haemodialysis showed a positive reaction with the antibodies for beta2 microglobulin as well as immunoglobulin lambda light chain. By the use of immunoblot assay of amyloid fibril proteins, polypeptides immunoreactive with antigamma light chain antibody (29 kDa) and with anti-beta2 microglobulin antibody (12 kDa) were detected. CONCLUSIONS: These results indicate that beta2 microglobulin is a component of amyloid fibrils in PASA.     

Formation of immunoglobulin light chain amyloid oligomers in primary cutaneous nodular amyloidosis

Background Primary cutaneous nodular amyloidosis (PCNA) is thought to be a plasma cell dyscrasia. The amyloid deposits are found in the dermis and subcutis, and they contain clonal immunoglobulin light chains, produced by a local proliferation of plasma cells. New insights into amyloid diseases have revealed that the pathology is due more to the presence of small, misfolded protein species termed oligomers than to the deposition of fibrillar material. Objectives To demonstrate the presence of amyloid oligomers in PCNA and to provide evidence that cutaneous amyloid diseases share a common pathogenic pathway similar to other amyloid diseases. Methods Immunohistochemical staining with conformation‐specific and sequence‐specific antibodies was used to localize different amyloid species of light chain immunoglobulins in a case of PCNA. Additionally, in vitro characterization of immunoglobulin oligomers and fibrils was performed to determine, through toxicity studies in a human keratinocyte cell line, which amyloidogenic form of the immunoglobulin is toxic in PCNA. Results Amyloid oligomers were identified in PCNA. Oligomers were mainly formed by lambda light chain immunoglobulins, and kappa light chain oligomers were detected in lesser amounts. Amyloid species were detected intra‐ and extracellularly. In addition, amyloid oligomers and fibrils, derived from unknown protein sources, were detected. This finding suggests that immunoglobulin amyloids can act as seeds capable of inducing the aggregation of heterogeneous proteins in the skin. Furthermore, cytotoxicity studies demonstrated that immunoglobulin oligomers, but not monomers or fibrils, are toxic to human keratinocytes. Conclusions These data indicate that PCNA has common pathways with other amyloid diseases with respect to protein misfolding and pathogenesis. Immunoglobulin oligomers may prove to be targets for the treatment of PCNA.     

Ultrastructural localization of amyloid P component in primary localized cutaneous amyloidosis

Amyloid P component (AP) was specifically localized to dermal amyloid deposits in the papular and nodular variants of primary localized cutaneous amyloidosis by an immunoperoxidase technique using an antibody to serum amyloid P component (SAP). Specific staining with anti-SAP of elastic fibre microfibrils which has previously been observed in normal skin, was also present and was noted in close proximity 10 deposits of amyloid material. AP associated with normal elastic fibre microfibrils may be involved in the deposition of amyloid fibrils in vivo.     

A study of apolipoproteins E and A-I in cutaneous amyloids

BACKGROUND: Apolipoprotein E (apoE) is present in a variety of biochemically different amyloid deposits, including Alzheimer's disease, systemic amyloidosis and primary cutaneous amyloidosis (PCA). Among the three closely related alleleic forms of apoE, the epsilon4 allele is linked to Alzheimer's disease. Apolipoprotein A-I (apoA-I), another apolipoprotein, is also found in senile plaques of Alzheimer's disease and in amyloid of aortic atherosclerotic plaques. Furthermore, apoA-I has recently been found to be associated with hereditary cutaneous and cardiac amyloidosis. OBJECTIVES: To determine whether the apoE epsilon4 allele is associated with increased risk of PCA and whether apoE and apoA-I are present in PCA and common secondary cutaneous amyloidosis (SCA) (i.e. basal cell carcinoma, Bowen's disease and seborrhoeic keratosis). METHODS: We examined the apoE genotype in 57 Chinese patients with PCA and 58 normal healthy control subjects of similar age. In addition, immunohistochemical staining was performed to determine the localization of apoE and apoA-I in skin tissues from 15 patients with SCA and 15 with PCA. RESULTS: The frequency of the epsilon4 allele in the PCA group was not significantly higher than that in the control group (8.8% vs. 6.9%, P > 0.05). ApoE was present in amyloid deposits in both PCA and SCA, but apoA-I was not detected in these cutaneous amyloid deposits. CONCLUSIONS: ApoE is also a component of amyloid deposits in SCA. Although the genetic susceptibility of certain apoE isoforms may not be a crucial factor in the development of PCA and, although apoA-I is not associated with amyloid deposits of PCA and SCA, the role of apolipoproteins in amyloidogenesis deserves further scrutiny.     

Discoid lupus erythematosus with secondary amyloidosis

BACKGROUND: Secondary localized cutaneous amyloidosis is a clinically unapparent phenomenon associated with various cutaneous pathologies, usually tumours of epidermal origin. The amyloid is thought to be derived from keratinocytes. OBJECTIVES: To characterize the amyloid deposition observed incidentally within lesional biopsies from three patients with discoid lupus erythematosus (DLE), and retrospectively to study the phenomenon within DLE skin samples. METHODS: Localized amyloid deposition was observed in three cases of DLE by immunofluorescence studies, and these cases were further studied by histology and immunohistochemistry using a monoclonal anticytokeratin antibody. Retrospective histological review of DLE tissue specimens archived over 12 months was performed to look for evidence of previously undetected amyloid. RESULTS: Amyloid deposition was confirmed histologically in the three index cases by staining with Congo red and thioflavin T. Positive staining with an anticytokeratin antibody demonstrated the epidermal origin of the amyloid protein. Of the 18 archived cases reviewed amyloid was retrospectively detected in one sample. CONCLUSIONS: Secondary cutaneous amyloidosis of keratinocyte origin can be seen in DLE lesions. It may be a not infrequent occurrence and may remain under-reported. We discuss the possible role of disease chronicity and colloid body degradation in the pathogenesis of amyloidosis.     

Extensive haemorrhagic-bullous skin manifestation of systemic AA-amyloidosis associated with IgGlambda-myeloma

In an 86-year-old woman with a multiple myeloma of the IgG lambda subtype a coinciding systemic amyloidosis manifested as a macroglossia, diffuse alopecia and generalized cutaneous involvement. The skin was affected by milium-like papules, petechial haemorrhages and an increased tissue fragility with subsequent blister formation. The typical histology and immunohistology pattern revealed large intradermal amyloid masses, reacting positively with anti-amyloid A antibodies, which surrounded cuff-like dilatated blood capillaries. The abundance of these amyloid deposits led to significant deflexibilization and fragility of the capillaries and the dermal matrix eventually resulting in the haemorrhagic-bullous eruptions. The peculiar feature of the present case is the intensity of bullous-haemorrhagic skin damage due to amyloid A deposition without any detection of cutaneous IgGl as the myeloma-derived paraprotein assumed to be causative for the development of systemic AA amyloidosis.     

Primary localized cutaneous nodular amyloidosis: case report and biochemical analysis of amyloid.

We report a patient with scalp lesions of primary localized cutaneous nodular amyloidosis. The extensive examination revealed no systemic involvement. Analysis of glycosaminoglycans (GAGs) in amyloid deposits showed a twofold increase as compared with normal skin, which was due to the increase in dermatan sulfate. Local disorders of GAG metabolism may be related to the amyloid fibril formation. Amyloid fibrils were purified and identified electron-microscopically, which consisted of two major 12,000- and 13,000-dalton and minor 29,000- and 48,000-dalton peptides. Western blotting analysis showed a minor 29,000-dalton peptide reactive with antibodies against both kappa and lambda light chains of immunoglobulin. There is a possibility that some components of amyloid in some cases of primary localized cutaneous nodular amyloidosis may consist of both kappa and lambda immunoglobulin light chains.     

A monoclonal anti-keratin antibody reactive with amyloid deposit of primary cutaneous amyloidosis

Specimens from cutaneous amyloidoses (lichen amyloidosis and macular amyloidosis) were stained immunohistochemically with monoclonal anti-keratin antibodies. One monoclonal antibody raised against hair keratin (HKN-6) reacted with the amyloids of both primary amyloidoses. Another monoclonal antibody, HKN-2, did not decorate the amyloid deposit. HKN-6 did not stain the interfollicular epidermis, but HKN-2 did. The possible explanations of these findings are 1) amyloid deposits contain keratin protein modulated to react with HKN-6; 2) amyloid deposits contain a protein unrelated to keratin protein, but reactive to HKN-6.     

Immunohistochemical studies on fibrillin in amyloidosis, lichen ruber planus and porphyria.

The pathogenesis of macular amyloidosis and lichen amyloidosis remains unsolved and the primary amyloid fibril protein(s) has not yet been identified. Ultrastructural association of skin amyloid with elastin associated microfibrils has been noted earlier. The presence of fibrillin in conjunction with such microfibrils was recently demonstrated immunohistochemically. The presence of fibrillin immunoreactivity in the amyloid deposits in skin biopsies from 3 patients with macular amyloidosis and 3 patients with lichen amyloidosis was studied, using monoclonal anti-fibrillin antibodies. For comparison, skin specimens were studied from five patients with lichen ruber planus, four patients with erythropoietic protoporphyria and from a patient with myeloma-associated cutaneous amyloidosis. Renal specimens from two cases of the amyloid A type of renal amyloidosis also were investigated. There was no immunostaining either of the keratin bodies in specimens of lichen ruber planus, the cutaneous PAS-positive vascular deposits in patients with erythropoietic protoporphyria, or the amyloid deposits in specimens of systemic amyloidosis and it was faint or absent in amyloid deposits in the specimens from patients with lichen amyloidosis. In contrast, distinct fibrillin immunoreactivity could be demonstrated in amyloid deposits in specimens from patients with macular amyloidosis. It was sometimes absent in deposits located in the upper part of the papillary dermis, close to the dermal epidermal junction zone, while consistently strong in deposits located lower down in the dermis. The results suggest that fibrillin or part of the fibrillin molecule may be present in some of the amyloid deposits in specimens of macular amyloidosis.     

Nodular primary cutaneous amyloidosis. Isolation and characterization of amyloid fibrils

A case of nodular cutaneous amyloidosis and Sj?gren's syndrome occurred in a 63-year-old woman. Nodules had been seen for the past ten years and Sj?gren's syndrome had accompanied amyloidosis for the last three years. The concomitant occurrence of nodular cutaneous amyloidosis and Sj?gren's syndrome may not be by chance, since four of 12 cases of nodular cutaneous amyloidosis that have been reported to date in Japan were in patients with both amyloidosis and Sj?gren's syndrome. The amyloid deposits in the tissue were stained with anti-lambda light-chain amyloid antibody. Amyloid fibrils were purified from the skin lesions in this patient and were characterized biochemically, immunologically, and ultrastructurally. The results indicated that the amyloid fibrils consisted of 29,000-, 20,000-, and 17,000- dalton peptides, the 29,000-dalton peptide of which was shown to react with the lambda light chain of immunoglobulin by immunoblot study.     

Histological and immunofluorescence findings of non-follicular papulopustular lesions in patients with Behçet''s disease

Background Papulopustular lesions (PPL), the most common type of cutaneous lesions in Behçet's disease (BD), clinically may not be differentiated from ordinary acne. Disagreement exists as to the exact nature of these acneiform and folliculitis‐like lesions and whether to include them as a major criterion. Objective We investigated whether PPL can be a useful tool for the diagnosis of BD when non‐follicular lesions over the trunk or extremities were selected, and were correlated with histological and/or immunofluorescence study. Methods Seventeen patients with BD (five women, 12 men; mean ± SEM age, 32 ± 7.9 years), were enrolled in the study with blind histopathological and immunofluorescence studies. Biopsies of the PPL and adjacent (approximately 2 cm distant) normal‐appearing skin were performed from the extremities and trunk. Follicle‐based acneiform lesions and those lesions over face were excluded. Histological evaluation primarily included epidermal and dermal alterations, cellular infiltration and vascular changes. We also performed direct immunofluorescence studies, using polyclonal antibodies for IgA, IgG, IgM, C3 and fibrin. Results Lesional specimens of the patients with BD revealed a significant leucocytoclastic vasculitis as compared with non‐lesional skin (P < 0.05). The vessels of the lesional skin showed a higher IgM deposition than non‐lesional skin (52.9% and 17.6%) (P < 0.05). IgG, C3 and fibrin deposits on the vessels of the lesional skin were also higher than non‐lesional skin (35.3, 11.8%; 41.2, 17.6%; and 47.1, 17.6%, respectively), but the differences were not statistically significant. Conclusions Our findings indicate that non‐follicular PPL over the trunk or extremities are more specific, and immune complex‐mediated vasculitis is likely to be the main feature of these lesions, as they are in other cutaneous lesions of BD.     

Poikiloderma-like Cutaneous Amyloidosis in an Ethnic Chinese Girl

Primary cutaneous amyloidosis is the deposition of amyloid in the skin without involvement of internal organs. It is easily diagnosed when presented in its typical manifestation. Atypical or rare clinical presentations can pose diagnostic difficulties. Poikiloderma-like cutaneous amyloidosis (PCA), a rare variant of primary cutaneous amyloidosis, was first reported in the literature in 1936 (1). It is characterised by: 1) poikilodermatous skin lesions; 2) lichenoid papules; 3) cutaneous amyloid deposit in the pigmented and lichenoid lesions; 4) light sensitivity; 5) short stature; and 6) other features such as blister formation or palmoplantar keratosis. Ogino coined the term PCA syndrome when these unusual features present early in life (2). We report a 26-year-old Chinese woman who presented with poikilodermatous skin lesions and was misdiagnosed as poikiloderma atrophica vasculare (PAV) on the basis of clinical appearance without any histological proof. The diagnosis of PCA was made after skin biopsy which showed amyloid deposits in the skin. This condition can easily be confused with other true poikiloderma skin diseases. Histology is important in confirming the diagnosis.     

Familial primary disseminated amyloidosis (a new clinical form?)

This report concerns two siblings we observed, one male the other female, who presented with primary disseminated amyloidosis. Repeated blood and urine examinations failed to demonstrate dysglobulinaemia. The brother developed, at the age of 51, extensive cutaneous amyloidosis with xanthochromia of the entire upper part of his body. His dermis contained a potassium permanganate-resistant amyloid substance. One year later, he presented with amyloid cardiomyopathy confirmed by biopsy. Owing to the intractable cardiac failure, heart transplantation was performed, but the patient died post-operatively. At autopsy, amyloid deposits were found to be present in the heart, liver, spleen and adrenal glands. His sister developed, at the age of 40, cutaneous amyloidosis in the form of yellowish and purpuric papules and plaques disseminated over the upper part of her body. Histological examination and electron microscopy of the skin showed large potassium permanganate-resistant amyloid deposits. In addition, endoscopy and histology demonstrated the presence of amyloid substance deposits in her larynx, oesophagus and rectum. Echocardiography revealed amyloid cardiomyopathy. She now has moderate cardiac failure, and heart transplantation is being contemplated. Like her brother, she has no renal of neurological amyloid lesions. There is no abnormality of serum or urinary globulins, and her SAA protein is present in normal concentrations. These cases do not fit in with the known nosological framework of amyloidosis. Clinically, both patients had disseminated amyloidosis of the AL type, and their disease clearly differed from familial systemic amyloidosis with neuropathy or nephropathy. To our knowledge, no case of familial primary amyloidosis of the AL type without dysglobulinaemia has yet been reported.     

Immunohistochemical demonstration of amyloid P component in skin of normal subjects and patients with cutaneous amyloidosis

The presence of amyloid P component (AP) in dermal deposits of cutaneous atnyloidosis was demonstrated by a direct immunofluorescence technique using an antibody to serum amyloid P component (SAP). AP was also shown, for the first time, to be a constituent of normal human skin. It was present at the periphery of dermal elastic tissue fibres, in basement membranes of dermal blood vessels and surrounding eccrine sweat glands but was absent from the dermo-epidermal basement membrane. The staining pattern in cutaneous amyloidosis was morphologically distinctive and readily distinguishable from staining of thickened vascular basement membranes in porphyria. Itnmunofluorescence with anti-SAP is simple and specific and may become the procedure of choice in the differential diagnosis of amyloidosis.     

Primary localized cutaneous amyloidosis: dermal amyloid deposits do not bind antibodies to amyloid A protein, prealbumin or fibronectin

The chemical nature of the dermal amyloid deposits in primary localized cutaneous amyloidosis (PLCA) was investigated using histochemical and immunohistochemical techniques. Deposits of macular, papular and nodular PLCA showed potassium permanganate resistance of their affinity for Congo red dye, unlike amyloid A protein (AA) deposits, and did not bind antibodies to AA, prealbumin or fibronectin. Despite the frequent presence of immunoglobulins in deposits of macular and papular PLCA, there is no evidence that the amyloid fibrils are composed of protein AL. Although previous studies have identified keratin-like material in deposits of macular and papular PLCA, the exact nature of the amyloid fibril protein in these conditions, and the mechanism by which tonofilaments of necrotic keratinocytes are trans formed into amyloid material in the papillary dermis, remain uncertain.     

An immunofluorescent study of cutaneous amyloidosis.

Twenty-four patients with primary localized cutaneous amyloidosis were studied by direct immunofluorescent techniques. Autofluorescence of the amyloid deposits was observed in two patients with very chronic lesions. IgG, IgA, IgM and C3 were found in association with the amyloid deposits in about two-thirds of the cases. The immune pathogenesis of amyloid formation is postulated.     

A study of cytokeratin profiles in localized cutaneous amyloids

The major component of localized cutaneous amyloids may be derived from cytokeratin (CK). However, the CK profiles of primary cutaneous amyloidosis (PCA) and secondary cutaneous amyloidosis (SCA) remain obscure. Paraffin-embedded sections of skin tissue from 64 patients with PCA, 111 with SCA and 3 with systemic amyloidosis were analyzed immunohistochemically using 12 different polyclonal or monoclonal anti-CK antibodies (34βE12, MNF116, LP34, AE1/AE3, anti-CK1, CK5, CK6, CK7, CK10, CK14, CK16 and CK17). In addition, frozen skin tissues from 12 patients with PCA were analyzed for comparison with the paraffin-embedded tissue. In all 64 PCA paraffin sections, the amyloid deposits were immunopositive for anti-CK5 antibody and 34βE12. In all 12 frozen sections of PCA, the amyloid deposits were immunopositive for anti-CK5 antibody, 34βE12, MNF116 and LP34, and seven (58.3%), three (25%) and one (8.3%) were immunopositive for anti-CK1, CK14, and CK10 antibodies, respectively. In all SCA sections, the amyloid deposits were immunopositive for CK5 and 34βE12. In addition, MNF116 immunolabeled amyloids of all sections from patients with basal cell carcinoma and trichoepithelioma, and MNF116 and LP34 immunolabeled amyloids of sections from patients with porokeratosis. Our results indicate that CK5 is the major CK present in the amyloid deposits of PCA and SCA, and “amyloid-K” is mainly derived from basal keratinocytes.