Ig light chains are secreted predominantly as monomers

Ig light (L) chains are secreted not only as part of assembled Ab molecules, but also as free L chains, the latter process being involved in the pathology of several diseases. The secretion competence of free L chains distinguishes them from free subunits of other oligomeric proteins, which are usually retained intracellularly. We used several techniques to test the idea that secretion of free L chains is dependent on dimerization. Coexpression of pairs of L chains, differing in only one amino acid, which alters the secretory phenotype, shows that these L chains behave independently: the wild-type chains are secreted, whereas the mutants are retained intracellularly. A survey of kappa- or lambda-producing cell lines by nonreducing gel electrophoresis shows that a negligible fraction of these L chains exists as disulfide-bonded dimers. Moreover, chemical cross-linking and density gradient centrifugation demonstrate that there is no significant pool of noncovalent L chain dimers. Noncovalent heterodimers can be detected readily between a kappa-chain and a chimera consisting of a heavy chain variable domain linked to the kappa-chain constant domain. This confirms that noncovalent L chain homodimers would have been detected if they were present. These findings about the association state of free L chains are independent of the host cell, as they are observed in both myeloma cells and COS fibroblasts. We conclude that L chain dimerization is a rare event that neither facilitates secretion nor is required for it.     

Fragments of the constant region of immunoglobulin light chains are constituents of AL-amyloid proteins

Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin light chain are a constituent of the AL-amyloid proteins of kappa type. A specific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The fragments are small and have been overlooked previously in the purification process. The significance of the constant part in AL-proteins is unclear, but adds new aspects to the discussion of pre- or post-fibrillogenic cleavage of the immunoglobulin light chains.     

Kinesin light chains are essential for axonal transport in Drosophila

The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.     

Localization of a single binding site for immunoglobulin light chains on human Tamm-Horsfall glycoprotein

Cast nephropathy is a severe complication of multiple myeloma. Binding of filtered monoclonal light chains (LC) with Tamm-Horsfall glycoprotein (THP) triggers heterotypic aggregation of these two proteins to form casts in the distal nephron of the kidney. To localize the LC binding site on THP, human THP was deglycosylated and underwent limited trypsin digestion in the presence or absence of a nephrotoxic LC known to bind THP. A 29.6-kD band was protected from trypsin digestion by the addition of LC. NH2-terminal amino acid sequence and amino acid analyses revealed this band was located between the 6th and 287th amino acid residues of THP. Six peptides located within this 29.6-kD fragment were synthesized and used as potential inhibitors of binding or aggregation of five different nephrotoxic LCs with THP. Peptide AHWSGHCCL (from amino acid 225 to 233) completely inhibited binding and aggregation of these proteins. Optimal inhibition required a cystine residue in this peptide. Truncation experiments demonstrated the entire sequence was necessary for ideal inhibition and the histidine residue explained the effects of pH on binding. These studies provided a basis for further study of LC-THP interaction and a potential approach toward the prevention of cast nephropathy.     

Extended analysis of AL-amyloid protein from abdominal wall subcutaneous fat biopsy: kappa IV immunoglobulin light chain

OBJECTIVE: HIV-associated nephropathy is an important cause of morbidity that is characterized clinically by uremia and proteinuria and histologically by focal segmental glomerulosclerosis. In the largest series yet analyzed to our knowledge, we describe new sonographic findings and record the prevalence of other findings. We review the sonographic findings in a large group of HIV-infected patients. MATERIALS AND METHODS: Seventy-six consecutive HIV-infected patients underwent renal sonography. Abnormalities seen on sonography were recorded. RESULTS: Of 152 kidneys imaged, sonography showed that 30 kidneys (20%) were enlarged. Abnormal echogenicity was present in 136 kidneys (89%). Eighty-one kidneys (53%) were globular; 58 (38%) had decreased corticomedullary definition; 74 (49%) had decreased renal sinus fat; and 66 (43%) had heterogeneous parenchyma, some with echogenic striations. CONCLUSION: Our data reveal several sonographic abnormalities that have not previously been described: decreased corticomedullary definition, decreased renal sinus fat, parenchymal heterogeneity, and globular renal configuration. These new findings were found mainly in patients with advanced HIV infection.     

Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic diseases--myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each samples was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence of low concentrations of urea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form high molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vivo results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid diagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregation and the treatment of those invariably fatal disorders associated with pathologic light-chain deposition.     

Intravenous immunoglobulin: implications for use in the neurological patient

The efficacy of intravenous immunoglobulin (IVIG) in the treatment of the autoimmune disease, idiopathic thrombocytopenic purpura (ITP), has been well documented in the literature. This has encouraged researchers to examine possible therapeutic uses of IVIG in other immune-mediated disorders. Recent clinical reports have suggested that IVIG may have a role in the treatment of neurological disorders with a possible immunopathogenic etiology. Intravenous immunoglobulin, a blood product which contains immunoglobulin G and a trace amount of immunoglobulin A, is believed to work as an immunomodulating agent. However, its mechanism of action is not well understood. Nurses involved with the administration of IVIG must be well informed about the manufacturing and regulation, proper dose and administration, adverse effects, appropriate assessments and related patient education.     

Two species of mRNAs for the fyn proto-oncogene are produced by an alternative polyadenylation

Two mRNA species with different sizes (3.8 kb and 2.8 kb) for the fyn proto-oncogene have been noticed during Northern hybridization analysis. However, the difference between the two mRNA species has not been resolved yet. By screening a phage expression library using the monoclonal antibody (mAb) B16-5 which recognizes Src homology 3 (SH3) domains of phospholipase C-gamma and Nck, we have cloned a cDNA encoding the larger species of fyn mRNA. The size of the clone was 3.5 kb and DNA sequencing analysis of the clone showed that it was fyn expressed mainly in T-cells, fyn (T), with an untranslated region 1 kb longer than the previously reported one. The 3'-end fragment of the clone hybridized only to the larger species (3.8 kb) of fyn mRNA but not to the smaller one (2.8 kb) on Northern blot analysis. Furthermore, an additional polyadenylation signal sequence was found at the end of this clone. These results indicate that the two mRNA species for fyn are produced by alternative polyadenylation.     

VH and VL gene complexes encoding an anti-spectrin antibody are defined by nucleotide sequencing of cDNA from a hybridoma generated from Hu-PBL-SCID mouse

Previously we reported that human imunocompetent cells engrafted into scid mice mount a sustained and vigorous humoral immune response to murine erythrocytes. One of the dominant and consistently observed reactivity pattern of these antibodies in immunoblot analysis is with the alpha and beta isoforms of spectrin. In order to define the human xenoreactive response more completely, a hybridoma was generated (from a hu-PBL-scid mouse) whose antibody reacted with two high molecular weight species 225 to 250 kDa. We report here that this conserved antibody species reacts with both the murine and human erythrocyte proteins and cDNA nucleotide sequence analysis of the light and heavy chain genes encoding this antibody reveals that the light chain variable region gene has been previously observed in association with an autoreactive antibody. In addition to characterizing a conserved human B cell clonotype this is the first report of a human monoclonal antibody being generated from the hu-PBL-scid model using the standard hybridoma technology.     

Risk assessment on the carcinogenic potential of hybridoma cell DNA: implications for residual contaminating cellular DNA in biological products

Fifty people (25 at risk for an eating disorder, 25 controls) performed a simple reaction-time (SRT) task and a negative-priming (NP) task. The two groups did not differ on the SRT task. For the NP task, the controls displayed the NP effect (responses on critical trials were slower than responses on control trials). At-risk participants, however, revealed no such NP effect. Although the pattern of NP performance in the at-risk participants may indicate that they as a group had deficiencies in their ability to inhibit irrelevant information, it is also possible that issues related to obsessionality, perfectionism, and restraint in the at-risk group affected the results.     

Rapid degradation of an unassembled immunoglobulin light chain is mediated by a serine protease and occurs in a pre-Golgi compartment

The immunoglobulin kappa light chain produced by the CH12 lymphoma is unusual because it is not secreted when expressed in the absence of a heavy chain. Instead, it undergoes rapid intracellular degradation. This degradation is selective, as another light chain expressed in the same cell is not degraded. It is also a property of the CH12 kappa chain itself, since it is degraded rapidly when expressed either in another myeloma cell or in COS-1 fibroblasts. When provided a heavy chain, this kappa chain assembles into IgM and is then protected from proteolysis. The degradation of kappa requires ATP, is sensitive to reduced temperature and to the thiol reagent diamide. Of all the proteolytic inhibitors tested, 3,4-dichloroisocoumarin, L-1-tosylamido-2-phenylethyl chloromethyl ketone, and to a lesser extent 1-chloro-3-tosylamido-7-amino-2-heptanone, inhibit kappa degradation, suggesting the involvement of a serine protease. The degradation of kappa does not require transport to the Golgi complex, nor is it sensitive to a variety of lysosomotropic agents. Both immunofluorescence and the observed association with the endoplasmic reticulum (ER) stress proteins GRP78/BiP and GRP94 indicate that the kappa chain is localized mostly in the ER. When a point mutation which blocks transport to the Golgi complex is introduced into this kappa chain, the association with the stress proteins is enhanced but the rate of degradation is not significantly decreased. We conclude that the CH12 kappa chain is a particularly good substrate for an ER degradation machinery, and that its sensitivity to the protease(s) is governed by its state of assembly. This ER degradation provides a possible quality control mechanism during the differentiation of B lymphocytes.     

Further investigation of the light chain shifting phenomenon: light chain replacement through secondary rearrangement induced by lectin stimulation in the hybridoma cell line HB4C5

We found that when the hybridoma cell line HB4C5 was stimulated with wheat germ agglutinin (WGA), loss of production of the original lambda light chain occurred, followed by production of new light chain, which mirrored the reaction when stimulated with concanavalin A (ConA). We previously reported that the RAG genes are expressed not only in HB4C5 and its ConA-treated variant subclones, but also in the in the parental Namalwa cells, which are known to be in the plasma state. However, the new lambda light chains were expressed only in the HB4C5 cells and not in the parental Namalwa cells. Here we found that the RAG genes are expressed in HB4C5 cells after continuous stimulation with WGA. To further investigate the mechanism of this loss of original lambda light chain production by stimulation with lectins in HB4C5 cells, which leads to a sIg-negative subpopulation, we analyzed the differences between HB4C5 and Namalwa cells. In this present study, we found that a 70 kDa phosphorylated protein in HB4C5 cells became undetectable after stimulation with lectins (WGA and ConA), and was not detected in Namalwa cells before or after lectin stimulation. It has been believed that the RAG genes and loss of original lambda light chain production are required to induce expression of a new lambda light chain in the HB4C5 cells. We suggested that the phosphorylated 70 kDa protein in HB4C5 cells play important roles in regulating the production of new lambda light chains which is induced by lectins.     

Peripheral T cell tolerance as a tumor escape mechanism: deletion of CD4+ T cells specific for a monoclonal immunoglobulin idiotype secreted by a plasmacytoma

Tumors could escape an immune attack by inducing peripheral T cell tolerance. To test this, T cell receptor (TCR)-transgenic mice were injected with plasmacytoma cells secreting a highly tumor-specific antigen, a monoclonal immunoglobulin (Ig), for which the transgene-encoded TCR is specific. The TCR recognizes a third hypervariable region idiotypic (Id) peptide of the Ig, presented by a class II molecule on host antigen-presenting cells. The TCR-transgenic mice have previously been shown to be protected against an Id+ plasmacytoma challenge. In the present experiments, the protection was deliberately overwhelmed by subcutaneous injection of large numbers of plasmacytoma cells. Such tumor mice, chronically exposed to increasing amounts of monoclonal Ig, delete Id-specific CD4+ T cells in their peripheral lymphoid organs and in the tumor. The residual CD4+ cells express endogenous, rather than transgene-encoded TCR alpha chains. Peripheral deletion, functional T cells unresponsiveness, and thymocyte deletion are all first detected at the same serum concentration of monoclonal Ig, approximately 50 micrograms/ml (0.3 microM), and become more and more profound as the tumor burden increases. The results suggest that peripheral T cell tolerance to Id could be a tumor escape mechanism in patients with B cell malignancies. In addition, the findings have implications for T cell tolerance to Ig V regions in normal individuals.     

Crohn's disease and ulcerative colitis mucosal T cells are stimulated by intestinal epithelial cells: implications for immunosuppressive therapy

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases, and their pathogenesis is attributed, in part, to alterations of the mucosal immune system. This study was designed to define the possible contribution of epithelial cells to the activation of lamina propria T lymphocytes (LPTs) in CD and UC. METHODS: LPTs isolated from CD, UC, and control surgical specimens were cocultured with freshly isolated allogeneic or autologous epithelial cells or epithelial cell lines. Resulting T-cell proliferation was evaluated by tritiated thymidine incorporation on day 5. RESULTS: When intestinal epithelial cells were used to stimulate mucosal T-cell proliferation, CD and UC LPTs were less responsive than control LPTs (p < 0.05 and p < 0.03, respectively). This difference between inflamed and control T cells was consistently observed by using a variety of different intestinal epithelial cell types. CONCLUSIONS: CD and UC mucosal T cells are hyporesponsive to activation by intestinal epithelial cells when compared with control LPTs. Elucidating the mechanism underlying the differential activation of CD and UC LPTs may help to better understand the immunopathogenesis of these conditions.     

On the specificity of assays to detect circulating immunoglobulin A-fibronectin complexes: implications for the study of serologic phenomena in patients with immunoglobulin A nephropathy

Immunoglobulin A (IgA)-fibronectin complexes have been proposed as specific serologic markers of IgA nephropathy. They have been detected by the use of ELISA composed of an immobilized antifibronectin antibody (or albumin as a negative control) and an enzyme-conjugated anti-IgA antibody (antifibronectin capture assay). By the use of this type of assay, plasma samples from 32 normal controls, 38 IgA nephropathy patients, and 81 patients with other types of glomerulonephritis were analyzed. Extinction values in IgA nephropathy patients were higher (P = 0.06) than in patients with other glomerulonephritis types and significantly higher than in normals. Markedly lower values were obtained when the plates were coated with albumin. However, when the antifibronectin antibody was replaced by normal IgG or F(ab')2 fragments, almost identical extinctions were measured. The use of different antifibronectin antibodies, IgG, ELISA plates, or blocking regimens did not modify these results. Extinction values could not be suppressed by the addition of exogenous fibronectin. Similar extinctions were observed when plasma samples were replaced by physiologic concentrations of fibronectin-free IgA. Extinction values measured in the plasma samples correlated significantly with IgA concentrations in plasma as analyzed by nephelometry. A collagen binding assay, a second type of assay used to measure IgA-fibronectin complexes, also allowed the detection of fibronectin-free IgA, and again, extinctions measured in plasma could not be suppressed by exogenous fibronectin. In conclusion, both antifibronectin capture ELISA and collagen binding assays do not specifically detect only IgA-fibronectin complexes, but also total plasma IgA, which is frequently, but nonspecifically, elevated in IgA nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two dogmas of conceptual empiricism: implications for hybrid models of the structure of knowledge

Concepts seem to consist of both an associative component based on tabulations of feature typicality and similarity judgments and an explanatory component based on rules and causal principles. However, there is much controversy about how each component functions in concept acquisition and use. Here we consider two assumptions, or dogmas, that embody this controversy and underlie much of the current cognitive science research on concepts. Dogma 1: Novel information is first processed via similarity judgments and only later is influenced by explanatory components. Dogma 2: Children initially have only a similarity-based component for learning concepts; the explanatory component develops on the foundation of this earlier component. We present both empirical and theoretical arguments that these dogmas are unfounded, particularly with respect to real world concepts; we contend that the dogmas arise from a particular species of empiricism that inhibits progress in the study of conceptual structure; and finally, we advocate the retention of a hybrid model of the structure of knowledge despite our rejection of these dogmas.