J. Radl

Organ involvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells in the C57BL/KaLwRij mouse

The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease.

Monoclonal gammapathies in patients undergoing immunosuppressive treatment after renal transplantation

The frequency of monoclonal gammapathies (MG) and their possible origin were investigated in renal graft recipients undergoing immunosuppressive treatment. In a cross-sectional study, homogeneous immunoglobulins were found in 30% of the patients investigated. This incidence was 10-times higher than that in a control group of patients with chronic renal failure on dialysis treatment. The increase in the frequency of homogeneous immunoglobulins in the renal transplant recipients was related to the age of the patients but not to the duration of the immunosuppressive treatment. A longitudinal study in 55 patients demonstrated that most of the MG reflected transient B-cell monoclonal proliferations, probably due to an immunodeficiency; however, the incidence of benign and malignant B-cell neoplasias seemed also to be unusually high. These findings indicate that the immunosuppressive treatment enhances and accelerates an immunodeficiency which develops spontaneously with aging of the immune system; it also may contribute to the development of other age-related, both benign and malignant monoclonal B-cell proliferative disorders.

Influence of treatment with APD-bisphosphonate on the bone lesions in the mouse 5T2 multiple myeloma

The effects of the treatment of multiple myeloma (MM) with APD‐bisphosphonate on bone destruction, the dissemination pattern of the MM, and toxicity for normal and malignant cells were investigated in an animal model, the 5T2 MM. This mouse MM very closely resembles the human disease, including the typical bone lesions. It was demonstrated by radiography, microradiography, and histologic investigation that the treatment of the 5T2 MM with APD‐bisphosphonate protected the mice against a loss of bone to a significant extent. It seemed that the treatment with APD not only diminished the bone destruction by the MM but also led to the formation of new bone in already‐affected bone tissue. The growth pattern of the MM was not substantially influenced by the treatment, even though there was an indication that APD exerts some cytotoxic effect on the MM cells.

Assay of human IgA subclass antibodies in serum and secretions by means of monoclonal antibodies.

Solid-phase radioimmunoassays have been developed for the detection and quantification of human serum and secretory IgA antibodies to a variety of food, bacterial and viral antigens. Monoclonal antibodies specific for IgA1 and IgA2 and capable of binding to serum and secretory IgA were used. The assays were calibrated by reference to standard serum or purified myeloma proteins bound to solid-phase anti-immunoglobulin reagents, and sigmoid calibration curves were constructed by means of computer programs using 4-parameter logistic or weighted logit-log principles. Polymeric and monomeric forms of IgA antibodies were assayed in fractions separated by high performance size exclusion chromatography. These techniques have demonstrated the expected predominance of IgA1 antibodies in serum, and these included polymeric forms. Saliva contained both IgA1 and IgA2 antibodies, and increased proportions of IgA2 antibodies to lipopolysaccharides and lipoteichoic acid were observed.

Intraperitoneal immunization of human subjects with tetanus toxoid induces specific antibody-secreting cells in the peritoneal cavity and in the circulation, but fails to elicit a secretory IgA response

Five patients on continuous ambulatory peritoneal dialysis (CAPD) were immunized intraperitoneally with tetanus toxoid (TT) through an indwelling catheter. Four control patients on CAPD received the same dose of TT intramuscularly. Before immunization, virtually no anti‐TT antibody‐secreting cells (AbSC) were detected by the enzyme‐linked immunospot (ELISPOT) assay in peripheral blood or peritoneal fluid from patients of either group. One to 2 weeks after immunization, high frequencies of TT‐specific AbSC were detected in the circulation and peritoneal cavity. More than 80% of those cells were of the IgG isotype, with IgA accounting for most of the remainder. Patients receiving TT by the i.p. route showed significantly higher frequencies of specific IgG and IgA AbSC in the peritoneal cavity than patients immunized intramuscularly. Frequencies of AbSC in peripheral blood did not significantly differ between the two groups. Immunization with TT by both routes resulted in a significant increase of IgG anti‐TT antibodies in serum, saliva and peritoneal fluid. A significant IgA antibody response was seen only in serum and peritoneal effluents. Therefore, i.p. immunization of human subjects with TT elicited both a localized response in the peritoneal cavity as well as a systemic response in serum, but did not induce a salivary IgA response.

Molecular forms of tear IgA and distribution of IgA subclasses in human lacrimal glands

Tears and sera from 14 subjects were analyzed for IgA levels by radioimmunoassay by use of secretory IgA standards for tear samples and monomeric IgA standards for serum samples. [corrected] Tears from subjects without conjunctival inflammation contained 93% polymeric IgA (pIgA) and 7% monomeric IgA; tears from subjects with conjunctival inflammation had less pIgA (79%). Biopsy specimens of 11 lacrimal glands and one accessory lacrimal gland were obtained from 12 additional subjects. Tissues were stained with polyclonal antisera for immunoglobulins, J chain, and secretory component. In addition, tissues were stained for IgA subclasses with monoclonal reagents specific for IgA1 and IgA2. IgA plasma cells predominated over other types of plasma cells, and most were J chain positive. Secretory component, although absent from the interstitium of the gland, was found in most acinar and ductal cells. The average proportion of IgA1 to IgA2 cells was 56% to 44%. We concluded that the lacrimal system of the human resembles other exocrine systems in the production of predominantly pIgA and the nearly equal occurrence of IgA1 and IgA2 plasma cells.

Immunoglobulin VH gene sequence analysis of spontaneous murine immunoglobulin-secreting B-cell tumours with clinical features of human disease

The 5T series of multiple myelomas (MM) and Waldenstrsöm’s macroglobulinaemia‐like lymphomas (WM), which developed spontaneously in ageing mice of the C57BL/KaLwRij strain, shows clinical and biological features that closely resemble their corresponding human diseases. In order to compare the patterns of somatic mutation in VH genes of mouse tumours with those of human counterparts, we have determined and analysed sequences of immunoglobulin VH genes in five cases of murine MM, two of WM and one of biclonal benign monoclonal gammopathy (BMG). Four of five MM and 2/2 WM cases used VH genes of the large J558 family; one MM used a gene of the VGAM3.8 family, and both clones of the BMG used genes of the 36‐60 family. N‐region insertions were observed in all cases, but D‐segment genes were only identified in 6/9 cases, which were all from the D‐SP family and translated in reading frame 3. Compared with human MM, in which the VH genes have been found to be consistently hypermutated (mean%±SD=8·8±3·2), the degree of somatic mutation in the murine tumours was significantly lower (mean%±SD=2·9±2·3). There was no significant evidence of clustering of replacement mutations in complementarity determining regions (CDR), a feature considered to be characteristic of antigen‐selected sequences. However, one clone of the biclonal BMG case showed intraclonal variation, a feature described in some cases of human BMG. These results indicate that murine VH genes in mature tumours differ from human counterparts in the level and distribution of somatic mutations, but support the concept that BMG may be distinct from MM.

Heterogeneity of carbohydrate moieties of IgA1 molecules from IgA nephropathy patients and normal individuals

Summary: IgA nephropathy (IgAN) is characterized by the deposition of IgA1 in kidney mesangia and the presence of IgA1‐containing immune complexes in the circulation. Structural studies of IgA1 isolated from sera of IgAN patients indicated a statistically significant decrease in the content of galactose (Gal). Using a combination of lectins specific for glycans in O‐ or N‐linked glycan side chains, this Gal deficiency was restricted to O‐linked glycans present in the hinge region of IgA1 molecules. Gal‐deficient IgA1 displayed a significantly higher binding to mesangial cells through a putative non‐internalizing receptor specific for N‐acetyl galactosamine (GalNAc) in O‐linked glycans. These data suggest that Gal deficiency results in diversion of IgA1 molecules from the usual degradative pathway and deposition of altered IgA1 in the mesangium.

IgA antibodies against phenolic glycolipid I from Mycobacterium leprae in serum of leprosy patients and contacts: Subclass distribution and relation to disease activity

The anti-PGL-I IgA response against phenolic glycolipid I (PGL-I) a specific surface antigen of Mycobacterium leprae, was demonstrated to be essentially of the IgA1 subclass in sera from leprosy patients and contacts. Anti-PGL-I IgA1 mean levels were found to increase significantly from the tuberculoid toward the lepromatous pole of the leprosy disease spectrum, thus resembling the predominating anti-PGL-I IgM response. Furthermore, anti-PGL-I IgA1 values were shown to increase significantly with increasing bacillary load, measured as bacillary index (BI) from skin biopsies. However, a number of BI negative leprosy patients recorded elevated anti-PGL-I IgA1 levels possibly reflecting a persistence of disease activity. Three of 28 household or family contacts of leprosy patients were detected seropositive for anti-PGL-I IgA1. Thus, our results suggest that anti-PGL-I IgA1 may be considered as an additional parameter for the early detection of infection with M. leprae.

STRUCTURAL DIFFERENCES AMONG SERUM IGA PROTEINS OF CHIMPANZEE, RHESUS MONKEY AND RAT ORIGIN

Asparagine-linked sugar chains were quantitatively released from chimpanzee, Rhesus monkey and rat IgA proteins as oligosaccharides by hydrazinolysis, converted to radioactive oligosaccharides by reduction with NaB3H4, and separated into neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. However, the content of N-acetyl and N-glycolyl neuraminic acids was different among three species. The neutral and sialidase-treated acidic oligosaccharides were fractionated by Bio-Gel P-4 column chromatography in combination with linkage-specific sequential exoglycosidase digestion. Although IgA molecules from these species have mainly biantennary complex-type sugar chains, the contents of fucose and bisecting N-acetylglucosamine residues displayed marked species differences. In addition to these sugar chains, a small amount of the high mannose-type sugar chains was detected in chimpanzee and rat, but not in Rhesus monkey IgA. These results indicated that the processing of asparagine-linked sugar chains of IgA is different in each species.