M components-a review of 1242 cases.

Among 1242 patients referred for immunologic investigation 1255 M components were detected in the serum. Of these patients 50.9% had multiple myeloma, 18.1% had nonmyelomatous malignant diseases such as macroglobulinemia, lymphoma, leukemia or cancer, 4.3% had connective tissue diseases, 2.5% had primary generalized amyloidosis (PGA) and the rest had various "benign" conditions. Whereas IgG was the commonest M component in multiple myeloma, connective tissue diseases and the other benign conditions, IgM was the commonest M component in lymphoma and leukemia; Bence Jones proteinemia was most frequently observed in PGA. The ratio of kappa to lambda light chains varied from 1.7:1 in IgG myeloma to 1:9 in IgD myeloma, and was 1:2.1 in PGA. Bence Jones protein was detected in 422 (66%) of 640 urine samples tested, the prevalence ranging from more than 70% in multiple myeloma and PGA to as low as 36% in various benign conditions. It is evident that the class and type of M components and the presence of Bence Jones proteinuria have no definite significance with regard to the diagnosis. Therefore, thorough investigation and follow-up at regular intervals are required when M components are detected.     

Anomalous Urinary Proteins in Patients with Serum M-Components

Abnormal urinary proteins were investigated in 365 patients with serum M-components; 250 urines were available for study. All specimens were concentrated and processed by electrophoresis and immunoelectrophoresis. An overall frequency of Bence Jones proteinuria was found in 63%, whereas other M-components were detected in 18%. In patients with multiple myeloma, Bence Jones proteinuria occurred in 71%, while M-components in the form of whole molecules of immunoglobulins or as Fc and F''c fragments were observed in 20%. Bence Jones globulin was found in 60% of patients with IgG myeloma, 69% of those with IgA myeloma and 100% of patients with IgD myeloma and light chain disease. In regard to other diseases, Bence Jones proteinuria was found in 44% of patients with macroglobulinemia, in 60% of patients with nonplasmacytic neoplastic diseases and in 28% of patients with non-malignant conditions. Other M-components were detected in the urine of 13%, 14% and 6% respectively.The detection of abnormal urinary proteins in patients with M-components in the serum is an important diagnostic tool and may have prognostic significance.     

Primary structure of the variable region of an amyloidogenic Bence Jones protein NIG-77

The complete amino acid sequence of the variable region of a Bence Jones protein NIG-77 from an individual with myeloma-associated systemic amyloidosis has been determined. This protein represents a complete light chain consisting of 216 residues and it has a sequence characteristic of V lambda I subgroup, which is closely homologous to that of another amyloidogenic V lambda I Bence Jones protein NIG-51, differing by 20 of 111 residues (82% homology). In contrast, it differs by 29 residues (74% homology) to that of non-amyloidogenic V lambda I light chain NIG-64. This finding shows that, in accordance with our previous report(1), the V lambda I-related light chains can further be divided into two distinct subsubgroups, V lambda I-1 and V lambda I-2, and the latter property seems to be more prone in association with the amyloid process.     

Refolding of the immunoglobulin light chain.

The kinetics of the refolding reactions of type lambda Bence Jones proteins from 4 M GuHCl were studied by CD, ultraviolet absorption, and fluorescence spectrophotometry. The kinetics were complex and consisted of at least three phases, an undetectable fast phase, a detectable fast phase, and a slow phase. The slow phase followed first-order kinetics and the three experimental methods used gave similar rate constants for all the Bence Jones proteins (about 3 X 10(-3) s-1). The refolding reaction of VL fragment was too fast to be measured in the present experiments. The refolding process of CL fragment was very similar to those of Bence Jones proteins except that the detectable fast phase was less significant. The rate constant of the slow phase observed for the CL fragment was similar to those of the slow phase observed for Bence Jones proteins. The activation energy of the slow phase was the same for a Bence Jones protein and its CL fragment. These results indicate that the refolding kinetics of the CL domain are very similar to those of isolated CL fragment and that refolding of the VL domain precedes refolding of the CL domain, even though both domains have similar immunoglobulin folds. However, the results of refolding experiments on Bence Jones proteins, and VL and CL fragments in the presence of ANS, as well as the other lines of evidence, indicate that the refolding kinetics of the Bence Jones protein molecule cannot be expressed as simple sum of the refolding reactions of isolated VL and CL fragments.     

Bence Jones proteins and light chains of immunoglobulins. XIII. Effect of elastase-like and chymotrypsin-like neutral proteases derived from human granulocytes on Bence Jones proteins.

Bence Jones proteins can be cleaved specifically by several types of endopeptidases into fragments corresponding to the amino-terminal, variant (VL) portion and to the carboxyl-terminal, constant (CL) portion of the light polypeptide chain. Two types of neutral proteases, designated elastase-like (ELP) and chymotrypsin-like (CLP), have been isolated and purified from human polymorphonuclear leukocytes. Because these proteases have defined proteolytic activity under physiologic conditions for several types of human proteins, we investigated their effect on human Bence Jones proteins. Incubation of kappa-type or lambda-type Bence Jones proteins with ELP or CLP under appropriate conditions resulted in cleavage of both types of light chains as evident by immunochemical and electrophoretic analyses. Treatment with ELP or CLP of one kappa Bence Jones protein resulted in the formation of a single component that had antigenic and electrophoretic properties similar to the VL fragment derived from pepsin digestion of the native protein. No component corresponding to the CL could be detected immunochemically or electrophoretically. Studies of isolated pepsin-labile (37 degrees C) and pepsin-stable (55 degrees C) CL fragments demonstrated the marked susceptibility of the carboxyl-terminal half of the light chain to proteolysis by the leukocyte-derived neutral proteases. Incubation with ELP of three other kappa Bence Jones proteins and three reduced-alkylated lambda Bence Jones proteins resulted, in each case, in the formation of a homogeneous component which was electrophoretically and immunochemically distinct from the pepsin-derived VL fragment. An identical component could also be formed by incubating a pepsin-derived VL fragment with ELP. In the ELP-treated samples, no CL-related material was detected electrophoretically or immunochemically with antisera possessing specificity for CL antigenic determinants present on the unfolded light polypeptide chain or on the isolated CL. The component formed by ELP or CLP treatment of certain Bence Jones proteins thus appears to be VL-related, but lacks the idiotypic antigenic determinant present on the native protein. In this respect, these neutral protease-derived light chain components are similar to the amyloid-like VL fragments generated in vitro from certain endopeptidase-treated Bence Jones proteins.     

Younger Age of Presentation and Extraosseous Tumour in IgD Myelomatosis

A study of 14 personal patients and 16 others in the literature shows that (1) IgD myelomatosis often presents at a significantly younger age than other forms of myelomatosis, and (2) during life extraosseous tumour can be detected in about two-thirds of these patients.The IgD form represents 1·5% of myelomatosis and shows an increased incidence of osteolytic lesions, hypercalcaemia, and renal failure, together with heavy Bence Jones proteinuria (90% type L).Like only Bence Jones myelomatosis, the IgD form seems to behave clinically in a more vicious manner.     

Clinical manifestations of multiple myeloma: relation to class and type M component.

From analysis of serum samples and clinical data from 632 patients with multiple myeloma it was determined that IgG was the commonest class of myeloma and that this type seemed to be the most benign. IgD myeloma and lambda light-chain disease were the most aggressive types; patients with these types were usually younger at diagnosis and more commonly had azotemia, osteolytic lesions and Bence Jones proteinemia. The sexes were equally represented in all but IgD myeloma, in which males predominated. Prognosis was more favourable when the M component had kappa light chains.     

Serologically defined V region subgroups of human lambda light chains

The availability of numerous antisera prepared against lambda-type Bence Jones proteins and lambda chains of known amino acid sequence has led to the differentiation and classification of human lambda light chains into one of five V lambda subgroups. The five serologically defined subgroups, V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI, correspond to the chemical classification that is based on sequence homologies in the first framework region (FR1). Proteins designated by sequence as lambda V react with specific anti-lambda II antisera and are thus included in the V lambda II subgroup classification. The isotypic nature of the five V lambda subgroups was evidenced through analyses of lambda-type light chains that were isolated from the IgG of normal individuals. Based on analyses of 116 Bence Jones proteins, the frequency of distribution of the lambda I, lambda II/V, lambda III, lambda IV, and lambda VI proteins in the normal lambda chain population is estimated to be 27%, 37%, 23%, 3%, and 10%, respectively. This distribution of V lambda subgroups was comparable to that found among 82 monoclonal Ig lambda proteins. Considerable V lambda intragroup antigenic heterogeneity was also apparent. At least two sub-subgroups were identified among each of the five major V lambda subgroups, implying the existence of multiple genes in the human V lambda genome. The V lambda classification of 54 Ig lambda proteins obtained from patients with primary or multiple myeloma-associated amyloidosis substantiated the preferential association of lambda VI light chains with amyloidosis AL and the predominance of the normally rare V lambda VI subgroup in this disease.     

Amino acid sequence of an amyloidogenic Bence Jones protein in myeloma-associated systemic amyloidosis

The complete amino acid sequence of an amyloidogenic Bence Jones protein (NIG-84) from an individual with myeloma-associated systemic amyloidosis has been determined. The protein, with a blocked N-terminus, represents a complete light chain consisting of 217 residues and it has a structural feature characteristic of the V lambda II subgroup. In addition to a two-residue insertion at positions 28 and 29, it has an additional rare insertion of alanine at position 100. NIG-84 is an example of the first complete sequence presented for the amyloidogenic Bence Jones protein of the V lambda II subgroup.     

Waldenstr?m's macroglobulinemia: review of 45 cases.

Of 168 patients with monoclonal IgM in the serum 45 (27%) had Waldenström''s macroglobulinemia. The mean age at diagnosis was 66 years. Generalized weakness, fatigue and bleeding manifestations were the usual chief complaints. Lymphadenopathy and hepatosplenomegaly were frequent. Moderate or severe anemia was noted in 29 patients, 9 had abnormal liver function, 8 had cold agglutinemia and 7 had cryoglobulinemia. Two patients had false-positive VDRL tests. The serum concentration of IgM usually exceeded 1000 mg/dl. Comparison of IgM values obtained by immunoquantitation and electrophoresis showed two types of discrepancy: readings were too high by immunoquantitation when IgM of low molecular weight was present, and readings were too low when, probably, IgM/IgG complexes were present or the serum was hyperviscous. Bence Jones protein was detected in 71% of the urine samples tested but the concentration was usually low, exceeding 200 mg/24 h in only nine instances. The mean survival time from the time of diagnosis for the 24 patients who died was 49.5 months and for the patients who are still alive, 43 months. Survival seemed to be related to the presence of azotemia, hypoalbuminemia and abnormal liver function.     

Characterization of a variable fragment of a human immunoglobulin lambda chain obtained by peptic cleavage in urea

The digestion of a human dimeric lambda Bence Jones protein with pepsin in 8 M urea without prior reduction and alkylation produced a fragment of molecular weight 11,000, designated as upV lambda, corresponding to almost the entire variable region of an immunoglobulin light chain. The C-terminal amino acid residue of fragment upV lambda was shown to correspond to leucine at position 104 of the immunoglobulin lambda light chain. Fragment upV lambda was isolated in the yield of 30% by ion exchange chromatography after gel filtration. When another lambda Bence Jones protein was treated in the same way, similar results were obtained.     

Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains

AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescence microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.     

IgD Plasma Cell Neoplasia: Clinical Manifestations and Characteristic Features

Three patients, one with plasma cell leukemia and clinically asymptomatic hypernephroma and meningioma, and two others with multiple myeloma, had M-components of IgD/λ type. In the first case, IgD globulin was found in the serum, ascitic and pleural fluids. Including our patients, 50 cases of IgD myeloma have been reported in the literature. A review of this group showed some significant differences from the other classes of multiple myeloma. IgD myeloma seems to involve a larger proportion of younger people, 66% being less than 59 years of age. The involvement of internal organs and renal damage were more frequent in IgD myeloma than in other classes. Serum total protein was frequently not increased, the relative concentration of M-component was often low and in 12% there was no spike in electrophoresis. The diagnosis therefore was sometimes difficult. In a quarter of the cases Bence Jones proteinemia was found and in 15% there were multiple spikes, both these manifestations being rare in IgG or IgA classes of myeloma. In 89%, IgD globulin had lambda type light chain, clearly contrasting with the figure of approximately 30% in other classes. Bence Jones protein was found in the urine in 91%. The survival time seemed to be shorter than in other myelomas.     

Serum amyloid A (SAA) protein-interaction with itself and serum albumin.

Serum amyloid A (SAA) protein is a 12,000 dalton protein that exists in serum under physiologic conditions as an 85,000 dalton complex and under certain conditions, as a 170,000 dalton component. To study the reason for this finding, the behavior of 125I-SAA was studied in the presence of cold SAA and several serum proteins. SAA caused a shift of some of the radioactivity to the region of albumin. Addition of normal human serum or albumin caused a shift of a significant fraction of the radioactivity to a peak eluting slightly ahead of albumin (80.000 daltons). This interaction could be blocked by the addition of cold SAA. No shift was noted when IgG or Bence Jones proteins were added. Thus, it appears that low molecular SAA protein has a tendency to aggregate with itself and to bind to albumin but not to human IgG or Bence Jones proteins.     

Homozygous M694V as a risk factor for amyloidosis in Turkish FMF patients

Secondary amyloidosis is the most severe complication of familial Mediterranean fever (FMF). Since the M694V mutation was associated with clinical severity, it was expected to be associated with amyloidosis as well. However, a number of contradicting reports have been published, especially pertinent to Turkish patients nearly 10 years ago. The aim of this study was to analyze recent data regarding the association between M694V mutation and amyloidosis among FMF patients in Turkey.We conducted a comprehensive review of the literature regarding the role of M694V mutation in the development of amyloidosis secondary to FMF. Twenty-seven papers from 20 centers including 3505 Turkish subjects were reviewed.Four-hundred patients had amyloidosis and homozygous M694V was detected in 189 (47%) of the 400 amyloidotic patients which was significantly higher than that in the FMF patients not developing amyloidosis (p < 0.0001).In the presented analysis we were able to reach a patient number of 400 which is much higher than all those published hitherto. Our findings confirmed that homozygous M694V is associated with amyloidosis in the Turkish population as well similar to Armenia, Israel, and Arabian countries. The necessity to treat asymptomatic or mildly symptomatic FMF patients with this genotype, even in countries where amyloidosis is rare, should be considered carefully.     

NADP-Agarose: an affinity adsorbent for tobacco extracellular nuclease and other nucleases.

A general method for the isolation of modified tryptophan-containing peptides is described, which takes advantage of the adsorption properties of tale for aromatic nitrocompounds. The method has been extensively assayed on tryptic and chymotryptic hydrolyzates of human Bence Jones proteins of k and λ type and of human serum albumin, and in preliminary experiments on egg white lysozyme and rabbit IgG light chains. All those proteins had been previously carboxymethylated and modified with 2,4 dinitrophenylsulfenylchloride, a selective reagent for tryptophan.     

Detection of high-molecular-weight amyloid serum protein complexes using biological on-line tracer sedimentation

The systemic amyloidoses are a rare but deadly class of protein folding disorders with significant unmet diagnostic and therapeutic needs. The current model for symptomatic amyloid progression includes a causative role for soluble toxic aggregates as well as for the fibrillar tissue deposits. Although much research is focused on elucidating the potential mechanism of aggregate toxicity, evidence to support their existence in vivo has been limited. We report the use of a technique we have termed biological on-line tracer sedimentation (BOLTS) to detect abnormal high-molecular-weight complexes (HMWCs) in serum samples from individuals with systemic amyloidosis due to aggregation and deposition of wild-type transthyretin (senile systemic amyloidosis, SSA) or monoclonal immunoglobulin light chain (AL amyloidosis). In this proof-of-concept study, HMWCs were observed in 31 of 77 amyloid samples (40.3%). HMWCs were not detected in any of the 17 nonamyloid control samples subjected to BOLTS analyses. These findings support the existence of potentially toxic amyloid aggregates and suggest that BOLTS may be a useful analytic and diagnostic platform in the study of the amyloidoses or other diseases where abnormal molecular complexes are formed in serum.     

Clinical features of common acute lymphoblastic leukemia antigen (CALLA)-positive myeloma: report of four cases

Four patients with common acute lymphoblastic leukemia antigen (CALLA)-positive myeloma are presented. The subclasses of monoclonal protein were IgD kappa (1 case), IgA lambda (1 case), and IgA kappa (2 cases). Bence Jones proteinuria was seen in all cases. The clinical stages were determined as IIA (2 cases) and IIIA (2 cases). All patients died with a median survival time after diagnosis of 62 days due to rapid development of renal failure (3 cases), and renal insufficiency and pneumonia (1 case). According to light microscopic evaluation, these myelomas corresponded to plasmablastic (1 case), immature (2 cases), and intermediate (1 case) types. Both CALLA and a cytoplasmic immunoglobulin identical with the serum monoclonal protein were simultaneously detected in single cells from all cases using immunofluorescent double labeling. These findings suggest that CALLA-positive and plasma-blastic myelomas constitute clinically a subgroup characterized by extremely poor survival but they represent cytologically different subcategories.     

Surface immunoglobulins on mouse myeloma cells.

Plasma cells from 22 different transplantable mouse myelomas (PCT) were tested by 14 different class-specific and type-specific anti-immunoglobulin antiserums for cytotoxicity effects using a trypan blue-exclusion method. Seven IgF,κ tumors were all sensitive to anti-IgF and anti-κ antiserums. None of the other antiserums showed a cytotoxic effect. Five IgG,κ and four IgH,κ tumors were lysed by anti-κ serums, but not by anti-IgG or anti-IgH or the others. One of two IgA,κ tumors was lysed by anti-κ, but neither was lysed by anti-IgA or the other serums.One IgM,λ tumor was lysed by anti-IgM and anti-λ. Two λ Bence Jones tumors were not lysed by anti-λ, but both of these and the IgM,λ tumor were lysed by anti-κ. One κ Bence Jones tumor was lysed only by anti-κ serum.Only the IgF tumors and an IgM tumor were lysed by the appropriate anti-heavy chain serum, and $\text{21}\text{22}$ lines had κ surface determinants.     

Conformational transitions of non-helical proteins effected by dodecyl sulfate. Circular dichroism of alpha1-acid glycoprotein, Bence Jones protein, carbonic anhydrase B, deoxyribonuclease, pepsinogen, and plasminogen.

Circular dichroism (CD) of serum alpha1-acid glycoprotein, urinary Bence Jones protein, human carbonic anhydrase B, deoxyribonuclease from bovine pancreas, porcine pepsinogen, and plasminogen from human serum was tested in the absence and presence of 0.005-0.05 M sodium dodecyl sulfate. It was found that in all cases the CD spectra of these proteins were modified by the dodecyl sulfate into spectra indicating the presence of a moderate content of alpha-helix. The transitions were enhanced by addition of acid (pH 2.1-4.4) in all cases tested. Comparison of the various proteins with respect to the amount of reconstruction of the main chain conformation showed that the amount of helix formed depended on the amino acid composition of the protein. Rigidity due to cross-linking by disulfide bridges is the strongest deterrant to the conformational change of the main chain. The CD bands of the native proteins in the 250-350 nm spectral zone were extinguished by sodium dodecyl sulfate, and new weak bands were observed the positions of which corresponded approximately to those of the native proteins. In all cases, except the carbonic anhydrase B, the bands of thus denatured proteins were negative.