The clinicopathologic characteristics of kidney diseases related to monotypic IgA deposits.

Monoclonal gammopathy of renal significance (MGRS) regroups renal disorders caused by a monoclonal immunoglobulin without overt hematological malignancy. MGRS includes tubular disorders, glomerular disorders with organized deposits, and glomerular disorders with non-organized deposits, such as proliferative glomerulonephritis with monoclonal IgG deposits. Since glomerular involvement related to monotypic IgA deposits is poorly described we performed retrospective analysis and defined clinico-biological characteristics, renal pathology, and outcome in 19 referred patients. This analysis allowed distinction between 2 types of glomerulopathies, α-heavy chain deposition disease (5 patients) and glomerulonephritis with monotypic IgA deposits (14 patients) suggestive of IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits in 12 cases. Clinicopathologic characteristics of α-heavy chain deposition disease resemble those of the γ-heavy chain disease, except for a higher frequency of extra-capillary proliferation and extra-renal involvement. IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits should be differentiated from diseases with polytypic IgA deposits, given distinct clinical, histological, and pathophysiological features. Similarly to IgG-proliferative glomerulonephritis with monoclonal immunoglobulin deposits, overt hematological malignancy was infrequent, but sensitive serum and bone marrow studies revealed a subtle plasma cell proliferation in most patients with IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits. Anti-myeloma agents appeared to favorably influence renal prognosis. Thus, potential progression towards symptomatic IgA multiple myeloma suggests that careful hematological follow-up is mandatory. This series expands the spectrum of renal disease in MGRS.

Unravelling the immunopathological mechanisms of heavy chain deposition disease with implications for clinical management.

Randall-type heavy chain deposition disease (HCDD) is a rare disorder characterized by tissue deposition of a truncated monoclonal immunoglobulin heavy chain lacking the first constant domain. Pathophysiological mechanisms are unclear and management remains to be defined. Here we retrospectively studied 15 patients with biopsy-proven HCDD of whom 14 presented with stage 3 or higher chronic kidney disease, with nephrotic syndrome in 9. Renal lesions were characterized by nodular glomerulosclerosis, with linear peritubular and glomerular deposits of γ-heavy chain in 12 patients or α-heavy chain in 3 patients, without concurrent light chain staining. Only 2 patients had symptomatic myeloma. By serum protein electrophoresis/immunofixation, 13 patients had detectable monoclonal gammopathy. However, none of these techniques allowed detection of the nephrotoxic truncated heavy chain, which was achieved by immunoblot and/or bone marrow heavy chain sequencing in 14 of 15 patients. Serum-free kappa to lambda light chain ratio was abnormal in 11 of 11 patients so examined. Immunofluorescence studies of bone marrow plasma cells showed coexpression of the pathogenic heavy chain with light chain matching the abnormal serum-free light chain in all 3 tested patients. Heavy chain sequencing showed first constant domain deletion in 11 of 11 patients, with high isoelectric point values of the variable domain in 10 of 11 patients. All patients received chemotherapy, including bortezomib in 10 cases. Renal parameters improved in 11 patients who achieved a hematological response, as assessed by normalization of the free light chain ratio in 8 cases. Tissue deposition in HCDD relates to physicochemical peculiarities of both variable and constant heavy chain domains. Early diagnosis and treatment with bortezomib-based combinations appear important to preserve renal prognosis. Thus, monitoring of serum-free light chain is an indirect but useful method to evaluate the hematological response.

Quantitative gingival crevicular fluid proteome in health and periodontal disease using stable isotope chemistries and mass spectrometry.

AIM: Application of quantitative stable isotope-labelling chemistries and mass spectrometry (MS) to determine alterations in gingival crevicular fluid (GCF) proteome in periodontal disease. MATERIAL AND METHODS: Quantitative proteome of GCF from 40 healthy individuals versus 40 patients with periodontal disease was established using 320 GCF samples and stable isotope-labelling reagents, ICAT and mTRAQ, with MS technology and validated by enzyme-linked immunosorbent methods. RESULTS: We have identified 238 distinct proteins of which 180 were quantified in GCF of both healthy and periodontal patients with additional 26 and 32 distinct proteins that were found only in GCF of healthy or periodontal patients. In addition, 42 pathogenic bacterial proteins and 11 yeast proteins were quantified. The data highlighted a series of proteins not quantified previously by large-scale MS approaches in GCF with relevance to periodontal disease, such as host-derived Ig alpha-2 chain C, Kallikrein-4, S100-A9, transmembrane proteinase 13, peptidase S1 domain, several collagen types and pathogenic bacterial proteins, e.g. formamidase, leucine aminopeptidase and virulence factor OMP85. CONCLUSIONS: The innovative analytical approaches provided detailed novel changes in both host and microbial derived GCF proteomes of periodontal patients. The study defined 50 host and 16 pathogenic bacterial proteins significantly elevated in periodontal disease most of which were novel with significant potential for application in the clinical arena of periodontal disease.

Renal crescentic alpha heavy chain deposition disease: a report of 3 cases and review of the literature.

Heavy chain deposition disease (HCDD) is a comparatively recently described entity characterized by glomerular and tubular basement membrane deposition of monoclonal heavy chains without associated light chains. To our knowledge, review of the literature shows only 24 previously reported cases of HCDD with unequivocal evidence of monoclonal heavy chain deposition in the kidney using immunofluorescence microscopic and electron microscopic studies. The predominant heavy chain subtype was γ. There has been a single case of µ HCDD and 2 previously reported cases of α HCDD. In this report, we describe 3 additional cases of α HCDD, all with a crescentic pattern of injury and one of which was associated with cutis laxa. We compare their clinicopathologic features with all previously reported cases of HCDD.

B-lymphocyte heterogeneity: ontogenetic development and organ distribution of B-lymphocyte populations defined by their density of surface immunoglobulin.

The density of total Ig and of IgM, IgG1, IgG2, and IgA on the surface of adult murine splenic B lymphocytes was measured using the technique of rapid flow microfluorometry. In addition, the density of total surface Ig and of IgM on B lymphocytes derived from adult bone marrow, lymph nodes, and Peyer's patches, and from neonatal spleen was determined. Adult spleen and lymph node B lymphocytes are characterized by the presence of a large population of cells with a low-to-intermediate density of total surface Ig, which is seen as a peak in the fluorescence profiles when these cells are labeled with fluorescein-conjugated (F1) anti-Ig. This peak is not detected when neonatal spleen or adult bone marrow are examined; the development of this peak among spleen cells occurs during the first 4 wk of life. Although the characteristic fluorescence intensity peak is not seen when adult spleen cells are labeled with Fl anti-mu, changes in the density of surface IgM do occur during the first few weeks of life and are detected as a decrease in the frequency of cells which have relatively large amounts of surface IgM. The differences seen in the fluorescence patterns of adult spleen cells labeled with Fl anti-Ig and Fl anti-mu may be due to the appearance of IgD on the surface of mature splenic B lymphocytes. This is supported by the similarity of the fluorescence profiles of adult bone marrow cells stained with Fl anti-Ig and Fl anti-mu, as the latter population of cells is reported to lack surface IgD.

Initial comparison of proteomic profiles of whole unstimulated saliva obtained from generalized aggressive periodontitis patients and healthy control subjects.

BACKGROUND AND OBJECTIVE: Salivary proteomics technology can be used to evaluate the disease progression of periodontitis and the systemic screening of proteomes of saliva from subjects with aggressive periodontitis has not been available. The objective of this preliminary study was to compare the proteomic profile of whole unstimulated saliva of subjects with generalized aggressive periodontitis (GAgP) with that of healthy volunteers to identify proteins, the levels of which were significantly altered between the two groups. MATERIAL AND METHODS: Whole unstimulated saliva was obtained from five subjects with GAgP and five healthy subjects, and proteins were separated using two-dimensional gel electrophoresis. Proteins, the levels of which were significantly different between the two groups, were identified by computer image analyses and subsequent electrospray ionization tandem mass spectrometry. RESULTS: Eleven proteins that exhibited a different level in the GAgP group vs. the control group were identified. Compared with whole saliva of healthy control subjects, the levels of serum albumin, immunoglobulin (Ig) gamma2 chain C region, Ig alpha2 chain C region, vitamin D-binding protein, salivary alpha-amylase and zinc-alpha2 glycoprotein were increased in whole unstimulated saliva of GAgP subjects, while those of lactotransferrin, elongation factor 2, 14-3-3 sigma, short palate, lung and nasal epithelium carcinoma-associated protein 2 precursor and carbonic anhydrase 6 were decreased. CONCLUSION: Comparison of the proteomic profile of whole unstimulated saliva of GAgP subjects with that of healthy control subjects revealed at least 11 differential proteins. The approach applied herein might be helpful to aid understanding of the etiology of GAgP.

Characterisation of protein composition and detection of IgA in cervicovaginal fluid by microchip technology.

In this paper the application of microchip electrophoresis to examine the protein profile of cervicovaginal fluid and the detection of IgA heavy and light chains is presented. This method is a fast growing field of technology and ensures high-speed analysis requiring only microliters of sample. Proteins with wide range of molecular masses could be separated within 1 min. Cervicovaginal specimens of healthy women showed a complex protein pattern-containing several peaks in the 15-70 kDa region. sIgA is considered to be an important protein constituent of all mucosal surfaces. Detection of sIgA in cervicovaginal samples was achievable by microchip technology. Under reduced circumstances (induced by mercaptoethanol, a component of the denaturating solution) the disulfide bonds connecting IgA heavy and light chains are broken up and chains can be detected as separate peaks during electrophoresis. In 82.5% of the cases only the light chain of IgA could be detected in the clinical samples. The intact IgA heavy chain could be demonstrated in only 12.5% of the cases. Based on our data some conclusions were provided about the correlation of these patterns with the age of patients, pH of the cervicovaginal fluid, operations performed before sample collection and usage of oral contraceptives.

Use of the ELISA to detect and quantify histocompatibility antigens and their subunits.

A solid phase enzyme immunoassay (ELISA) has been developed for the detection and quantification of human histocompatibility antigens and their subunits. The assay involves the binding to a microELISA plate of a mouse monoclonal antibody reacting with a common antigenic determinant to all HLA (A, B, C) antigens. The standard conditions for the assay and the curves obtained for the quantification of total HLA, free beta 2m, and free heavy chain subunit (alpha) present in a biological sample are described and the sensitivity and potential uses of the method are discussed.

Middle East lymphoma and alpha-chain disease. An immunohistochemical study.

An immunoperoxidase study of the small intestinal mucosa of three patients with alpha-chain disease showed heavy infiltration of the mucosa by plasma cells containing alpha-heavy chain and J-chain but no light chains. An additional band-like and nodular mucosal infiltrate was also present and consisted of cells showing no evidence of cytoplasmic immunoglobulin synthesis. The cells comprising this infiltrate invaded and destroyed intestinal crypts, and immunoperoxidase staining showed them to be sharply distinct from the alpha-chain-containing plasma cells. In two cases of Middle East lymphoma, immunohistochemistry revealed a normal plasma cell population in the lamina propria of the small intestine. These results show that alpha-chain disease can be diagnosed in routine paraffin sections which should permit clarification of its true incidence in Middle East lymphoma. The demonstration of sharp distinction between the two types of mucosal infiltrate in alpha-chain disease is in contrast to previous immunofluorescence results and enables more ready identification of mucosal changes that may be important in the management of the disease.

Primary upper small-intestinal lymphoma and alpha-chain disease. Report of 10 cases emphasizing pathological aspects.

Ten cases of primary upper small-intestinal lymphoma associated with alpha-chain protein in serum were discovered in a prospective study of the sera of patients with immunoproliferative small-intestinal disease (IPSID). Patients were mostly young males presenting with abdominal pain, weight loss, and diarrhea and showing laboratory evidence of carbohydrate, fat, and vitamin B12 malabsorption and hypoalbuminemia. The more frequently encountered pathologic abnormality was a diffusely nodular jejunal mucosa produced by a plasmacytic infiltrate of variable cell maturity involving a varible depth of small bowel wall with or without involvement of the mesenteric or para-aortic-lymph node complex and, in one instance, the liver. A less frequent picture included circumferential ulcerative and constrictive transmural tumors of the upper small intestine produced by a malignant lymphoma with involvement of abdominal lymph nodes. Small-intestinal surface epithelial abnormalities, a dense mantle of mature plasma cells overlying the lymphoma, a pronounced follicular lymphoid hyperplasia adjacent to and at distances from the lymphoma were other features of note in our IPSID cases associated with alpha-chain protein.

Two-dimensional gel electrophoresis as an aid in the analysis of monoclonal gammopathies.

Two-dimensional gel electrophoresis is the most powerful protein separation technique currently available. In the authors' laboratory it has proved useful in the analysis of specimens from patients with monoclonal gammopathies when those specimens were otherwise difficult to diagnose. Examples of problematic specimens include biclonal gammopathies, heavy chain diseases, and monoclonal gammopathies that show a small or invisible spike on serum protein electrophoresis on cellulose acetate. In addition, two-dimensional electrophoresis is being used to investigate the pathophysiologic features of myeloma kidney disease, especially regarding potential interactions of Bence Jones proteins and kidney proteins.

Distribution of immunoglobulin heavy chains in diseased synovia.

Synovium from 142 patients with 12 different arthropathies was examined for the distribution of alpha, delta, gamma, and mu immunoglobulin heavy chains. A high proportion of plasma cells in the superficial subintima in all diseases reacted for alpha heavy chains. Only in rheumatoid disease did the synovium contain more than 10% of plasma cells reacting for mu heavy chains.

Middle Eastern intestinal lymphoma: a morphological and immunohistochemical study.

A total of 31 cases of Middle Eastern gastrointestinal lymphoma (from Mosul, Iraq) has been analysed by conventional histology, and also by immunoperoxidase staining of trypsinised paraffin sections with anti-Ig and anti-J chain antisera. Histologically these neoplasms fell into three categories: undifferentiated lymphoma of Burkitt type (8 cases); follicle centre cell (FCC) lymphoma, resembling European lymphomas of FCC origin (15 cases); and Mediterranean lymphoma (MTL) (3 cases). Immunohistological staining of the FCC neoplasms showed that these tumours resembled their European counterpart in that cytoplasmic Ig and J chain could be demonstrated in a proportion of cases. However there was a striking difference in that alpha chain alone, light chain alone, or J chain alone were detected in several cases (in contrast to the predominance of mu plus kappa or lambda light chains found in European cases). Furthermore, prominent intracellular inclusions of alpha chains were found in two cases. Staining of the MTL cases revealed that the characteristic mucosal plasma cell infiltrate was positive for alpha chain (weakly) and J chain (strongly) but that the invasive cells which morphologically resembled FCCs were negative for both constituents. It is argued that these three histological categories constitute the major types of gastrointestinal lymphoma in the Middle East; and that in MTL the invasive lymphoma is of FCC type.

Cytogenetic studies in four cases of alpha chain disease.

Cytogenetic studies were performed in four cases of alpha chain disease. Chromosomal abnormalities were found in the lymphoid cells of the mesenteric lymph nodes of three patients, two of whom had not reached the stage of overt malignant lymphoma. In two instances, a rearrangement of 14q32, resulting from a t(9;14)(p11;q32) and a t(2;14)(p12;q32) was observed. One case showed complex rearrangements including t(5;9). No abnormalities were found in the intestinal tumor of the fourth case with immunoblastic lymphoma. It is concluded that alpha chain disease is a clonal proliferation with frequent alteration of chromosome #14 at band q32 resulting from translocations that differ from those observed in the vast majority of other non-Hodgkin lymphomas.

The determination of antibody to Streptococcus mutans serotypes in saliva for children ages 3 to 7 years.

The saliva of 29 children ages 3 to 7 years was followed by indirect immunoflourescence to determine the antibody reacting with the 5 different serotypes of S mutans. Fluorescent antisera specific for alpha chain and gamma chain were used. Statistical analysis of the data demonstrated a significant negative correlation between antibody of immunoglobin class (IgA) to S mutans type b and the decayed, extracted and filled surfaces of deciduous teeth.

Immune components in dental plaque.

Acquired pelicle appears to contain primarily IgA and other proteins of salivary origin. With the increased time necessary for plaque formation, gingival crevicular fluid contributes proteins to the growing plaque accumulation. However, secretory IgA is still the major intact immunoglobulin in plaque samples since appreciable portions of the molecules bearing IgG determinants appear to be degraded to small fragments. Nevertheless, the amount of IgA present in plaque, which could be considered antibody, is too little to account for most of the plaque interactions. Since secretory IgA appears to be resistant to proteolytic degradation by a mixture of plaque enzymes, and IgA fragments are not prominent in plaque extracts, degradation of secretory IgA probably cannot explain the relatively low IgA levels in plaque. It has been shown that salivary IgA antibody can interfere with enzymes responsible for the plaque-forming potential of certain organisms. All the preceding evidence is consistent with our current contention that secretory IgA functions as blocking antibody to interfere with the formation of dental plaque. This could occur by direct inhibition of bacterial polymer formation or by direct or indirect inhibition of bacterial interaction with salivary constituents by secretory IgA. Less than 1% of the plaque interactions can probably be attributed to secretory IgA antibody. IgG may contribute even less since it is degraded.

Elevated serum IgA associated with immunoproliferative enteropathy of Basenji dogs: lack of evidence for alpha heavy-chain disease or enhanced intestinal IgA secretion.

Immunoglobulin isotypes in serum and intestinal secretions of Basenji dogs with chronic diarrhea, asymptomatic Basenji dogs, and healthy control dogs were quantitated and their molecular sizes characterized to detect alpha-chain, gamma-chain, or mu-chain fragments. Quantitation of immunoglobulin isotypes in serum showed that affected Basenjis have significantly elevated serum IgA values as compared to asymptomatic Basenjis and normal control dogs. However, IgA concentrations in intestinal wash fluids were not significantly different for the three groups. Immunoelectrophoresis (IEP) and polyacrylamide gel electrophoresis (PAGE) demonstrated that virtually all IgA was in the dimeric form. Using IEP and immunoselection, we were unable to detect evidence for the presence of alpha-chain or other heavy-(H)-chain fragments. Hyperimmune serum obtained from rabbits immunized with serum or a globulin fraction of affected Basenjis also failed to detect H-chain fragments. The results of this study indicate that immunoproliferative enteropathy of Basenjis resembles closely the nonsecretory form of human immunoproliferative small intestinal disease (IPSID).

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor.

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.

Monoclonal immunoglobulins.

Precise immunologic analysis of monoclonal immunoglobulin components is essential to allow proper diagnosis, monitoring of therapy and a clearcut understanding of the biosynthetic derangements in plasma cell neoplasms. Such analyses have resulted in the delineation of a number of well-defined clinical syndromes, such as the group of heavy chain diseases.

Immunoperoxidase study in alpha-chain disease.

Peroxidase-antiperoxidase technique was applied to search for the presence of alpha-chain protein in cells of the infiltrate from six cases of alpha-chain disease (one immunoblastic sarcoma and five plasmacytosis cases). Cases of poorly differentiated lymphocytic and Burkitt's type lymphomas, IgA myeloma, and tuberculous enteritis served as controls. Infiltrate cells from alpha-chain disease showed heavy and diffuse staining with anti-alpha-chain and not light-chain antisera. The myeloma reacted with both anti-alpha and anti-kappa antisera. Plasma cells from tuberculous enteritis showed variable staining with anti-heavy- and anti-light-chain antisera, while control lymphomas did not stain at all. We suggest that immunoenzyme histochemistry is a useful tool in demonstrating intracellular alpha-chain protein.