In vitro apolipoprotein B mRNA editing activity can be modulated by fasting and refeeding rats with a high carbohydrate diet.

Apolipoprotein B mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the metabolic modulation of apo B mRNA editing activity can be assayed in vitro using rat liver extracts. The editing activity in extracts from 48h-fasted rats was suppressed relative to that of normal chow-fed rats. Refeeding with a high-sucrose fat-free chow for 48h stimulated liver in vitro editing activity to approximately three times that of control liver extracts. The physical properties of editosomes assembled in extracts from fasted/refed rats differed from those assembled in control or fasted rat liver extracts. Polypeptide analysis revealed quantitative alterations of several proteins in each treatment group suggesting a complex regulatory process. The data corroborate those from in vivo studies and suggest the potential of the in vitro system in studying factors responsible for metabolic regulation of apo B mRNA editing.     

Influence of avidity and idiotope recognition on the modulation of surface immunoglobulin on malignant human B cells by rat monoclonal anti-idiotype antibodies

Immunoglobulin (Ig) was obtained from the tumor cells of patients with B cell malignancies by somatic cell hybridization to mouse-human heteromyeloma cells. The human Ig secreted by one of these hybridomas was used as an immunogen for the production of rat monoclonal antibodies (mAb). A panel of mAb specific for the idiotype (Id) was produced and characterized. Competitive binding studies that made use of [Se]-labeled anti-Id mAb (MAID) demonstrated several distinct yet topographically related Id on the Id-bearing Ig. These antibodies were shown to have avidities ranging from 0.38 to 45.3 X 10(8) l/mol. Additional studies demonstrated varying degrees of antigenic modulation of surface Id in vitro by MAID. The degree of modulation correlates with antibody avidity.     

Naturally occurring antibodies to liposomes. II. Specificity and electrophoretic pattern of rabbit antibodies reacting with sphingomyelin-containing liposomes

Sera obtained from rabbits after immunization with a variety of unrelated antigens contain antibodies that induce complement- (C) mediated lysis of sphingomyelin-containing liposomes in the absence of the relevant antigen from the membrane. Absorption or inhibition with dimyristoyl-phosphatidyl choline-containing liposomes were less effective than with sphingomyelin-containing liposomes in decreasing or abolishing C-dependent lysis of target-liposomes. Phosphoryl choline chloride inhibited the C-dependent lysis mediated by these antibodies, but only when used in high molar excess and in the presence of low antibody concentrations. Purified anti-liposome antibodies displayed an isoelectric focusing pattern consistent with a polyclonal response. The findings confirm the antibody nature of the anti-liposome activity of rabbit sera and indicate that their predominant specificity is directed against conformations of the phospholipid molecule in which the polar (phosphoryl choline) group does not have a major contribution.     

Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.     

Mouse monoclonal antibodies to chicken VH idiotypic determinants reactivity with B and T cells

We have prepared mouse monoclonal antibodies against idiotypic (Id) determinants on chicken antibodies to N-acetylglucosamine (NAGA) and p-aminobenzoic acid (PABA) made by inbred line EL 6(3) birds. The monoclonal anti-NAGA Id antibody, termed CId-1, reacted with affinity purified antibodies to NAGA, but not with antibodies specific for PABA, arsanilic acid (Ars), phosphorylcholine (PC), or with normal chicken IgG and IgM. The monoclonal anti-PABA ID antibody, termed CId-2, reacted with anti-PABA antibodies and to a lesser extent with anti-Ars antibodies, but not with anti-NAGA, anti-PC, and normal IgG and IgM. The Id determinants were found among antibodies to NAGA and PABA made by outbred and inbred lines of White Leghorn chickens. The binding of the CId-1 and CId-2 antibodies to intact homologous anti-NAGA and anti-PABA antibodies, respectively, was not hapten-inhibitable in either case. Both anti-Id antibodies reacted specifically with isolated homologous heavy chains, suggesting VH Id specificities. The monoclonal CId-1 and CId-2 antibodies were reactive by immunofluorescence with approximately 0.9 and 0.2%, respectively, of the circulating lymphocytes and with approximately 0.4 and 0.15 of plasma cells. CId-1+ and CId-2+ bursal cells were first detected on the 16th and 14th days of incubation, respectively; both reached maximal frequencies by the 17th day of incubation. The CId-2 antibody reacted exclusively with immunoglobulin-positive cells. The CId-1 antibody also reacted with a subpopulation (0.4%) of immunoglobulin-negative lymphocytes from normal and agammaglobulinemic chickens, and thus would appear to recognize an idiotypic determinant expressed by certain clones of B and T cells.     

SV 40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes

Cell-based antitumor immunity is driven by CD8(+) cytotoxic T cells bearing TCR that recognize specific tumor-associated peptides bound to class I MHC molecules. Of several cellular proteins involved in T cell:target-cell interaction, the TCR determines specificity of binding; however, the relative amount of its contribution to cellular avidity remains unknown. To study the relationship between TCR affinity and cellular avidity, with the intent of identifying optimal TCR for gene therapy, we derived 24 MART-1:27-35 (MART-1) melanoma Ag-reactive tumor-infiltrating lymphocyte (TIL) clones from the tumors of five patients. These MART-1-reactive clones displayed a wide variety of cellular avidities. alpha and beta TCR genes were isolated from these clones, and TCR RNA was electroporated into the same non-MART-1-reactive allogeneic donor PBMC and TIL. TCR recipient cells gained the ability to recognize both MART-1 peptide and MART-1-expressing tumors in vitro, with avidities that closely corresponded to the original TCR clones (p = 0.018-0.0003). Clone DMF5, from a TIL infusion that mediated tumor regression clinically, showed the highest avidity against MART-1 expressing tumors in vitro, both endogenously in the TIL clone, and after RNA electroporation into donor T cells. Thus, we demonstrated that the TCR appeared to be the core determinant of MART-1 Ag-specific cellular avidity in these activated T cells and that nonreactive PBMC or TIL could be made tumor-reactive with a specific and predetermined avidity. We propose that inducing expression of this highly avid TCR in patient PBMC has the potential to induce tumor regression, as an "off-the-shelf" reagent for allogeneic melanoma patient gene therapy.     

Identification of GRY-RBP as an apolipoprotein B RNA-binding protein that interacts with both apobec-1 and apobec-1 complementation factor to modulate C to U editing

C to U editing of apolipoprotein B (apoB) mRNA involves the interaction of a multicomponent editing enzyme complex with a requisite RNA sequence embedded within an AU-rich context. This enzyme complex includes apobec-1, an RNA-specific cytidine deaminase, and apobec-1 complementation factor (ACF), a novel 65-kDa RNA-binding protein, that together represent the minimal core of the editing enzyme complex. The precise composition of the holo-enzyme, however, remains unknown. We have previously isolated an enriched fraction of S100 extracts, prepared from chicken intestinal cells, that displays apoB RNA binding and which, following supplementation with apobec-1, permits efficient C to U editing. Peptide sequencing of this most active fraction reveals the presence of ACF as well as GRY-RBP, an RNA-binding protein with approximately 50% homology to ACF. GRY-RBP was independently isolated from a two-hybrid screen of chicken intestinal cDNA. GRY-RBP binds to ACF, to apobec-1, and also binds apoB RNA. Experiments using recombinant proteins demonstrate that GRY-RBP binds to ACF and inhibits both the binding of ACF to apoB RNA and C to U RNA editing. This competitive inhibition is rescued by addition of ACF, suggesting that GRY-RBP binds to and sequesters ACF. As further evidence of the role of GRY-RBP, rat hepatoma cells treated with an antisense oligonucleotide to GRY-RBP demonstrated an increase in C to U editing of endogenous apoB RNA. ACF and GRY-RBP colocalize in the nucleus of transfected cells and, in cotransfection experiments with apobec-1, each appears to colocalize in a predominantly nuclear distribution. Taken together, the results indicate that GRY-RBP is a member of the ACF gene family that may function to modulate C to U RNA editing through binding either to ACF or to apobec-1 or, alternatively, to the target RNA itself.     

The murine IL 2 receptor. I. Monoclonal antibodies that define distinct functional epitopes on activated T cells and react with activated B cells

The properties of three distinct rat monoclonal antibodies, designated 3C7, 7D4, and 2E4, to the murine IL 2 receptor have been compared in binding, biochemical, and functional assays. 3C7 appears to define an epitope near or identical to the IL 2-binding site of the receptor, because 3C7 inhibited the binding of radiolabeled IL 2 to CTL-L cells and because unlabeled IL 2 inhibited the binding of FITC-3C7 to CTL-L cells. 7D4 and 2E4 had no effect on IL 2 binding. Competitive antibody-binding studies confirmed that the epitope seen by 3C7 was distinct from the epitope(s) seen by 7D4 and 2E4. Sequential immunoprecipitation studies demonstrated that all three antibodies were reactive with the same molecular species, and that each precipitated identical components of 20,000 to 25,000 daltons, 50,000 to 60,000 daltons, and 100,000 to 120,000 daltons from the surface of CTL-L. FACS studies demonstrated a quantitatively and qualitatively identical cell distribution for the antigen defined by each antibody. They failed to stain more than 95% of resting lymphocytes, but were strongly reactive with Con A T blasts and substantially less reactive with LPS B blasts. Unlabeled IL 2 was also able to inhibit the binding of FITC-3C7 to LPS B cell blasts, suggesting the presence of IL 2-binding sites on activated B cells. Each antibody inhibited IL 2-driven proliferation of HT2 or CTL-L cells. 3C7 and 7D4 were more potent inhibitors of proliferation than was 2E4, and the combined use of 3C7 and 7D4 resulted in greater levels of inhibition of proliferation than that shown from the use of either antibody alone. Collectively, the results support the hypothesis that these antibodies detect two distinct functional regions of the IL 2 receptor.     

Frequency of B cells committed to the production of antibodies to insulin in newly diagnosed patients with insulin-dependent diabetes mellitus and generation of high affinity human monoclonal IgG to insulin

Circulating autoantibodies to insulin can be detected in patients with insulin-dependent (type I) diabetes mellitus (IDDM) at the onset of the clinical disease. To characterize the autoantibody response in IDDM patients, we determined the frequency of circulating B cells committed to the production of IgM, IgG, and IgA to insulin in 12 newly diagnosed IDDM patients and, for comparison, in 9 healthy subjects and 17 insulin-treated IDDM patients. We found that B cells committed to the production of anti-insulin IgG, but not IgM, autoantibodies are present at much higher frequency in the circulation of newly diagnosed IDDM patients before insulin treatment (0.209 +/- 0.142%, mean value +/- SD of total IgG-producing cell precursors) as compared with age-matched healthy controls (0.032 +/- 0.030% of total IgG-producing cell precursors). In IDDM patients who had been treated with insulin, cells producing IgG antibody to insulin were 0.177 +/- 0.139% of total IgG-producing cell precursors. Generation of IgG mAb from B cells of IDDM patients revealed that they were monoreactive, i.e., they bound to insulin, but to none of the other Ag tested, and displayed a high affinity for insulin (Kd approximately 10(-7) moles/liter). In contrast, the IgG mAb derived from healthy subjects were polyreactive, i.e., they bound to all Ag tested, and displayed a low to moderate affinity for insulin (Kd approximately 10(-5) to 10(-6) moles/liter). These findings show that lymphocytes committed to the production of high affinity IgG autoantibodies to insulin are common in the B cell repertoire at the onset of IDDM.     

Mutations of Epstein-Barr virus gH that are differentially able to support fusion with B cells or epithelial cells

The core fusion machinery of all herpesviruses consists of three conserved glycoproteins, gB and gHgL, suggesting a common mechanism for virus cell fusion, but fusion of Epstein-Barr virus (EBV) with B cells and epithelial cells is initiated differently. Fusion with B cells requires a fourth protein, gp42, which complexes with gHgL and interacts with HLA class II, the B-cell coreceptor. Fusion with an epithelial cell does not require gp42 but requires interaction of gHgL with a novel epithelial cell coreceptor. Epithelial cell fusion can be inhibited by gp42 binding to gHgL and by antibodies to gH that fail to block B-cell fusion. This suggests that regions of gHgL initiating fusion with each cell are separable from each other and from regions involved in fusion itself. To address this possibility we mapped the region of gH recognized by a monoclonal antibody to gH that blocks EBV fusion with epithelial cells but not B cells by making a series of chimeras with the gH homolog of rhesus lymphocryptovirus. Proteins with mutations engineered within this region included those that preferentially mediate fusion with B cells, those that preferentially mediate fusion with epithelial cells, and those that mediate fusion with neither cell type. These results support the hypothesis that the core fusion function of gH is the same for B cells and epithelial cells and that it differs only in the way in which it is triggered into a functionally active state.     

CpG drives human transitional B cells to terminal differentiation and production of natural antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.     

Production and analytical properties of antibodies with high specificity to zearalenone

The time course of production, specificity, and analytical potential of anti-zearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; alpha-zearalenol, 0.15%; and beta-zearalenol, < 0.02%. With synthetic analogues: zearalanone, 31.7% and alpha-zearalanol, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Establishment of permanent human endothelial cells achieved by transfection with SV40 large T antigen that retain typical phenotypical and functional characteristics

Summary The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan, was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE). In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted interleukins (IL-1β and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability to express the functional specific amino acid Na⁺-independent system Y⁺ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF, nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation, and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1β and IL-6 secretions, and gene regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary endothelial cells both from human and animal origin.     

CD22 is a recycling receptor that can shuttle cargo between the cell surface and endosomal compartments of B cells

CD22 is a member of the sialic acid-binding Ig-like lectin (Siglec) family that is known to be a regulator of B cell signaling. Its B cell-specific expression makes it an attractive target for immunotoxin-mediated B cell depletion therapy for the treatment of B cell lymphomas and autoimmune diseases. Although CD22 is well documented to be an endocytic receptor, it is believed that after internalization, it is targeted for degradation. We show in this study that CD22 is instead constitutively recycled to the cell surface. We also find that glycan ligand-based cargo is released from CD22 and accumulates intracellularly as CD22 recycles between the cell surface and endosomal compartments. In contrast, Abs to CD22 do not accumulate but remain bound to CD22 and recycle to the cell surface. The results have implications for development of agents that target CD22 as an endocytic receptor for delivery of cytotoxic cargo to B cells.     

Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans

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