Charge differences between in vivo deposits in immunoglobulin light chain amyloidosis and non-amyloid light chain deposition disease

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders that may occur in plasma cell dyscrasias with dysproteinemia. In systemic AL, the deposits are fibrillar and patchy in distribution, within and amongst different organs, whereas in NALCDD, the deposits are granular and diffusely distributed in systemic basement membranes, suggesting different mechanisms of aggregation and deposition. Previous evidence, that charge differences between the light chains in AL and NALCDD might account for their different phenotypes, prompted the present study, which compared the isoelectric points (pIs) of AL and NALCDD protein deposits extracted from human tissues. The pI profiles (5.2-8.8) of polypeptides in AL deposits were heterogenous in four cases, with a spread of both anionic and cationic isoforms; in contrast, in three of NALCDD the pI profiles (8.2-8.8) were homogeneous and restricted in the cationic range. These in vivo findings in human disease, together with other reported in vitro and in vivo experimental data, suggest that the fibrillar deposits in AL may form by electrostatic interaction between oppositely charged polypeptides, whereas the granular deposits in NALCDD form by the binding of cationic polypeptides to anionic proteoglycans sites in basement membranes.     

Free light chains in plasma of patients with light chain amyloidosis and non-amyloid light chain deposition disease. High proportion and heterogeneity of disulfide-linked monoclonal free light chains as pathogenic features of amyloid disease

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders accompanied by a monoclonal gammopathy. Monoclonal free light chains (FLCs) are precursors of the pathological light chain tissue deposits that are fibrillar in AL and granular in NALCDD. However, direct biochemical examination of plasma FLC precursors, which would allow comparison and better understanding of these two diseases, is still lacking. In this study, we examined FLCs in plasma of patients with AL and NALCDD by employing separation on Sep-PaK C18 cartridges, micro-preparative electrophoresis, Western blotting and mass spectrometry. Comparative analysis of AL versus NALCDD and control plasma samples showed new evidence of increased level and heterogeneity of circulating disulfide-bound FLC species in AL. In addition to full length monomers comprising the disulfide-linked FLCs, the monoclonal disulfide-bound FLC fragments were typically revealed in AL plasma. We hypothesized that enhanced disulfide binding of FLCs in AL interferes with their normal clearance and metabolism, which in turn might play a role in amyloid formation. The applied methods might be useful to diagnose or predict the pathological form of the disease and shed light on the mechanisms involved in light chain aggregation in tissues.     

Serum levels of immunoglobulin free light chains in patients with chronic hepatitis C presenting cryoglobulinemia

Hepatitis C virus (HCV) infects B-lymphocytes, provokes cellular dysfunction and causes lymphoproliferative diseases such as cryoglobulinemia and non-Hodgkin's B-cell lymphoma. In the present study, we investigated the serum levels of kappa and lambda free light chains (FLC) of immunoglobulins and the kappa/lambda FLC ratio in Brazilian patients with chronic HCV infection and cryoglobulinemia. We also analyzed the immunochemical composition of the cryoglobulins in these patients. Twenty-eight cryoglobulinemic HCV patients composed the target group, while 37 HCV patients without cryoglobulinemia were included as controls. The median levels of kappa and lambda FLC were higher in patients with cryoglobulinemia compared to controls (p = 0.001 and p = 0.003, respectively), but the kappa/lambda FLC ratio was similar in patients with and without cryoglobulinemia (p > 0.05). The median FLC ratio was higher in HCV patients presenting with advanced fibrosis of the liver compared to HCV patients without fibrosis (p = 0.004). Kappa and lambda FLC levels were strongly correlated with the IgA, IgG and IgM levels in the patients with cryoglobulinemia. In patients without cryoglobulinemia, the kappa FLC level was only correlated with the IgG level, whereas the lambda FLC were weakly correlated with the IgA, IgG and IgM levels. An immunochemical pattern of mixed cryoglobulins (MC), predominantly IgM, IgG, IgA and kappa light chain, was verified in these immune complexes. We concluded that HCV-infected patients presenting cryoglobulinemia have vigorous polyclonal B-lymphocyte activation due to chronic HCV infection and persistent immune stimulation.     

Capillary electrophoresis/immunosubtraction as a better alternative to immunofixation for detecting and immunotyping serum monoclonal proteins in patients with immunoglobulin light chain (AL) amyloidosis

Abstract

Capillary electrophoresis/immunosubtraction (CE/IS) is a simple method for detecting and immunotyping serum or urine monoclonal proteins. To our knowledge, there are no previous reports of the use of CE/IS for characterizing patients with Immunoglobulin light chain (AL) amyloidosis, and there are no convincing data available to compare CE/IS with serum immunofixation electrophoresis (IFE) and free light chain (FLC) assay. The aim of this study was to evaluate the clinical utility of CE/IS in patients with AL amyloidosis as a diagnostic accuracy study. This study included 50 patients with AL amyloidosis (17 newly diagnosed and 33 undergoing treatment). Serum IFE identified monoclonal proteins in 15/50 (30%) of all cases and in 7/17 (41%) of newly diagnosed cases. CE/IS identified monoclonal proteins in 16/50 (32%) of all cases and in 7/17 (41%) of newly diagnosed cases. The FLC assay detected an abnormal ratio of kappa and lambda light chains in 26/50 (52%) of all cases and in 15/17 (88%) of newly diagnosed cases. IFE and CE/IS combined with FLC assay identified monoclonal proteins more sensitive than IFE alone and CE/IS alone, in newly diagnosed patients (p?=?0.002 and 0.002, respectively) and in patients undergoing treatment (p?=?0.031 and 0.016, respectively). CE/IS is an acceptable alternative to IFE.     

Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases

Deposition of immunoglobulin light chains is a result of clonal proliferation of monoclonal plasma cells that secrete free immunoglobulin light chains, also called Bence Jones proteins (BJP). These BJP are present in circulation in large amounts and excreted in urine in various light chain diseases such as light chain amyloidosis (AL), light chain deposition disease (LCDD) and multiple myeloma (MM). BJP from patients with AL, LCDD and MM were purified from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results show that LCDD and MM proteins have the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJP at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. The aggregates formed under in vitro conditions suggested that the various proteins derived from patients with different light chain diseases might follow different aggregation pathways.     

Aberrant expression of immunoglobulin light chain isotype in B lymphocytes from patients with monoclonal gammopathies: isotypic discordance and clonal B-cell excess

A study was made of 29 patients with monoclonal gammopathies to detect aberrations in immunoglobulin (Ig) light chain isotype expression in lymphocytes at various levels of B-cell differentiation, namely, circulating surface Ig positive (SIg+) cells, Ig-secreting cells (plaque forming cells, PFC) and mitogen-induced PFC. By using kappa-lambda analysis, two major phenotypes of aberrant Ig light chain isotype expression were found in circulating B cells at these three levels of differentiation: an absolute increase in B cells bearing the same Ig light chain isotype as that of monoclonal protein (clonal B-cell excess), and a relative decrease in those B cells (isotypic discordance). Isotypic discordance (ID) was found to be essentially negative in patients with monoclonal gammopathy of undetermined significance (MGUS) provided that they were in a stable condition. In myeloma patients, ID was found only in stage I, except for a remission case of stage III (4/7 in stage I, 0/8 in stage II, and 1/6 in stage III). ID was not restricted to a circulating SIg+ cell level but was also demonstrable at a spontaneous or pokeweed mitogen-induced PFC level. However, ID was negative at a PFC level induced by Staphylococcus aureus Cowan I. Clonal B-cell excess (CE) was frequently found in patients with active myeloma but not in stable patients (0/8 in MGUS, 1/7 in stage I, 8/8 in stage II, and 4/6 in stage III). CE was positive not only at a circulating SIg+ cell level but also at a circulating PFC level. Furthermore, patients with CE at a PFC level were found to have a higher proliferating capacity, defined as a percentage labelling index of marrow myeloma cells, than those without CE at a PFC level (P less than 0.02). ID and CE can therefore be considered as useful markers for discriminating between MGUS and myeloma, evaluating the clinical stability and predicting the clinical course.     

轻链沉淀病肾脏损害1例报道及文献回顾

报告了１例轻链沉淀病（ＬＣＤＤ）合并多发性骨髓瘤（ＭＭ）具有典型肾脏损害的患者。临床表现为肾病综合征、肾功能损害、异常浆细胞增生、低γ血症等。肾活检病理示肾小球系膜结节、肾小管基膜增厚、免疫酶标法抗ｋ轻链阳性。ＬＣＤＤ与异常浆细胞增生有关，多合并ＭＭ，肾脏损害为其首发和突出表现，肾功能受累明显肾脏病理有特异性的改变，免疫荧光和免疫酶标法可检出轻链的沉积，是明确诊断的重要依据。     

Antibody reactivities to skeletal muscle proteins in a patient with λ light chain secreting multiple myeloma, generalised amyloidosis and rhabdomyolysis

Rhabdomyolysis is a rare complication in haematological malignancies, and a diverse range of factors has been implicated in the etiology of the syndrome. In the present study we analysed muscle morphology and antibody reactivities to skeletal muscle proteins in a patient diagnosed with lambda (lambda) light chain-secreting multiple myeloma (MM) and amyloidosis, who developed a progressive rhabdomyolysis. The muscle tissue analysis showed focal amyloid depositions and a low degree of atrophy and inflammation. Antibody reactivities against muscle proteins of approximately 42, 51 and 66 kD, respectively, were present in the patient's serum. The antibody specificities were revealed by lambda light chain- or IgM-specific antibodies. The results indicate a possible etiologic link between antibody reactivities towards muscle proteins and muscle tissue disorder in a patient with the unique combination of rhabdomyolysis, amyloidosis and MM of the light chain type.     

Reconstitution of the T-cell repertoire following treatment with alemtuzumab (anti-CD52 monoclonal antibody) in patients with B-cell chronic lymphocytic leukaemia

In this pilot study, T-cell receptor B-variable (TCR-BV) gene usage in CD4 and CD8 T cells was assessed, by real-time polymerase chain reaction, as well as complementarity-determining region 3 (CDR3)-length polymorphism, before and after therapy in five patients with B-cell chronic lymphocytic leukaemia who received alemtuzumab (anti-CD52 monoclonal antibody) as first-line therapy. A decline in expression of most BV family genes in both CD4 and CD8 T cells was observed after alemtuzumab treatment, which was followed by a gradual increase in most BV families during long-term follow-up. After treatment, CDR3-length polymorphism showed an even more restricted pattern in CD4 T cells compared with pretreatment, with a shift towards a monoclonal/oligoclonal pattern. The clonally restricted pattern was significantly reduced in CD4 (P < 0.01) but not in CD8 T cells. This was followed by a gradual increase in the number of peaks within the CDR3 region of the different TCR-BV families, i.e. a polyclonal repertoire, during long-term follow-up. A restricted CDR3 pattern became even more restricted after treatment, but normalised during unmaintained follow-up. These results indicate that perturbations in the T-cell alterations following alemtuzumab are complex and include not only changes in CD4/CD8 T-cell numbers but also a highly restricted T-cell repertoire especially in CD4 T cells.     

一例以胸闷气短为首发症状的系统性轻链（AL）型淀粉样变性个案报道

轻链型(AL)系统样变性是由单克隆免疫球蛋白轻链错误折叠形成淀粉样蛋白，沉积于气管，造成组织结构破坏、气管功能障碍并进行性进展的疾病，主要与克隆性浆细胞异常增殖有关。免疫球蛋白轻链（AL）型淀粉样变性是浆细胞病的一种罕见类型，个体差异大，疾病的首发起病症状不一，且舌体肥大和眶周紫癜是AL型淀粉样变性的特异性表现[3]。这是我们报道这例以胸闷气短为首发症状的免疫球蛋白轻链（AL)型淀粉样变性的主要原因。病例报告：我们报告一例59岁女性患者，以活动后乏力、气短4年余，双下肢水肿，夜间不能平卧起病，患者病史多年，且一直以“胸闷气短”为主要症状，并伴有舌体肥大，在按照心内科常规治疗情况下未见好转，经过多学科会诊及讨论，建议行舌体活检及骨髓穿刺考虑为系统性轻链型（AL）型淀粉样变性。明确诊断后启动化疗，现患者胸闷气短症状明显好转，且舌体较前缩小。结论:以“胸闷气短”为首发症状，且症状反复，心内科治疗效果欠佳，并伴有多系统累及，应考虑该病的诊断且尽快行相关的专科检查，主要依靠组织病理学检查。     

Free immunoglobulin light-chain serum levels in the follow-up of patients with monoclonal gammopathies: correlation with 24-hr urinary light-chain excretion

In patients with light-chain myeloma or primary AL-amyloidosis, 24-hr light-chain excretion in the urine is considered an essential marker of the tumor mass. However, 24-hr urine collection and analysis may be cumbersome and prone to inaccuracy. Recently, a sensitive immunonephelometric assay for immunoglobulin free light chains (FLC) in the serum was developed. We sought to determine whether the serum level of monoclonal FLC could be used as an indicator of urinary excretion and disease evolution. Seven patients with light-chain myeloma and AL-amyloidosis were studied, all of which had a monoclonal FLC that could be detected in the urine using standard methods. In four of these patients, follow-up revealed a remarkable correlation between FLC serum levels and daily urinary excretions. The ratio of serum level to urinary light-chain excretion, although stable in a given patient, was extremely variable between patients. In the three remaining cases featuring hardly measurable amounts of light chain in the urine, the serum FLC assay proved sensitive enough for correlation with clinical events. Thus, immunonephelometric measurement of serum FLCs is a reliable method for the follow-up of patients with light-chain secreting monoclonal gammopathies.     

Diagnostic and prognostic utility of the serum free light chain assay in patients with AL amyloidosis

Background: Organ dysfunction in AL amyloidosis is related to the production and deposition of amyloidogenic monoclonal light chains. These pathological light chains can now be quantified using the recently developed serum free light chain assay. Methods: We retrospectively reviewed 31 patients with AL amyloidosis to determine the frequency of abnormal free light chain assay results at diagnosis and whether changes in the serum free light chain assay predict outcome after therapy. Results: An abnormal free light chain assay was found in 30 of 31 patients (97%) at the time of diagnosis. In the subset of our patients who received treatment for AL amyloidosis, a >50% reduction of the pathological free light chain following treatment was shown to predict improved overall survival. In our series of analyses, achievement of greater magnitudes of reduction of the free light chain result did not appear to provide additional prognostic information, nor did the baseline free light chain result predict outcome. Conclusion: Our findings support the use of the free light chain assay in the diagnostic work‐up of patients with suspected AL amyloidosis, and also as a sensitive biomarker of response to therapy.     

系统性红斑狼疮患者外周血单个核细胞微管相关蛋白1轻链3 mRNA表达水平及其意义

目的 通过检测系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)中微管相关蛋白1轻链3 (LC3)表达,探讨其与SLE发病的关系.方法 选56例SLE患者(SLE组)和45例健康体检者(对照组),应用实时定量PCR检测2组受试者PBMCs中LC3 mRNA表达水平,同时采用流式细胞术检测2组受试者PBMCs自噬发生情况,进一步分析LC3 mRNA表达水平与自噬发生、SLE患者临床特点及病情活动度的相关性.结果 SLE患者PBMCs中LC3 mRNA表达水平为1.30±0.10,低于对照组(1.35 ±0.09;P =0.029).SLE患者PBMCs自噬发生率为(2.21±1.07)％,显著低于对照组[(9.91±4.01)％;P=0.047].PBMCs中LC3 mRNA表达水平与PBMCs自噬发生率存在相关性(r=0.735,P=0.003).PBMCs中LC3 mRNA表达水平＜1.351者SLE疾病活动指数为10.7±2.8,85.2％(23/27)的患者出现肾脏受累,而关节炎、浆膜炎和血液系统损害发生率虽有增高趋势,但差异无统计学意义.结论 SLE患者PBMCs中LC3 mRNA表达减少,导致自噬形成障碍,致使不能有效清除衰老、变性的生物分子,提示自噬在SLE发病及病情进展中发挥重要作用.     

Heterogeneous intracellular expression of B-cell receptor components in B-cell chronic lymphocytic leukaemia (B-CLL) cells and effects of CD79b gene transfer on surface immunoglobulin levels in a B-CLL-derived cell line

B-cell chronic lymphocytic leukaemia (B-CLL) cells display low amounts of surface immunoglobulins (sIg). To investigate the mechanisms underlying this phenomenon, we performed a thorough study of surface and intracellular expression of the B-cell receptor (BCR) components in B-CLL cells using flow cytometry. There was an heterogeneous pattern of expression. Overall, 20 of 22 samples showed reduced sIgM levels, compared with normal B cells. Among them, three (15%) had very low to undetectable intracellular IgM levels and variable amounts of CD79a and CD79b; nine (45%) had low intracellular CD79b levels but appreciable levels of IgM and CD79a; and eight (40%) had relatively normal intracellular levels of all BCR components. To investigate whether surface BCR levels could be controlled by the rate of CD79b synthesis, adenoviral vectors encoding CD79b were generated and used for gene transfer experiments. Delivery of CD79b to non-B cells transfected with IgM and CD79a lead to high-level expression of a functional BCR. Moreover, CD79b gene transfer in a B cell line derived from a B-CLL patient and characterised by low intracellular levels of endogenous CD79b consistently increased sIgM levels. These findings indicate that the phenotype of B-CLL cells in a subset of patients may depend primarily on poor CD79b expression, and suggest that upregulation of CD79b expression may correct the phenotype of these cells.     

CASE REPORT: Light Chain Deposition Disease of the Liver Without Renal Involvement in a Patient with Multiple Myeloma Related to Liver Failure and Rapid Fatal Outcome

We describe a 36-year-old man with advanced multiple myeloma (Salmon and Durie stage III) who developed jaundice and severe cholestasis after a first cure with systemic chemotherapy of vincristine, doxorubicin, and oral dexamethasone (VAD). Serology for hepatitis A, B, and C and for CMV was negative. A liver ultrasound and CT scan showed mild hepatomegaly without evidence of extrahepatic or intrahepatic biliary tree dilatation. A percutaneous liver biopsy revealed perisinusoidal deposits of an abundant slightly eosinophilic, PAS-positive amorphous substance. Immunohistochemistry showed positivity for kappa-light chains and was negative for lambda-light chains, for IgA, IgG, IgM, and IgD immunoglobulins as well as for AA and AL proteins and for amyloid P component. A diagnosis of light chain deposition disease (LCDD) of the liver was made. The patient developed rapid deterioration of liver function, leading to a multisystem dysfunction and death. The occurrence of LCDD in multiple myeloma is close to 5% and myeloma is the underlying disease in two thirds of patients with LCDD. The kidneys are involved in almost all cases of LCDD and renal dysfunction usually reveals the disease. Only three patients with LCDD of the liver without overt renal involvement have been reported so far. This is the first observation of LCDD presenting with jaundice and severe cholestasis shortly after the diagnosis of high tumor mass myeloma, without overt renal involvement, leading rapidly to the patient's death.     

Serum levels of macrophage inflammatory protein-1 alpha (MIP-1alpha) correlate with the extent of bone disease and survival in patients with multiple myeloma

The role of serum macrophage inflammatory protein-1 alpha (MIP-1alpha) in bone disease and survival was evaluated in 85 newly diagnosed multiple myeloma (MM) patients. MIP-1alpha was elevated in MM patients and correlated with the extent of bone disease, bone resorption markers and levels of soluble receptor activator of nuclear factor-kappaB (RANK) ligand. MIP-1alpha was also associated with survival; the 3-year probability of survival was 85% and 44% for MIP-1alpha levels below and above 48 pg/ml respectively (P = 0.021). This suggests that MIP-1alpha contributes to the pathogenesis of bone disease in MM and possibly in tumour growth, as reflected by its impact on survival.     

Systemic immunoglobulin (AL) amyloidosis in a cat: complete primary structure of a feline lambda light chain

A tarsal mass removed from a ten year old cat proved to be an extramedullary plasmacytoma largely composed of amyloid. Systemic amyloidosis was indicated when histological examination at necropsy identified amyloid also in the lymph nodes, spleen, and liver. The amyloid subunit protein was isolated by molecular sieve chromatography of reduced and alkylated fibrils extracted from the mass, and its amino acid sequence was determined by sequence analysis of peptides isolated after digestion with trypsin or Staphylococcus protease. The sequence of the C-terminal half of the protein was homologous to the constant region of immunoglobulin lambda light chains of other species establishing the immunoglobulin origin of the amyloid. The N-terminal half of the protein was homologous to the variable region of human lambda light chains and contained several rare residues in the framework regions. To our knowledge, this is the first complete sequence determination of a feline immunoglobulin light chain, and the first complete sequence determination of an amyloid immunoglobulin light chain from a species other than man.