Proteomic Characterization of Immunoglobulin Content in Dermal Interstitial Fluid.

Microneedles have been demonstrated to be a minimally invasive technique for sampling dermal interstitial fluid (ISF). Shotgun quantitative proteomics has already identified hundreds of proteins in ISF and quantitatively compared the proteome to matching serum and plasma. Interstitial fluid was determined to be a viable minimally invasive alternative to blood-derived fluids. In this communication, we re-examined the proteomic data from previous work to determine the diversity of immunoglobulins present compared with serum and plasma. Similar to our previous findings regarding the proteomic content across fluid types, ISF had a similar composition of IgG, IgA, IgD, and IgE antibodies as plasma or serum and lower quantities of IgM, which reflects the relative concentrations of dermal tissue T-cell and B-cell populations, indicating that the Ig's were likely locally derived. This work has significant implications for the utility of measuring Ig's in ISF for the clinical diagnosis of immunological diseases and skin infections. Data are available via ProteomeXchange with identifier PXD012658.

Characterization of the Cell Surface Phenotype and Regulatory Activity of B-Cell IgD Low (BDL) Regulatory B Cells.

B-cell IgD Low (BDL) B cells have been shown to promote immunological tolerance by inducing proliferation of CD4+Foxp3+ T-regulatory cells (Treg) in a glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL, Tnfsf18)-dependent manner. BDL cells constitute a small subset of splenic B lymphocytes that, in mice, are characterized by the B220+IgMintCD21intCD23+CD93-IgDlow/- cell surface expression profile. In this chapter, we show the flow cytometry gating strategy developed to identify and purify BDL. In addition, we describe an in vitro assay and two in vivo assays to assess BDL regulatory activity by quantitating Treg expansion/proliferation and indicate how they can be used in mouse models of disease. Collectively, these methods are useful to track and quantitate BDL and Treg numbers and assess their regulatory activity in inflammatory disease models.

Cloning and expression of partial Japanese flounder (Paralichthys olivaceus) IgD.

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

Purification of IgD from human serum--a novel application of recombinant M. catarrhalis IgD-binding protein (MID).

Moraxella catarrhalis IgD-binding protein (MID) is a multimeric outer membrane protein belonging to the family of autotransporters. The IgD-binding domain of MID is located between amino acids MID 962-1200 and binds to amino acids 198-224 of the IgD C(H)1 region. In the present study, we describe a method to purify IgD from serum with high levels of IgD using a two-step affinity chromatography process. The first step involves depletion of MID-specific antibodies of all classes from serum using the non-IgD-binding fragment MID(1000-1200). This step is followed by selective capture of IgD with MID(962-1200). Furthermore, we demonstrate that the eluted IgD is pure, intact and functional for use in downstream applications. Our approach reduces the non-specificity commonly associated with lectin-based IgD purification regimes that rely on glycosylation of the IgD molecule.

Semi-extended solution structure of human myeloma immunoglobulin D determined by constrained X-ray scattering.

Human immunoglobulin D (IgD) occurs most abundantly as a membrane-bound antibody on the surface of mature B cells (mIgD). IgD possesses the longest hinge sequence of all the human antibody isotypes, with 64 residues connecting the Fab and Fc fragments. A novel rapid purification scheme of secreted IgD from the serum of an IgD myeloma patient using thiophilic (T-gel) and lectin affinity chromatography gave a stable, homogeneous IgD preparation. Synchrotron X-ray scattering and analytical ultracentrifugation of IgD identified the solution arrangement of its Fab and Fc fragments, and thereby its hinge structure. The Guinier X-ray radius of gyration R(G) of 6.9(+/-0.1)nm showed that IgD is more extended in solution than the immunoglobulin subclass IgA1 (R(G) of 6.1-6.2nm). Its distance distribution function P(r) showed a single peak at 4.7nm and a maximum dimension of 23nm. Velocity experiments gave a sedimentation coefficient of 6.3S, which is similar to that for IgA1 at 6.2S. The complete IgD structure was modelled using molecular dynamics to generate IgD hinge structures, to which homology models for the Fab and Fc fragments were connected. Good scattering curve fits were obtained with 18 semi-extended best fit IgD models that were filtered from 8500 trial models. These best-fit models showed that the IgD hinge does not correspond to an extended polypeptide structure. The averaged solution structure arrangement of the Fab and Fc fragments in IgD is principally T-shaped and flexible, with contribution from Y-shaped and inverted Y-shaped structures. Although the linear sequence of the IgD hinge is much longer, comparison with previous scattering modelling of IgA1 and IgA2(m)1 suggests that the hinge of IgA1 and IgD are more similar than might have been expected, Both possess flexible T-shaped solution structures, probably reflecting the presence of restraining O-linked sugars.

A rapid technique for the isolation of human IgD myeloma proteins employing ultragel AcA34.

A relatively rapid technique for the isolation of human IgD myeloma proteins from whole sera is described. It is based on the use of the newly available Ultragel AcA34 gel filtration medium which yields a very substantially purified IgD fraction from whole serum. The absence of IgG from this fraction allows further purification on DEAE cellulose under conditions where the IgD protein is not absorbed but other protein contaminants are retained. The overall yield of IgD protein is estimated at greater than 90% and the technique is particularly applicable to the isolation of IgD from small serum volumes.

A rapid one step purification procedure for murine IgD based on the specific affinity of Bandeiraea (Griffonia) simplicifolia-1 for N-linked carbohydrates on IgD.

The alpha-D-galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M D-galactose. The sugar eluted product is 95-99% pure as determined by SDS-PAGE and represents 90-95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains.

Detection of IgD 'hidden' lambda light chain.

Immunoelectrophoresis and immunodiffusion of some IgD myeloma proteins fail to demonstrate precipitin bands between the light chain and anti-light chain serum. Reduction and alkylation of purified IgD preparation was found to render the molecule reactive to anti-lambda serum. Iodinated IgD myeloma protein was successfully precipitated by both anti-delta and anti-lambda antibodies. Both antisera precipitated heavy and light chains of a MW of 70,000 and 24,000 daltons respectively. In addition, a band of 39,000 daltons was also evident. Reduction and alkylation of the iodinated molecule, followed by precipitation with anti-delta serum demonstrated only the 70,000 and 39,000 daltons bands. The present report indicates that by sensitive techniques, such as iodination and immunoprecipitation, non-reactive hidden light chains can be detected in IgD lambda molecules.

Jacalin, the human IgA1 and IgD precipitating lectin, also binds IgA2 of both allotypes.

The lectin jacalin from jackfruit seeds shows a human IgA-subclass specificity by gel precipitation and Western blotting. However, its reactivity with IgA2 is a matter of controversy. We further studied the immunoglobulin isotype specificity of jacalin by affinity chromatography with myeloma sera and by inhibition of jacalin binding to solid-phase IgA1 by purified monoclonal immunoglobulins. The lectin proved to bind IgA2 of both allotypes with a lower apparent affinity than for IgA1 and IgD.

Involvement of a rapamycin-sensitive pathway in CD40-mediated activation of murine B cells in vitro.

Activation of resting B cells requires an initial triggering of the B cell antigen receptor (BCR) and secondary stimuli through various cytokine receptors and B cell activation molecules including CD40. We found that activation of B cells through CD40 is selectively inhibited by an immunosuppressant drug, rapamycin. This effect of rapamycin on anti-CD40-mediated activation of B cells was observed using three different in vitro assays. Rapamycin suppressed the anti-CD40-induced proliferation of splenic B cells, suppressed differentiation to surface IgMhigh/IgDlow B cells, and inhibited an anti-CD40-mediated prevention of apoptosis induced by BCR cross-linkage of WEHI-231 cells. We next examined several known CD40 signal transduction pathways to identify the target of rapamycin in stimulated B cells. Rapamycin did not inhibit the activation of c-Jun N-terminal kinases (JNKs) induced by anti-CD40 stimulation nor the activation of immediate nuclear transcription factors of NF-kappaB. Therefore, rapamycin affects a novel element of the CD40 signal transduction pathway which influences the proliferation, differentiation, and prevention of apoptosis of B cells.

Surface immunoglobulins in human chronic leukemia cells.

By means of the cytotoxic and immunofluorescence tests, frequency of various classes of immunoglobulins (IgG, IgA, IgM, IgD, IgE) and light chains was examined in peripheral pathologic blood cells of patients with chronic granulocytic and lymphatic leukemia. The dominant immunoglobulins were of the IgD and IgE classes. Light chains of both types were present in cells of chronic granulocytic leukemia, and kappa type in chronic lymphatic leukemia. Use of the method of resynthesis of digested immunoglobulins in vitro confirmed the monoclonal origin of chronic lymphatic leukemia in humans.

Identification of Toxoplasma gondii antigens involved in the IgM and IgG indirect hemagglutination tests for the diagnosis of toxoplasmosis.

Crude Toxoplasma gondii antigens represent raw material used to prepare reagents to be employed in different serologic tests for the diagnosis of toxoplasmosis, including the IgM and IgG indirect hemagglutination (IgG-HA and IgM-HA) tests. So far, the actual antigenic molecules of the parasite involved in the interaction with agglutinating anti-T. gondii antibodies in these tests are unknown. The absorption process of serum samples from toxoplasmosis patients with the IgG-HA reagent (G-toxo-HA) demonstrated that red cells from this reagent were coated with T. gondii antigens with Mr of 39, 35, 30, 27, 22 and 14 kDa. The immune-absorption process with the IgM-HA reagent (M-toxo-HA), in turn, provided antibody eluates which recognized antigenic bands of the parasite corresponding to Mr of 54, 35 and 30 kDa, implying that these antigens are coating red cells from this reagent. The identification of most relevant antigens for each type of HA reagent seems to be useful for the inspection of the raw antigenic material, as well as of reagent batches routinely produced. Moreover the present findings can be used to modify these reagents in order to improve the performance of HA tests for the diagnosis of toxoplasmosis.

[The diagnosis of IgD-Myelomatosis (author's transl)]. / Zur Diagnose des IgD-Plasmozytoms

Two patients with IgD-lambda myelomatosis are presented and the differential diagnosis is discussed. Typical features of this disease are the high incidence of Bence-Jones proteinuria, osteolytic lesions, amyloidosis and the predominance of male patients. Furthermore, an augmentation of serum IgD level to 165 mg% was observed in a 22-year-old female patient with presumed Coxsackie myocarditis. The theories in regard to IgD function are discussed.

[Isotachophoretic isolation of IgD and IgE and production of monospecific antisera against them]. / Vydelenie metodom izotakhoforeza IgD i IgE i poluchenie k nim monospetsificheskikh antisyvorotok.

The method of preparative isotachophoresis in acrylamide gel ensuring a high yield of IgD and IgE with insignificant admixtures of IgG, etc. was used for the isolation of IgD and IgE from the blood sera of myeloma patients. As a result of immunization with these antigens, monospecific IgD and IgE antisera were obtained. These antisera, alongside with specific antibodies, contained antibodies to admixtures; the latter were eliminated by the method of immune absorbtion carried out with the use of a sorbent based on Sepharose activated with bromo-cyanogen and conjugated with normal human blood serum. Ig D antisera were also shown to contain antibodies to idiotypical IgD determinants located in the Fab fragment of this immunoglobulin.