Meibomian Gland Dysfunction Is Associated with Low Levels of Immunoglobulin Chains and Cystatin-SN.

Meibomian gland dysfunction (MGD) is a highly prevalent condition and the most common cause of evaporative dry eye disease. Studying the proteome of MGD can result in important advances in the management of the condition. Here, we collected tear film samples from treatment naïve patients with MGD (n = 10) and age-matched controls (n = 11) with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography-tandem mass spectrometry. The proteins were considered differentially expressed if p < 0.05. A total of 88 proteins were significantly regulated. The largest change was observed in cystatin-SN, which was downregulated in MGD and correlated negatively with tear meniscus height. The downregulation of cystatin-SN was confirmed with targeted mass spectrometry by single reaction monitoring (SRM). Eighteen immunoglobulin components involved in B cell activation, phagocytosis, and complement activation were downregulated in MGD including Ig alpha-1 chain C region, immunoglobulin J chain, immunoglobulin heavy variable 3-15, and Ig mu chain C region. The changes in cystatin-SN and immunoglobulin chains are likely to result from the inflammatory changes related to tear film evaporation, and future studies may assess their association with the meibum quality.

Systemic amyloidosis as a cause of chronic diarrhea after gastric surgery. Literature review.

Amyloidosis with gastrointestinal involvement is a rare cause of chronic diarrhoea, and should be considered especially in adult patients with intestinal malabsorption and extra GI manifestations. We present the case of a male patient who, after an oncological gastrectomy, presented with chronic diarrhea that did not respond to treatment and, after study, the cause of the diarrhea was finally found. Primary systemic light chain amyloidosis (AL) initially presents as chronic diarrhea and weight loss after gastrectomy. Immunohistochemistry was completely negative for amyloid A, which virtually rules out AA amyloidosis. With regard to the immunoglobulin chains, an amyloid signal was observed for both light chains, with a predominance of lambda light chain but not entirely conclusive. This situation is not uncommon since amyloid, whatever its chemical nature, can annex serum proteins, including immunoglobulin chains. In the case of chronic diarrhea, the possibility of amyloidosis should be kept in mind, especially in the case of weight loss.

Implications of detecting serum monoclonal protein by MASS-fix following stem cell transplantation in multiple myeloma.

Measurable residual disease (MRD) assessment by marrow-based next-generation flow cytometry (NGF) following autologous stem cell transplantation (ASCT) may lead to false-negative results due to patchy marrow involvement and extramedullary disease in patients with multiple myeloma. We assessed the value of simultaneous MRD evaluation with NGF and serum matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MASS-FIX). Of all 61 complete responders who were NGF-negative for MRD, around day-100 post ASCT, 59% were MASS-FIX-positive. At median follow-up of 26 months, 69% of MASS-FIX(+)/NGF(-) patients were alive and progression-free versus 96% of MASS-FIX(-)/NGF(-) patients, P = 0·02. MASS-FIX, a simple peripheral blood-based assay complements marrow-based NGF to accurately prognosticate patients with myeloma.

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry.

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.

A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry.

Monoclonal antibodies (mAbs) are heterogeneous macromolecules that display a complex isoform profile as a result of the large series of modifications they can undergo. Product-related charge variants that are associated with a loss of biological activity or affected half-life and immunogenicity are especially important. Consequently, they are often considered critical quality attributes such that acceptance criteria and controls should be established. The characterization of mAbs charge variants has long been a time and resource consuming task. Recent successes in the use of salt mediated pH gradient ion exchange chromatography with volatile mobile phases have shown there to be significant promise in using online mass spectrometric (MS) detection to facilitate peak detection. In this study, a newly developed 3 µm non-porous cation exchange column technology was investigated for its capability to be hyphenated to MS for the purpose of characterizing mAb charge variants. A 2 mm ID format was selected for the ease of configuring it to classical MS ESI ion sources. A monoclonal antibody reference material from NIST (RM 8671; NISTmAb) was used in its intact and IdeS/IgdE-digested forms to test for column performance and MS sensitivity. Furthermore, three different mAbs with highly basic isoelectric points (pI) were analyzed in their native and proteolyzed forms to demonstrate the straightforward application of the developed technique even with mAbs having strong retention on cation exchange media. The MS detection of low-abundance charge variant species (<0.1%) demonstrated there to be acceptable sensitivity and dynamic range even from routine analyses. The capability of the column to separate different mAbs having high basic pI was demonstrated, and it was found that slight adjustment of ammonium acetate concentration in the eluent can be a convenient way to rapidly optimize a separation if necessary. Linearity was shown to exist between protein mass loads of 2.5 and 50 µg while an optimal balance between chromatographic resolution and MS sensitivity was observed between 5 and 10 µg. Excellent run-to-run and column-to-column repeatability was achieved in terms of retention times, resolution and recovery.

Development of a mouse IgA monoclonal antibody-based enzyme-linked immunosorbent sandwich assay for the analyses of RBP4.

Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R2 = 0.859). After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.

The Biochemical Properties of Antibodies and Their Fragments.

Immunoglobulins (Ig) or antibodies are powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen-binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this class of protein will provide users with the knowledge necessary to select the appropriate antibody composition to maximize immunoassay results. Here we define the general biochemical properties of antibodies and their similarities and differences, explain how these properties influence their functional relationship to an antigen target, and describe a method for the enzymatic fragmentation of antibodies into smaller functional parts.

Two cases of concurrent development of essential thrombocythemia with chronic lymphocytic leukemia, one related to clonal B-cell lymphocytosis, tested by array comparative genomic hybridization.

We present two cases of concurrent development of essential thrombocythemia (ET) with chronic lymphocytic leukemia (CLL) and one related to clonal B-cell lymphocytosis (CBL). Both patients were referred for lymphocytosis and thrombocytosis. A bone marrow biopsy revealed infiltration of small, mature lymphocytes and megakaryocytic hyperplasia. Flow cytometric immunophenotyping and immunoglobulin (IG) gene clonality tests revealed clonal B lymphocytes. Both patients were positive for the JAK2 V617F mutation in whole bone marrow aspirate. The JAK2 V617F mutation was present in isolated B lymphocytes of patient 1, but not patient 2. Cytogenetics were normal in both patients. An array comparative genomic hybridization (CGH) analyses of B cells revealed a gain of 4q28.3, which is reported in non-Hodgkin's lymphoma, in patient 1, and deletion 22q11.22, which is associated with CLL, and a gain of Xp22.31 in patient 2. In both patients, B cells showed no myeloproliferative neoplasm (MPN)-specific genetic abnormalities. These results suggest that different oncogenic mechanisms in each cell lineage may underlie the concurrent development of ET and CLL (or CBL). Array CGH may be helpful in identifying the pathogenic mechanism in cases of concurrent development of lymphoid neoplasm and MPN.

Defining the recognition elements of Lewis Y-reactive antibodies.

Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1-4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.

Visual circuit assembly requires fine tuning of the novel Ig transmembrane protein Borderless.

Establishment of synaptic connections in the neuropils of the developing nervous system requires the coordination of specific neurite-neurite interactions (i.e., axon-axon, dendrite-dendrite and axon-dendrite interactions). The molecular mechanisms underlying coordination of neurite-neurite interactions for circuit assembly are incompletely understood. In this report, we identify a novel Ig superfamily transmembrane protein that we named Borderless (Bdl), as a novel regulator of neurite-neurite interactions in Drosophila. Bdl induces homotypic cell-cell adhesion in vitro and mediates neurite-neurite interactions in the developing visual system. Bdl interacts physically and genetically with the Ig transmembrane protein Turtle, a key regulator of axonal tiling. Our results also show that the receptor tyrosine phosphatase leukocyte common antigen-related protein (LAR) negatively regulates Bdl to control synaptic-layer selection. We propose that precise regulation of Bdl action coordinates neurite-neurite interactions for circuit formation in Drosophila.

Early postnatal B cell ontogeny and antibody repertoire maturation in the opossum, Monodelphis domestica.

Marsupials are a lineage of mammals noted for giving birth to highly altricial young, which complete much of their "fetal" development externally attached to a teat. Postnatal B cell ontogeny and diversity was investigated in a model marsupial species, the gray short-tailed opossum, Monodelphis domestica. The results support the initiation of B cell development late in gestation and progressing into the first two weeks of postnatal life. Transcription of CD79a and CD79b was detected in embryonic tissue prior to birth, while immunoglobulin heavy chain locus transcription was not detected until the first postnatal 24 hours. Transcription of the Ig light chains was not detected until postnatal day 7 at the earliest. The predicted timing of the earliest appearance of mature B cells and completion of gene rearrangements is consistent with previous analyses on the timing of endogenous antibody responses in newborn marsupials. The diversity of early B cell IgH chains is limited, as has been seen in fetal humans and mice, but lacks bias in the gene segments used to encode the variable domains. Newborn light chain diversity is, from the start, comparable to that of the adult, consistent with an earlier hypothesis that light chains contribute extensively to antibody diversity in this species.

Computer-aided antibody design.

Recent clinical trials using antibodies with low toxicity and high efficiency have raised expectations for the development of next-generation protein therapeutics. However, the process of obtaining therapeutic antibodies remains time consuming and empirical. This review summarizes recent progresses in the field of computer-aided antibody development mainly focusing on antibody modeling, which is divided essentially into two parts: (i) modeling the antigen-binding site, also called the complementarity determining regions (CDRs), and (ii) predicting the relative orientations of the variable heavy (V(H)) and light (V(L)) chains. Among the six CDR loops, the greatest challenge is predicting the conformation of CDR-H3, which is the most important in antigen recognition. Further computational methods could be used in drug development based on crystal structures or homology models, including antibody-antigen dockings and energy calculations with approximate potential functions. These methods should guide experimental studies to improve the affinities and physicochemical properties of antibodies. Finally, several successful examples of in silico structure-based antibody designs are reviewed. We also briefly review structure-based antigen or immunogen design, with application to rational vaccine development.

Antibody repertoires in humanized NOD-scid-IL2Rγ(null) mice and human B cells reveals human-like diversification and tolerance checkpoints in the mouse.

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ(null) engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγ(null) mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential D(H)-J(H) pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.

Hexameric immunoglobulin M in humans: desired or unwanted?

Immunoglobulin M (IgM) is the first antibody produced upon infection, and is often suggested as the first line of defense of human immune system. In addition to being present on the surface of naïve B cells as a monomeric molecule, IgM is always secreted as a polymer. The most abundant IgM polymer in humans is pentamer, composed of five monomeric units, joined together by so-called joining or J chain. On the other hand, it is well known that hexameric IgM can be also found in human sera. Its presence is often related to different dissorders (Waldenström's macroglobulinemia, cold agglutinin, and recurrent urinary bacterial infections), although it is believed that small amounts of hexamer are present in normal human sera as well. Unlike pentamer, IgM hexamer contains six monomeric blocks and completely lacks J chain. Although it has been decades since its discovery, the precise function of IgM hexamer is still unknown. Since it was documented that hexamer is very potent in activating complement, it is suggested that its production in humans must be under strict control, and that it is produced in special conditions, when strong activation of complement is absolutely needed. However, the question is whether hexameric IgM is really a secret weapon or just an undesirable molecule in humans. According to structural and known functional characteristics of both pentamers and hexamers of IgM, it can be concluded that hexamers are, in addition to being maybe too reactive to be around, probably not that efficient in protecting us from bacterial and viral infections.

[Screening of single-chain variable fragment (scFv) to bone sialoprotein].

AIM: To select single-chain variable fragment(scFv) antibody specific for bone sialoprotein(BSP) from Human Single Fold scFv Libraries. METHODS: Human Single Fold scFv Libraries were panned against immobilized BSP in a microtiter plate, after three rounds of panning, 96 clones were determined specific to BSP. The specificity of each scFv clone was determined by ELISA. The coding gene for BSP protein scFv has been sequenced. RESULTS: Phage antibody for BSP protein had a specific combination character. There were 368 bp, 527 bp, 935 bp which werer light chain, heavy chain and joint gene fragment with the resuLt of PCR. The DNA sequence data showed that there were 11 differences of the amino acids in the light chain, while there were only 3 differences in the heavy chain of scFv. CONCLUSION: scFv specific to BSP has been identified by means of phage display technology.

Determinism and stochasticity during maturation of the zebrafish antibody repertoire.

It is thought that the adaptive immune system of immature organisms follows a more deterministic program of antibody creation than is found in adults. We used high-throughput sequencing to characterize the diversifying antibody repertoire in zebrafish over five developmental time points. We found that the immune system begins in a highly stereotyped state with preferential use of a small number of V (variable) D (diverse) J (joining) gene segment combinations, but that this stereotypy decreases dramatically as the zebrafish mature, with many of the top VDJ combinations observed in 2-wk-old zebrafish virtually disappearing by 1 mo. However, we discovered that, in the primary repertoire, there are strong correlations in VDJ use that increase with zebrafish maturity, suggesting that VDJ recombination involves a level of deterministic programming that is unexpected. This stereotypy is masked by the complex diversification processes of antibody maturation; the variation and lack of correlation in full repertoires between individuals appears to be derived from randomness in clonal expansion during the affinity maturation process. These data provide a window into the mechanisms of VDJ recombination and diversity creation and allow us to better understand how the adaptive immune system achieves diversity.

Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry.

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is essential for Fc-receptor-mediated activities. Changes in IgG Fc glycosylation have been found to be associated with various diseases. Here we describe a high-throughput IgG glycosylation profiling method. Sample preparation is performed in 96-well plate format: IgGs are purified from 2 microL of human plasma using immobilized protein A. IgGs are cleaved with trypsin, and the resulting glycopeptides are purified by reversed-phase or hydrophilic interaction solid-phase extraction. Glycopeptides are analyzed by intermediate pressure matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Notably, both dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) matrixes allowed the registration of sialylated as well as nonsialylated glycopeptides. Data were automatically processed, and IgG isotype-specific Fc glycosylation profiles were obtained. The entire method showed an interday variation below 10% for the six major glycoforms of both IgG1 and IgG2. The method was found suitable for isotype-specific high-throughput IgG glycosylation profiling from human plasma. As an example we successfully applied the method to profile the IgG glycosylation of 62 human samples.

Cell-secreted Gp96-Ig-peptide complexes induce lamina propria and intraepithelial CD8+ cytotoxic T lymphocytes in the intestinal mucosa.

Induction of mucosal immunity is critical for protection from enteric pathogens. Heat shock protein gp96 is one of the primary peptide and protein chaperones located in the endoplasmic reticulum. We reported previously that a cell-secreted gp96-Ig fusion protein (gp96-Ig) mediated strong systemic, antigen-specific CD8-CTL expansion in vivo. We now evaluate the mucosal immune response to stimulation by secreted gp96 using allogeneic NIH-3T3 transfected with ovalbumin (OVA) and gp96-Ig. A single intraperitoneal NIH-3T3-OVA-gp96-Ig immunization caused significant homing of OVA-specific TCR transgenic CD8 cells (OT-I) to Peyer's patches, to the intraepithelial compartment and to the lamina propria. Intraperitoneal immunization with cells secreting gp96-Ig provided stronger mucosal immunity than the same dose instilled vaginally or rectally or injected subcutaneously or intradermally. Our results provide the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce mucosal immunity.

How antibodies fold.

B cells use unconventional strategies for the production of a seemingly unlimited number of antibodies from a very limited amount of DNA. These methods dramatically increase the likelihood of producing proteins that cannot fold or assemble appropriately. B cells are therefore particularly dependent on 'quality control' mechanisms to oversee antibody production. Recent in vitro experiments demonstrate that Ig domains have evolved diverse folding strategies ranging from robust spontaneous folding to intrinsically disordered domains that require assembly with their partner domains to fold; in vivo experiments reveal that these different folding characteristics form the basis for cellular checkpoints in Ig transport. Taken together, these reports provide a detailed understanding of how B cells monitor and ensure the functional fidelity of Ig proteins.