IgG Light Chain Research Articles

Cryptosporidium p30, a Galactose/N-Acetylgalactosamine-specific Lectin, Mediates Infection in Vitro

Cryptosporidium sp. cause human and animal diarrheal disease worldwide. The molecular mechanisms underlying Cryptosporidium attachment to, and invasion of, host cells are poorly understood. Previously, we described a surface-associated Gal/GalNAc-specific lectin activity in sporozoites of Cryptosporidium parvum. Here we describe p30, a 30-kDa Gal/GalNAc-specific lectin isolated from C. parvum and Cryptosporidium hominis sporozoites by Gal-affinity chromatography. p30 is encoded by a single copy gene containing a 906-bp open reading frame, the deduced amino acid sequence of which predicts a 302-amino acid, 31.8-kDa protein with a 22-amino acid N-terminal signal sequence. The p30 gene is expressed at 24-72 h after infection of intestinal epithelial cells. Antisera to recombinant p30 expressed in Escherichia coli react with an approximately 30-kDa protein in C. parvum and C. hominis. p30 is localized to the apical region of sporozoites and is predominantly intracellular in both sporozoites and intracellular stages of the parasite. p30 associates with gp900 and gp40, Gal/GalNAc-containing mucin-like glycoproteins that are also implicated in mediating infection. Native and recombinant p30 bind to Caco-2A cells in a dose-dependent, saturable, and Gal-inhibitable manner. Recombinant p30 inhibits C. parvum attachment to and infection of Caco-2A cells, whereas antisera to the recombinant protein also inhibit infection. Taken together, these findings suggest that p30 mediates C. parvum infection in vitro and raise the possibility that this protein may serve as a target for intervention.

Conversion of a mouse Fab into a whole humanized IgG antibody for detecting botulinum toxin

Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives. However, the lack of the Fc region of the antibody can be difficult in many standard diagnostic platforms. Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated conversion to human IgG with no traces of mouse Fab as determined by Western blot analysis. In addition, the humanized IgG performed better as both a detection and capture reagent in an ELISA format and detected the botulinum toxoid at a lower concentration than the parental murine Fab. This technique offers the ability to convert various species of antibodies or antibody fragments into humanized antibodies with improved characteristics in immunodiagnostic assays, for use as human controls in serological assays, or for possible therapeutic benefit.

Focused antibody response in plasma cell-infiltrated non-medullary (NOS) breast cancers

Breast tumors with prominent plasma cell (PC) infiltrates often have a more favorable natural course that may plausibly be mediated by anti-tumor activity of the elicited antibodies. These breast tumor-associated PCs are typically IgG dominant in contrast to normal breast PCs, which are mainly IgA. It is our hypothesis that this PC infiltration represents a host immune response that is driven by one or more tumor antigens. Previously, we and others showed that medullary carcinoma (MC) had a focused repertoire and features suggestive of a protein antigen driven response. Infrequently, non-MC, not otherwise specified (NOS) breast tumors may exhibit heavy PC infiltrations, also of IgG isotype. In this first characterization of this favorable prognosis NOS subgroup, IgG heavy chain (Hc) and light chain (Lc) variable (V) regions from three PC-infiltrated NOS tumors were randomly cloned and sequenced. We found biased (V) gene usage by the infiltrating PCs and somatic hypermutation in the rearranged Ig Hc and Lc V regions that were compatible with antigenic selection of the progenitor B cells. The antibody response of NOS infiltrated breast cancer is repertoire-focused, with 13-68% of isolates being clonally reiterated in the samples. Each NOS patient used distinct Hc V-D-J and Lc V-J rearrangements, with her own immune response "footprint," but the overall pattern of gene usage followed that typical of exogenous antigen-induced immune responses. The data are consistent with the hypothesis that PC infiltrates infrequently arising in NOS tumors, as previously inferred for MC, are in response to one or more breast cancer-associated protein tumor antigens.

Solitary Extramedullary Plasmacytoma of the Liver without Systemic Monoclonal Gammopathy

Extramedullary plasmacytoma of the liver is a very rare tumor. Although a few cases of extramedullary plasmacytoma of the liver have been reported, we could not find any report on truly localized extramedullary plasmacytoma of the liver in the literature. The patient was a 63-yr-old man who exhibited a solitary liver mass on dynamic computed tomography and magnetic resonance imaging. Histologically, the tumor was composed of mature plasma cells with mild atypia. Immunohistochemistry demonstrated monoclonal IgG and Kappa light chain expression. Bone marrow examination revealed no abnormalities. There was no evidence of a monoclonal protein in the serum and urine, lytic bone lesions, anemia, renal insufficiency, and hypercalcemia. The patient was treated with 5,000 cGy of radiotherapy, and the tumor disappeared 6 months after treatment.

Distinctive T Cell-suppressive Signals from Nuclearized Type 1 Sphingosine 1-Phosphate G Protein-coupled Receptors

Sphingosine 1-phosphate (S1P) generated by cells of innate immunity and the type 1 S1P G protein-coupled receptor (S1P(1)) on mobile T cells constitute a major system for control of lymphoid organ traffic and tissue migration of T cells. Now we show that T cell activation mediated by the T cell antigen receptor translocates plasma membrane S1P(1) to nuclear envelope membranes for association there with G(i/o), Erk (1/2), and other proteins that plasma membrane S1P(1) uses to signal T cell proliferation. However, nuclear S1P(1) and plasma membrane S1P(1) transduce opposite effects of S1P on T cell proliferation and relevant signaling as exemplified by respective decreases and increases in T cell nuclear concentrations of both phospho-Erk and active (phosphorylated) c-Jun. T cell antigen receptor-mediated activation of T cells therefore both eliminates migration responses to S1P by down-regulation of plasma membrane S1P(1) and translocates the S1P-S1P(1) axis into the nuclear domain where signals are directed to transcriptional control of immune functions other than migration.

The Uncertain Significance of Anti–Glomerular Basement Membrane Antibody Among HIV-Infected Persons With Kidney Disease

Glomerular lesions that complicate patients with human immunodeficiency virus (HIV) infection include HIV-associated nephropathy, membranous glomerulopathy, and immune-complex glomerulonephritides. This case series presents 3 patients with clinically significant renal disease and positive test results for anti-glomerular basement membrane (anti-GBM) antigen. Characteristic histological findings that would suggest anti-GBM antibodies have a significant role in the pathological state of each patient's kidney disease were absent. In addition, each patient recovered without specific treatment for anti-GBM disease. This case series suggests that anti-GBM antibodies likely are related to the B-cell expansion previously described in patients with HIV infection. We propose that clinicians interpret results of anti-GBM antibody tests carefully for patients with HIV infection, considering biopsy before empiric therapy, particularly in a clinical presentation that is atypical for Goodpasture disease.

Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins

Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.

Urine from Scrapie-Infected Hamsters Comprises Low Levels of Prion Infectivity

The question of whether prion diseases can be transmitted by body fluids has important epidemiological, environmental and economical implications. In this work, we set to investigate whether urine collected from scrapie-infected hamsters can transmit fatal or subclinical infectivity to normal hamsters. After prolonged incubation times ranging from 300 to 700 days, a small number of animals inoculated with scrapie urine succumbed to scrapie disease, and several asymptomatic hamsters presented low levels of PrP^Sc in their brains. In addition, most of the asymptomatic hamsters inoculated with scrapie urine, as opposed to those inoculated with normal urine, presented extensive gliosis as well as protease-resistant light chain IgG in their urine, a molecule shown by us and others to be a surrogate marker for prion infection. Our results suggest that urine from scrapie-infected hamsters can transmit a widespread subclinical disease that in some cases develops into fatal scrapie.

A Series of Collaborations Between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis

An international project team (including members from US, Canada and UK) has been formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results from this exercise demonstrate the robustness of CE-SDS across eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology. Data generated from the analysis of a series of molecular weight markers showed very good precision with regards to relative migration time (RMT) both within and between organisations. The apparent molecular weight of bovine serum albumin (BSA) was measured with good precision to within approximately 2% RSD across the participants. A representative IgG sample showed similar results with regards to relative migration time of its 3 main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain. Fractional peak area for each peak also showed good agreement, with less than 9% RSD for all peaks. This exercise will facilitate both increased regulatory and industrial opinion of CE for biopharmaceutical analysis.

Ovine placental eluate immunoglobulins recognise isologous and third party acid-treated trophoblast microvesicle antigens in vitro

Placental microvesicles were prepared from ovine placentae and immunoglobulins eluted with 0.5 M glycine buffer pH 2.5. The ability of eluate immunoglobulins to re-associate with isologous (self) and third party acidified microvesicles was tested by ELISA. Ovine placental immunoglobulins re-associated with isologous and third party acidified microvesicles suggesting that at least 2 types of antigenic epitopes I and II maybe expressed on the ovine placentae. Type I antigens may be present on placentae of all ovines while type II epitopes may be paternally derived, hence unique to each pregnancy. Analysis by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27 kDa, respectively, together giving a relative molecular weight of 158 kDa. Results suggest that immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the trophoblast, possibly defining a mechanism by which the foetus evades maternal immunological rejection.

ERK8 Down-regulates Transactivation of the Glucocorticoid Receptor through Hic-5

Extracellular signal-regulated kinase 8 (ERK8) is the most recently identified member of the ERK subfamily of MAPKs. Although other members of the ERK subfamily are established regulators of signaling pathways involved in cell growth and/or differentiation, less is known about ERK8. To understand the cellular function of ERK8, a yeast two-hybrid screen of a human lung library was performed to identify binding partners. One binding partner identified was Hic-5 (also known as ARA55), a multiple LIM domain containing protein implicated in focal adhesion signaling and the regulation of specific nuclear receptors, including the androgen receptor and the glucocorticoid receptor (GR). Co-immunoprecipitation experiments in mammalian cells confirmed the interaction between Hic-5 and both ERK8 and its rodent ortholog ERK7. The C-terminal region of ERK8 was not required for the interaction. Although the LIM3 and LIM4 domains of Hic-5 were sufficient and required for this interaction, the specific zinc finger motifs in these domains were not. Transcriptional activation reporter assays revealed that ERK8 can negatively regulate transcriptional co-activation of androgen receptor and GRalpha by Hic-5 in a kinase-independent manner. Knockdown of endogenous ERK8 in human airway epithelial cells enhanced dexamethasone-stimulated transcriptional activity of endogenous GR. Transcriptional regulation of GRalpha and interaction with its ligand binding domain by ERK8 were dependent on the presence of Hic-5. These results provide the first physiological function for human ERK8 as a negative regulator of human GRalpha, acting through Hic-5, and suggest a broader role for ERK8 in the regulation of nuclear receptors beyond estrogen receptor alpha.

Evaluation of canine serum for the presence of antiretinal autoantibodies in sudden acquired retinal degeneration syndrome

To determine the presence of serum antiretinal antibodies in sudden acquired retinal degeneration syndrome (SARDS) affected dogs and the size of the antigen to which these antibodies bind via the use of enzyme-linked immunosorbent assay (ELISA) and Western blot immunoassays. Serum was collected from 13 dogs affected by SARDS and five dogs with normal ocular examinations. All serum samples were subjected to ELISA with saline-soluble canine retinal tissue and Western blot analyses with SDS solubilized normal canine retinal tissue as the antigen. Antirecoverin (23 kDa) and antiheat shock cognate (65 kDa) antibodies were used as positive controls for both procedures. Affinity-purified goat antidog IgG and IgM labeled with horseradish peroxidase were used for all clinical samples and goat antirabbit IgG was used as the secondary antibody for the positive controls. ELISA demonstrated antibody reaction with all samples. Western blot immunoassays identified multiple bands in all canine serum samples, as well as in negative controls. Approximate sizes of the bands were 25 and 50 kDa, corresponding to IgG light and heavy chains, respectively. No antiretinal autoantibodies were identified in the serum of dogs affected by SARDS as compared to normal canine patients.

Identification of disialic acid-containing glycoproteins in mouse serum: a novel modification of immunoglobulin light chains, vitronectin, and plasminogen

Serum glycoproteins are involved in various biologic activities, such as the removal of exogenous antigens, fibrinolysis, and metal transport. Some of them are also useful markers of inflammation and disease. Although the amount of sialic acid increases following inflammation, little attention has been paid to the presence of linkage-specific epitopes in serum, especially the alpha2,8-linkage. In a previous study, we demonstrated that four components in mouse serum contain alpha2,8-linked disialic acid (diSia), based on immunoreactivity with monoclonal antibody 2-4B, which is specific to N-glycolylneuraminic acid (Neu5Gc)alpha2-->(8Neu5Gc alpha2-->)(n-1), n > or = 2 [Yasukawa et al., (2005) Glycobiology, 15, 827-837]. In this study, we purified three components, 30-, 70-, and 120-kDa gp, and identified them as an immunoglobulin (Ig) light chain, vitronectin, and plasminogen, respectively, using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses. Modifications of these proteins with alpha2,8-linked diSia were chemically confirmed by fluorometric C7/C9 analyses and mild acid hydrolysates-fluorometric anion-exchange chromatography analyses. We also demonstrated that the IgG, IgM, and IgE light chains are commonly modified with alpha2,8-linked diSia. In addition, both mouse and rat vitronectin contained diSia, and the amount of disialylation in vitronectin dramatically decreased after hepatectomy. These results indicate that a novel diSia modification of serum glycoproteins is biologically important for immunologic events and fibrinolysis.

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate beta-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in beta1,3- or both beta1,3- and beta1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular beta-glucans and compete with zymosan binding and triggering of tumor necrosis factor-alpha secretion by a Dectin-1-expressing macrophage cell line.

Generation of monoclonal antibodies using simplified single-cell reverse transcription-polymerase chain reaction and cell-free protein synthesis

The single-step PCR amplification of IgG Light chain (Lc) and Heavy chain (Hc) (Fd portion) from the cDNAs of a single cell was facilitated using a low concentration of cDNA-specific primers with 5' homotags in the presence of a homotag-specific primer. This method was found to be successful in generating a functional antibody with an antigen-binding activity and useful for the high-throughput generation or screening of monoclonal antibodies.

Proteomic analysis of the extent and distribution of immunogenic proteins in xenogeneic bioscaffolds

Objective Decellularized xenogeneic bioscaffolds offer the possibility of serving as a platform for tissue engineering. However, the full extent and distribution of xenoantigens in these materials have not been explored. We used a proteomic approach to identify immunogenic proteins in a candidate xenogeneic biomaterial. Methods - Cytoplasmic and membrane proteins were extracted from bovine pericardium (BP) using differential solubility and precipitation techniques. Extracted proteins were separated by either 1d or 2d gel electrophoresis. Western blots were performed using serum from rabbits collected before and after immunization with homogenized BP. Blots were probed with anti-rabbit IgG light chain specific secondary antibody (Jackson ImmunoResearch). Target proteins were digested with trypsin and assessed using MALDI-TOF/TOF mass spectroscopy. Protein identifications were made using MASCOT software to compare MS data to various databases (eg NCBInr, SWISS-PROT). Results - IgG specific anti-BP antibodies were found in both pre and post immunization serum. The distribution of these antibodies was mainly towards cytoplasmic proteins in the pre-immunization serum. Rapid and persistent development of anti-BP antibodies was found post-immunization, directed mainly at membrane proteins. Conclusion Rabbit serum contains pre-existing IgG antibodies directed towards several cytoplasmic proteins in BP. Following exposure to BP, rabbits exhibit a rapid and persistent development of IgG anti-BP antibodies, which are mainly directed towards membrane proteins.

Prion urine comprises a glycosaminoglycan-light chain IgG complex that can be stained by Congo red

Light chain IgG, a known amyloidotic protein, is present in the urine of prion disease affected individuals in a protease resistant form. In addition, it was shown recently that prion urine samples comprise a significant excess of glycosaminoglycans. Since amyloidotic proteins and glycosaminoglycans are the major components of amyloid aggregates, a Congo red dot blot assay was developed for detection of Creutzfeldt–Jacob disease (CJD) in urine. This assay was also positive for about 10% of patients suffering from diseases such as Alzheimer disease, cerebrovascular attacks and multiple sclerosis, but negative for healthy controls. Both glycosaminoglycans and proteins such as light chain IgG were required for the binding of Congo red to the urine fractions, as shown by the fact that Proteinase K digestion of the samples either after guanidine or after choindrotinase abolished the Congo red signal from the CJD samples.

Role of Apoptosis Signal-Regulating Kinase 1 in Stress-Induced Neural Cell Apoptosis in Vivo

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in oxidative stress-induced apoptosis. In the present study, we used ASK1 knockout (KO) mice to examine the possibility that ASK1 is involved in the neural cell apoptosis that occurs during retinal development and ischemic injury. ASK1 was expressed in retinal neurons, including retinal ganglion cells (RGCs), but retinal structure and extent of cell death during development were normal in ASK1 KO mice. On the other hand, the strain was less susceptible to ischemic injury, and the number of surviving retinal neurons was significantly increased compared with that in wild-type mice. Interestingly, ischemia-induced phosphorylation of p38 mitogen-activated protein kinase (p38), which mediates RGC apoptosis, was almost completely suppressed in ASK1 KO mice. In such retinas, the numbers of cleaved caspase-3- and TUNEL-positive neurons were apparently decreased compared with those in wild-type mice. Furthermore, cultured RGCs from ASK1 KO mice were resistant to H(2)O(2)-induced apoptosis. Our findings suggest that ASK1 is involved in the neural cell apoptosis after various kinds of oxidative stress. Thus, inhibition of the ASK1-p38 pathway could be useful for the treatment of neurodegenerative diseases including glaucoma.

Fibrillary glomerulonephritis associated with monoclonal gammopathy of undetermined significance showing lambda-type Bence Jones protein

A 79-year-old woman was admitted to our hospital because of leg edema due to a nephrotic syndrome. Urinary and serum immunoelectrophoresis showed positive for the lambda type of Bence Jones protein. A bone marrow aspiration test revealed mild plasmacytosis (6.4% of the total cells). These findings confirmed her diagnosis of monoclonal gammopathy of undetermined significance (MGUS). Her renal biopsy specimen revealed mild mesangial cell proliferation and an increase in the mesangial matrix. Immunofluorescence studies showed positive staining for IgG, IgA, C3, and kappa and lambda light chains in the capillary wall and mesangium area. Electron microscopy showed that the electron deposits in the thickened basement membrane were formed by randomly arranged 16- to 18-nm nonbranching fibrils. A Congo red stain for amyloid was negative. These findings corresponded with the diagnosis of fibrillary glomerulonephritis. Therefore, this case showed a rare combination of fibrillary glomerulonephritis and MGUS.

Macrocytosis associated with monoclonal gammopathy

To report the potential association between unexplained macrocytosis and monoclonal gammopathy. We retrospectively reviewed the medical records of patients who had monoclonal protein detected by serum electrophoresis and immunofixation from October 1999 until September 2003 at our institution. Patients with concomitant macrocytosis were included in this study. We collected data on patient demographics, evaluations performed for macrocytosis, pertinent laboratory tests relevant to the diagnosis of monoclonal gammopathy and presence of associated hematologic disorders. We identified 258 patients with monoclonal gammopathy. Thirty-one (12%) of them had concomitant macrocytosis. Of the latter group, 14 (5%) patients had no identifiable cause of macrocytosis after thorough evaluation and were considered to have macrocytosis associated with monoclonal gammopathy. The median values for mean cell volume and serum monoclonal protein were 103.9 fL (range 100.8-109.8) and 1.95 gm/dL (range 0.8-4.3), respectively. Most patients had IgG (71%) and kappa light chain (79%). All of the 11 (of 14) patients who underwent a bone marrow biopsy as part of the initial evaluation had megaloblastoid maturation of the erythroid precursors. No correlation was found between the level of serum monoclonal protein and the degree of macrocytosis (r = +0.48, P = 0.08). After a median follow-up of 22.5 month (range 3-60+), all but one patient had persistent but stable macrocytosis. Macrocytosis can be a manifestation of monoclonal gammopathy. Disorders associated with monoclonal gammopathy should be considered in the differential diagnoses during evaluation of macrocytosis.