Variability of plasma viscosity in monoclonal IgM gammopathies.

Hyperviscosity syndromes can caused by both plasmatic and cellular factors. We have studied 20 patients affected by IgM gammopathy of different origin and 12 healthy subjects matched for sex and age, in order to evaluate the relation between paraprotein levels and plasma viscosity. We have observed a significant plasma viscosity increase only in 14 patients with monoclonal IgMk gammopathy. In the same patients was also evident an hyperviscosity syndrome. In the other 6 patients, with monoclonal IgM or polyclonal gammopathy and without clinical symptoms, plasma viscosity was only slightly increased. We have also observed a significant correlation between IgM and light chains (kappa, lambda) serum level and increased plasma viscosity. These results suggest that one can't consider all IgM gammopathies as cause of hyperviscosity syndrome.     

Analysis of paraprotein transport into the saliva by using anti-idiotype antibodies

To determine the extent of clonal involvement of the secretory immune system and the origin of salivary immunoglobulins (Ig) in monoclonal gammopathy patients, saliva and serum samples were collected from five affected individuals (two IgA myelomas, one IgG myeloma, one IgG benign monoclonal gammopathy, and one IgM lymphoma) and were assayed for the presence of monoclonal Ig. Purified polyclonal or monoclonal anti-idiotype (Id) antibodies were prepared against each of the isolated serum paraproteins. In all five individuals, the patient saliva samples inhibited the binding of 125I-labeled homologous Ig to the corresponding anti-Id antibodies, but normal saliva did not. The concentration of Id in patients' saliva varied from 1 to 400 micrograms/ml; i.e., 0.004 to 1.0% of the corresponding serum values. Saliva of a lymphoma patient whose IgM kappa protein exhibited rheumatoid factor (RF) activity also contained RF. The salivary Id-bearing molecules were found to have the same Ig isotype as the serum paraproteins. The myeloma IgA represented a minor component (0.4 and 3.9%) of the total salivary IgA. The salivary IgA myeloma proteins were associated at least in part with secretory component, but the salivary IgG paraproteins were not. In an IgA myeloma patient, a minority (17%) of the IgA+ plasma cells found in the lacrymal gland biopsy specimen were Id+, whereas the great majority (98%) of bone marrow IgA plasma cells were Id+. The results suggest active transport rather than passive transudation of myeloma IgA into the patients' saliva, and the integrity of the secretory immune system was not compromised by the neoplastic process.     

Changes in serum anion gap and sodium level in monoclonal gammopathies

In a group of patients with monoclonal gammopathies a decrease in the serum anion gap was seen with increasing serum concentrations of monoclonal IgG and IgM but not monoclonal IgA. This was probably related to the fact that IgG and IgM are cationic but IgA is a anionic at a physiologic pH. The serum sodium level decreased by 0.7 mmol/l for every increase of 1 g/dl in the serum level of the monoclonal immunoglobulin, likely because of the volume displacement effect of the monoclonal protein.     

IgA protease activity of microbes in the genus Bordetella

Monoclonal IgA paraproteins of subclasses 1 and 2, isolated from the sera of myeloma patients, were incubated for 4, 24, 48 and 72 hours with B. pertussis, B. parapertussis, B. bronchiseptica cultures, as well as Haemophilus influenzae strain. The fragmentation of IgA was studied by immunielectrophoresis with antisera to alpha-chain, to Fab alpha + Fc alpha, to Fab alpha and with antisera to light chains corresponding to the type of paraprotein. B. pertussis and B. parapertussis were found to have subclass-unspecific IgA protease which splitted off a cathode fragment, similar to Fab-fragment and, probably, corresponding to the variable domain of alpha-chain (Fv), after 48-hour incubation. Similar IgA protease was detected in H. influenzae, found to have classical IgA1 protease as well. All Bordetella species under study splitted off anode components from IgA paraproteins of both subclasses. These components, containing the determinants of heavy and light IgA chains, were either IgA - alpha I-antitrypsin complexes or some IgA fragments with high electrophoretic motility. None of the strains under study splitted monoclonal IgG.     

Activity of rheumatoid factors of different molecular sizes: comparison of autologous monomeric and polymeric monoclonal IgA rheumatoid factors

Differences in rheumatoid factor (RF) activity among the molecular species of IgA were investigated with the use of monomeric and polymeric monoclonal IgA RF paraprotein from the serum of a patient (PS) with idiopathic hyperviscosity syndrome. After fractionation by gel chromatography in acidic buffer, RF activity as determined by latex fixation and solid-phase radioimmunoassay (RIA) specific for IgA RF was confined to the high m.w. (greater than 7S) fractions. However, after adsorption into polystyrene wells, fractions containing monomeric (7S) IgA, as well as those containing polymeric IgA, bound 125I-labeled heat-aggregated human IgG. These observations were confirmed after further purification of the IgA fractions by passage through a protein A-Sepharose CL-4B column followed by precipitation of the IgA proteins with ammonium sulfate at 50% saturation. After "cross-linkage" by a hybridoma anti-human alpha-chain antibody, the activity of the monomeric IgA preparation in the IgA RF RIA approached that of the polymeric IgA preparation. Gel filtration studies verified that this activity was not due to contamination by polymeric IgA RF. Further, classic RF specificity was confirmed for both the monomeric and polymeric IgA RF by reaction with human Fc-coated but not Fab-coated wells. A control monomeric IgA myeloma protein and normal serum IgA did not react in the RF RIA when analyzed in the presence or absence of the hybridoma anti-alpha-chain antibody. Moreover, the activity of the polymeric IgA RF preparation from patient PS in the RIA was minimally influenced by the hybridoma antibody. These results indicate that IgA RF can coexist in both polymeric and monomeric forms, demonstrate that monomeric IgA RF may escape detection by previously described RIA techniques, and suggest an approach for its detection.     

The wasted mutant mouse. I. An animal model of secretory IgA deficiency with normal serum IgA

The wasted (wst) mutation was recently described as a spontaneous, recessive mutation leading to pathologic changes affecting both the neurologic and the immune systems of wst/wst homozygotes, which presented symptoms analogous to those observed in patients with ataxia-telangiectasia (A.T.). We studied the IgA system of wst/wst mutants and their normal littermates to determine whether IgA deficiency commonly found in A.T. patients was also affecting these mutants. Interestingly, although IgA plasma cells were totally absent from their entire (small and large) intestine, their serum contained a normal level of IgA with a normal ratio of monomeric vs polymeric IgA. The absence of gut IgA plasma cells was not due to malnutrition and was not compensated by the appearance of cells secreting any other isotypes. Studies at the precursor cell level showed the absence of IgA-specific B cell precursors in the Peyer's patches, whereas sIgA B cells and IgA plasma cells were found in normal numbers in the spleen of wasted mice. These data suggest that secretory and serum IgA may comprise distinct systems and that the wasted mutant mouse is a potential model for the study of the physiology and regulation of IgA production.     

"Benign" monoclonal IgE gammopathy.

So far IgE monoclonal paraproteins have been found only in patients with malignant diseases, though there are benign monoclonal paraproteins of other immunoglobulin classes. A patient with osteoporosis first seen in Paris in 1965 was found to have a paraprotein type lambda. In 1977 immunoelectrophoresis identified this as IgE lambda paraprotein, and immunodiffusion studies showed precipitin bands identical with those in patients with IgE myeloma. This patient seemed to have a benign monoclonal IgE gammopathy which had existed for 14 years. Though the possibility of transition into multiple myeloma cannot be excluded, this case suggests that a monoclonal expansion of IgE lymphocytes need not produce malignant change.     

Significance of Abnormal Protein Bands in Patients with Multiple Myeloma following Autologous Stem Cell Transplantation

Aim

We studied the characteristics of small abnormal protein bands (APB) (including oligoclonal bands and new apparent monoclonal bands) that are frequently detected by serum protein electrophoresis (SPEP) and isoelectric focusing (IEF) in the post-autologous stem cell transplant setting.

Methods

In a retrospective analysis of patients with multiple myeloma undergoing transplantation, paraprotein identity and quantification were performed using standard immunofixation electrophoresis. The nature of any new bands was determined by IEF which distinguished between oligoclonal bands and apparent monoclonal bands.

Results

Of 49 myeloma cases, the median follow-up was 33.7 months (range, 5.6–97.5 months) and 24 patients had relapsed. Thirty six (73%) developed APB. 22 patients had more than one episode of APB and 6 patients had more than 2 episodes resulting in a total of 69 episodes of APB observed post-transplant. IEF demonstrated 54 of these APB were oligoclonal bands and 15 appeared to be monoclonal. Of the 15 episodes of apparent monoclonal bands, 10 had differing heavy or light chain restriction compared to the original myeloma paraprotein and 5 had the same heavy and light chain restriction but different band location in the SPEP lane. Ten of these apparent monoclonal bands resolved, 5 persisted, and only one represented true disease progression. The presence of APB impacted favourably on event-free survival (p=0.05).

Conclusion

Small APB are very frequent post-transplant for myeloma, and IEF can identify these APB as oligoclonal or monoclonal. Apparent monoclonal bands may represent relapsed disease, but in the vast majority of cases it does not, and most likely represents a transient phenomenon representing regeneration of a limited immune response.     

Assessing the efficiency of free light chain assay in monitoring patients with multiple myeloma before and after autologous stem cell transplantation along with serum protein electrophoresis and serum protein immunofixation

Monoclonal gammopathies are a group of disorders, referred to as paraproteinaemias, dysproteinaemias or immunoglobulinopathies, associated with monoclonal proliferation of plasma cells. Monoclonal immunoglobulin secreted by these cells is an indicator of clonal proliferation. The aim of this study is to analyze the efficiency of three methods: serum protein electrophoresis (SPE), serum protein immunofixation (IFE) and FLC (free light chain) assay for the diagnosis and monitoring of the tumor burden in multiple myeloma. In this study we have presented the dynamic evolution of 7 patients with intact immunoglobulin multiple myeloma (IIMM) (2 IgG, kapa; 3 IgG, lambda; 1 IgA, kappa; 1 IgA, lambda) and 2 patients with light chain multiple myeloma before and after autologous peripheral blood stem cell transplantation (PBSCT). All 7 patients fulfilled the four criteria for the diagnosis of IIMM: bone marrow plasma cells exceeding 20%, lytic bone lesions, identification and quantification of M protein by scanning densitometry of electrophoresis gels, IFE (immunofixation protein electrophoresis) confirmed and typed the M protein. All patients had been given cytotoxic chemotherapy (VAD or VELCADE) before autologous (PBSCT). In two of the patients with IIMM both SPE and kappa/lambda ratio fell towards normal range after autologous PBSC and both reported a relapse of the disease after 23 months and 19 months respectively. SPE could not normalize after chemotherapy and transplantation in three patients with IIMM, the kappa/lambda ratio being the only marker used to monitor the tumor kill. In one patient the kappa/lambda ratio could not normalize even after PBSCT still indicating the presence of plasma cell disorder at the time when IFE was still negative. 16 months after PBSCT both SPE and FLC indicated a relapse of the disease. Classical SPE failed to demonstrate the presence of M-protein in light chain multiple myeloma, the diagnosis being established by using IFE and the FLC assay. Because IFE is a qualitative method and its interpretation may be sometimes subjective, FLC was the only method used to follow the disease course. The measurement of kappa/lambda ratio proved to be more sensitive than SPE, IFE and the levels of free light chains kappa or lambda individually indicating whether the treatment is effective or not.     

Disturbances of platelet function in patients with multiple myeloma

Coagulation disturbances in patients with multiple myeloma are presented. Platelet disfunction was especially observed, but only in patients with marked hyperproteinemia and a high level of immunoglobulins in plasma, whereas normal adhesion and aggregation were noticed in 3 out of 17 patients without protein disturbances. Chemotherapy as well as plasma exchange caused not only a decrease of the paraprotein level in plasma, but also a normalization of the failure previously observed in platelet function. Our experiments confirm the opinion that protein M in plasma is responsible for the disturbances observed in multiple myeloma.     

Plural light chains in a single plasma cell of a monoclonal gammopathy undetermined significance case: An ultrastructural study

A 44-year-old man was found to have M-proteins of IgG consisting of kappa- and lambda-chains in serum without lymphadenopathy or splenomegaly. The serum concentrations of IgG, IgA and IgM were within normal limits. Bone marrow examination showed normal cellular marrow containing 6.3% of plasma cells with no abnormal features. No chromosomal abnormality was observed at all. The patient was diagnosed as having monoclonal gammopathy of undetermined significance. The bone marrow plasma cells possessed free kappa- and lambda-chains in Golgi apparatus, rough endoplasmic reticula and cytoplasmic matrices. Plural light chains were simultaneously produced with the same heavy chain in a plasma cell by immunoelectron microscopy. This is the first report in the world of a monoclonal gammopathy of undetermined significance producing plural light chains with the same heavy chain.     

Detection of IgA defects in blood donors using an enzyme immunoassay

An enzyme immunoassay is presented for screening blood donors for IgA-defects using horseradish peroxidase conjugated IgA. Fife of about 4,700 screened donors was found to be IgA-deficient. With another EIA using horseradish peroxidase conjugated monoclonal anti-IgA and IgA coated microplates the IgA-concentration in serum of these selected donors was determined.     

Frequent association of idiopathic myelofibrosis with plasma cell dyscrasias

In a retrospective analysis of 199 cases of myeloproliferative diseases a concomitant plasma cell dyscrasia was found in three out of 46 patients with idiopathic myelofibrosis. Chronic myeloid leukemia, polycythemia vera or unclassifiable myeloproliferative disorders were in no case associated with monoclonal gammopathy. One patient with idiopathic myelofibrosis had primarily coexistent IgG-lambda paraproteinemia and increasing osteolytic lesions; histologic evidence of multiple myeloma, however, was insufficient. In the second patient the interval between diagnosis of idiopathic myelofibrosis and IgG-kappa paraproteinemia was 11 years. After a stable period of 9 years' duration the paraprotein level rapidly increased, associated with depression of normal background immunoglobulins and progressive bone marrow failure. The exact nature of this patient's malignant plasma cell dyscrasia remained uncertain. In the third case benign monoclonal gammopathy of the IgM-lambda type was diagnosed 13 years after idiopathic myelofibrosis. A review of the literature confirms a remarkably high incidence of monoclonal gammopathies in idiopathic myelofibrosis. Benign monoclonal gammopathy seems to occur in at least 8% of the patients while only a few cases of concomitant multiple myeloma have been reported. It may be speculated that plasma cell dyscrasias in idiopathic myelofibrosis reflect involvement of the lymphoid lineage in the neoplastic stem cell disorder.     

A synthetic ligand for IgA affinity purification

We reported previously that TG19318, a synthetic ligand deduced from the screening of combinatorial libraries, displays specific and selective recognition properties for immunoglobulins of the G class and can be used conveniently for affinity chromatography purification of monoclonal and polyclonal antibodies. In this study we have extended the ligand characterization, examining its ability to bind IgA from cell culture supernatants and from IgG-deprived serum. Affinity columns prepared by immobilizing TG19318 on Sepharose allowed convenient one-step purification of monoclonal IgA directly from crude feedstocks, in high yield and with full recovery of immunoreactivity. Optimal column adsorption occurred with phosphate buffer at neutral pH, while elution of adsorbed IgA could be accomplished by a buffer pH change to acidic or basic conditions. Column capacity was close to 7 mg IgA/ml support.     

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.     

Subclass distribution of antigen-specific IgA antibodies in normal donors and individuals with homozygous C alpha 1 or C alpha 2 gene deletions

To analyze the subclass restriction of Ag-specific IgA, sera and saliva from healthy blood donors and from IgA class or subclass deficient individuals were studied. The latter included donors with or without C alpha 1 or C alpha 2 gene deletions. Monoclonal human IgA1 and a genetically engineered IgA2 antibody, normal human serum and colostrum IgA were used as standards to estimate serum and saliva levels of Ag-specific antibodies. In normal individuals, there was a strong IgA1 preference of naturally acquired antibodies in serum against both polysaccharide Ag (PPS 6A, PPS 23, pneumococcal C-polysaccharide, and LPS from Escherichia coli) and protein Ag (Staphylococcus aureus alpha-toxin and HSV). Specific IgA2 in serum against the tested Ag were frequently not measurable. In contrast, most of the individuals with homozygous C alpha 1 gene deletions displayed substantial amounts of specific IgA2 against protein as well as polysaccharide Ag. The median levels of specific IgA in serum against protein Ag were approximately one-third as compared to normal individuals and one-fifth, or less, against polysaccharide Ag. Normal serum levels of IgA against the tested Ag, restricted to the IgA1 subclass, were noted in two individuals with IgA2 deficiency, one of whom carried a homozygous C alpha 2 gene deletion. Median values of specific IgA, against the tested Ag S. aureus alpha-toxin, HSV, and pneumococcal C-poly-saccharide, from normal healthy donors were approximately four to eight times higher in serum as compared to saliva. Individuals with homozygous C alpha 1 gene deletions displayed increased levels of the various specific IgA2 antibodies in saliva. In conclusion, the individuals with homozygous C alpha 1 gene deletions displayed decreased median levels of specific IgA antibodies in serum despite normal levels of total IgA. Normal levels of both specific IgA and total IgA in saliva were found.     

解毒通络生津颗粒联合羟基氯喹对原发性干燥综合征患者血液流变学及血清IgG、IgA、CRP水平的影响

目的:探讨解毒通络生津颗粒联合羟基氯喹对原发性干燥综合征(pSS)患者血液流变学及血清免疫球蛋白G(Ig G)、免疫球蛋白A(Ig A)、C-反应蛋白(CRP)水平的影响。方法:选取2017年1月-2017年9月期间我院收治的60例pSS患者为研究对象,采用随机数字表法将患者分为对照组(n=30)和观察组(n=30)。对照组给予硫酸羟基氯喹治疗,观察组在此基础上联合解毒通络生津颗粒治疗,比较两组患者的血液流变学指标、Ig G、Ig A和CRP水平,同时评价两种治疗方案的疗效和不良反应。结果:治疗前两组患者血液流变学指标相比差异均无统计学意义(P0.05)。治疗后观察组全血高切粘度、全血低切粘度、血浆粘度和红细胞沉降率(ESR)均较治疗前降低,且观察组全血低切粘度和血浆粘度低于对照组(P0.05)。治疗前两组患者Ig G、Ig A和CRP水平相比差异均无统计学意义(P0.05)。治疗后观察组Ig G、Ig A和CRP水平均较治疗前降低,且低于对照组(P0.05)。观察组的不良反应发生率为13.33%,低于对照组的46.67%,差异有统计学意义(P0.05)。对照组总有效率为43.33%,低于观察组的为70.00%,差异有统计学意义(P0.05)。结论:解毒通络生津颗粒和硫酸羟基氯喹联合应用能够有效改善pSS患者血液流变学及免疫学指标,增强患者免疫力,且安全性较好,值得进一步推广使用。     

Plasma exchange in the long-term management of Waldenstr?m's macroglobulinemia.

Two patients with Waldenström''s macroglobulinemia (WM), which had become resistant to cytotoxic drugs, were treated for features of the hyperviscosity syndrome by repeated plasma exchange with the continuous-flow blood-cell separator over periods of 36 and 28 months, respectively. After four initial weekly plasma exchanges the procedure was carried out every 4 to 6 weeks and both patients tolerated it well. Relative viscosity of the serum was maintained within the normal range in one patient, and both patients remained free of symptoms of the hyperviscosity syndrome. The results suggest that treatment of WM by long-term "maintenance" plasma exchange alone should be considered in any patient with complications due to chemotherapy or whose disease fails to respond to chemotherapy.     

Idiotypic analysis of a B cell clone with anti-intermediate filament specificity in a patient with Sj?gren's syndrome: involvement of five subpopulations producing different immunoglobulin isotypes

An IgM paraprotein from patient LP with Sj?gren's syndrome exhibited an antibody activity to intermediate filaments (IMF) of cells from all vertebrates examined, and appeared to recognize several classes of IMF (i.e., vimentin, desmin, and keratin). A mouse monoclonal anti-idiotype (Id) antibody, K4A, was prepared against the IgMk (LP) and used as a specific probe in two-color immunofluorescence to examine the extent of clonal involvement in the patient's blood and bone marrow mononuclear cells (MNC). Twenty to 30% of MNC in her blood samples were IgMk+ plasmablasts with morphologic similarity to Waldenstr?m's macroglobulinemia cells. IgG+ and IgA+ plasmablasts were demonstrated in lower frequencies (approximately 2%). Almost all of the IgM+ cells and approximately 80% of the IgG+ cells and IgA+ cells in the blood were reactive with the K4A anti-Id antibody. Immunoglobulin (Ig) subclass analysis revealed that the K4A Id was expressed by IgG1+, IgG3+, IgA1+ and IgA2+ plasmablasts. Similar observations were obtained with bone marrow samples, although the proportion of Id+ cells among IgG+ or IgA+ cells was lower in marrow than in blood. IgG and IgA fractions isolated from the patient's serum were also shown to contain anti-IMF activity. Ig biosynthetic analysis of blood MNC revealed that the K4A anti-Id antibody precipitated not only IgM but also IgG and IgA. Because cells simultaneously producing two different Ig isotypes were not detected, these results indicate the presence of five separate subpopulations of the K4A Id+ neoplastic clone. The data thus suggest the occurrence of a neoplastic or pre-neoplastic transformation event before the switching of Ig heavy chain isotypes, and imply a role for the IMF antigen in the exaggerated proliferation and differentiation along five of the nine potential intraclonal pathways.     

The molecular organization of the protein HC-IgA complex (HC-IgA)

Complexes of protein HC and monoclonal IgA1 or IgA2 or polyclonal IgA were isolated from human blood plasma. Dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that all complexes contain three types of chains: two light immunoglobulin chains, one regular IgA alpha-chain, and one chain with Mr = 90,000 carrying both alpha-chain and protein HC epitopes. The complexes were split into Fab alpha and Fc alpha fragments by bacterial IgA proteases. The protein HC epitopes were linked to the Fc fragments. Complexes of protein HC and an alpha-chain devoid of the variable region and the first heavy chain constant domain could also be demonstrated to be present in the blood plasma of a patient with alpha-heavy chain disease. Pepsin digestion of HC-IgA released a fragment containing all the protein HC epitopes and the C-terminal nonapeptide of the IgA alpha-chain. The light immunoglobulin chains, the regular alpha-chain, and the 90,000-Da chain from monoclonal HC-IgA1 were isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis and by repeated gel filtration in dodecyl sulfate-containing buffer. The N-terminal amino acid sequence of the alpha-chain was identical with that of a regular human heavy immunoglobulin chain of subgroup III. Subtractive degradations of the 90,000-Da chain displayed 2 amino acid residues in each position in a pattern suggesting simultaneous degradations of a chain identical with the regular alpha-chain of HC-IgA and of uncomplexed, low molecular weight, protein HC. All the results are compatible with a model for HC-IgA in which a single low molecular weight protein HC polypeptide chain is covalently linked, side by side, to the C-terminal nonapeptide of one of the two alpha-chains of a regular monomeric IgA unit.