The murine IgH locus contains a distinct DNA sequence motif for the chromatin regulatory factor CTCF.

Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP-chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.

Effect of lactoferrin on taste and smell abnormalities induced by chemotherapy: a proteome analysis.

Cancer patients receiving chemotherapy often experience taste and smell abnormalities (TSA). To date, the underlying molecular mechanisms of this frequent side-effect have not been determined and effective treatments are not available. This study assessed the feasibility of lactoferrin (LF) supplementation as a treatment for TSA and investigate the related mechanisms through salivary proteome analysis. Nineteen cancer patients with established TSA following chemotherapy administration were enrolled in this study. Cancer patients and additional 12 healthy subjects took LF supplements, 3 tablets per day (250 mg per tablet), for 30 days. Saliva was collected at three timepoints: baseline, 30-day LF supplementation, and 30-day post-LF supplementation. Patient's TSA level, salivary proteome, and salivary minerals at each LF treatment stage were analyzed. High TSA level was associated with high concentration of salivary Fe and loss of critical salivary immune proteins. LF supplementation significantly decreased the concentration of salivary Fe (P = 0.025), increased the abundance (P < 0.05) of salivary α-amylase and Zn-α-2-GP, and led to an overall increase of expression (≥2-fold changes) of immune proteins including immunoglobulin heavy chain, annexin A1, and proteinase inhibitor. Abundance of α-amylase and SPLUNC2 were further increased (P < 0.05) at 30-day post-LF supplementation in cancer patients. At the same time, total TSA score was significantly reduced (P < 0.001) in chemotherapy patients. This study demonstrated the feasibility of developing lactoferrin supplementation as a treatment to reduce TSA caused by chemotherapy and improve cancer patient's oral immunity.

YY1 Is Required for Germinal Center B Cell Development.

YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction.

Development of antiricin single domain antibodies toward detection and therapeutic reagents.

Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.6 ng/mL was obtained. One sdAb pair in particular was found to be highly specific for ricin. While polyclonal antibodies cross react strongly with RCA120, the sdAb pair had minimal cross reactivity. In addition, the binders were found to be thermal stable, regaining their ricin binding activity following heating to 85 degrees C for an hour. Cycles of thermally induced unfolding of the sdAb and their subsequent refolding upon cooling was monitored by circular dichroism. As several of the sdAb were observed to bind to ricin's A chain, cell free translation assays were performed to monitor the ability of the sdAbs to inhibit ricin's biological activity. One of the sdAb (C8) was particularly effective and blocked ricin's biological activity with an effectiveness equal to that of a mouse antiricin antibody. These results indicate that antiricin sdAb have great potential for both diagnostic and therapeutic applications.

Improved anti-IgG and HSA affinity ligands: clinical application of VHH antibody technology.

Large scale, highly specific purification of valuable proteins from blood and removal of undesirable components promise to have wide therapeutic applications. Moreover, depletion of bulk proteins from blood is a prerequisite for clinical proteomics. Here we describe the development of specific, high affinity Camelid antibody fragments (VHH) derived from immune libraries for purification and depletion of the bulk protein HSA and IgG from human serum and plasma for therapeutic and research purposes. The anti-IgG VHH substantially improved depletion of IgGs from blood over the classical method based on protein A. To demonstrate the improved performance of VHH based IgG depletion, we analyzed the presence of auto-antibodies in human plasma before and after depletion from two groups of patients with auto-immune disease: Goodpasture syndrome (GP) and systemic lupus erythematosus (SLE). VHHs can be produced efficiently and cost effectively in Saccharomyces cerevisiae, a genetically regarded as safe (GRAS) microorganism. A good manufacturing process (GMP) for purification of these VHHs has also been developed. Moreover, as VHHs are single protein chains, they can be coupled relatively easily to solid matrices. These three factors are important for developing affinity purification medication.

Early B-cell factor-associated zinc-finger gene is a frequent target of retroviral integration in murine B-cell lymphomas.

The early B-cell factor (EBF)-associated zinc-finger protein (EBFAZ) binds to and negatively regulates EBF, a basic helix-loop-helix transcription factor required for B-cell lineage commitment and development of the olfactory epithelium. It also binds to SMA- and MAD-related protein 1 (SMAD1) and SMAD4 in response to bone morphogenic protein 2 (BMP2) signaling. It is highly related to ecotropic viral integration site 3 (EVI3), a protein that, like EBFAZ, contains 30 Krüppel-like zinc-finger repeats. In previous studies, we showed that Evi3 is a frequent target of retroviral integration in AKXD27 B-cell lymphomas. Here, we show that EBFAZ is also a frequent target. Integrations at Ebfaz and Evi3 are mutually exclusive, suggesting that they function in the same tumor pathway. Lymphomas with integrations at Ebfaz or Evi3 express the pre-B-cell-specific marker immunoglobulin lambda chain 5, and contain immunoglobulin heavy-chain rearrangements, suggesting that they are blocked at an early B-cell stage. Unlike Evi3, which is expressed at low levels in normal B cells, or Ebfaz, which is not expressed in B cells, both genes are highly expressed following viral integration. Collectively, our results suggest that ectopic expression of Ebfaz can substitute for the upregulated expression of Evi3 in B-cell disease and highlight the importance of this gene family in hematopoietic cancer.

NF-kappa B and Oct-2 synergize to activate the human 3' Igh hs4 enhancer in B cells.

In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3' Igh enhancers, which are located downstream of the Calpha gene(s) in both mouse and human. In vivo experiments have implicated murine 3' enhancers, hs3B and/or hs4, in class switching and somatic hypermutation. We previously reported that murine hs4 was regulated by NF-kappaB, octamer binding proteins, and Pax5 (B cell-specific activator protein). In this study we report that human hs4 is regulated differently. EMSAs and Western analysis of normal B cells before and after stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern formed by NF-kappaB, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1) binding was also observed. We have confirmed the in vivo association of these transcription factors with hs4 in B cells by chromatin immunoprecipitation assays. The diffuse large B cell lymphoma cell lines had a distinctive slow-migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not with other NF-kappaB family members. Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NF-kappaB and octamer binding proteins. In addition, human hs4 enhancer activity required Oct-2 and correlated with expression of Oct coactivator from B cells (OCA-B).

Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage.

The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (mu(0)) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F(1) (DQ52(+/-)) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52(-) allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM(+) B cells of bone marrow and spleen used the DQ52(-) allele less frequently. On DJ joints of the DQ52(-) allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52(-) allele. In splenic B cells of the DQ52(-/-) mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3' JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.

Induction of glucose-regulated protein 78 in rat uterine glandular epithelium during uterine sensitization for the decidual cell reaction.

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.

Cytogenetic analysis of chimeric antibody-producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure.

Previously, the highest producing (HP) recombinant CHO subclones isolated at various methotrexate (MTX) levels showed different antibody production stability during long-term culture, although they were clonally derived from CS13 transformant. In this study, genetic basis for their difference in antibody production stability was investigated using southern blot hybridization and fluorescence in situ hybridization (FISH) techniques. Southern analysis of HP subclones revealed that light-chain (LC) and heavy-chain (HC) cDNAs were located closely within 23 kb on an amplification unit, and the configuration of LC and HC cDNAs within this amplification unit was not disrupted during long-term culture in the absence of MTX. However, when LC and HC genes were localized on the metaphase chromosomes of HP subclones using FISH, the amplified sequences were present as an extended array on diverse marker chromosomes. HP subclones selected at higher MTX level had more kinds of marker chromosomes. CS13*-002 isolated at 0.02 microM MTX had only one marker chromosome (m002), whereas CS13*-1.0 isolated at 1 microM MTX had five different ones (m10A, m10B, m10C, m10D, and m10E). Each marker chromosome showed different fate during long-term culture of HP subclones in the absence of MTX, resulting in different degrees of stability among the HP subclones. The m10A and m10B remained unchanged, whereas the others disappeared or evolved to variants with shortened amplified arrays. The cells containing stable marker chromosomes constituted dominant subpopulations in CS13*-1.0, and thereby CS13*-1.0 became most stable in regard to antibody production during long-term culture. Furthermore, our dual-color FISH showed that the telomeric ends of amplified arrays on the stable marker chromosomes were always surrounded by (TTAGGG)(n) sequences, indicating that (TTAGGG)(n) sequences are closely related to the stability and evolution of amplified sequences. Taken together, our data show that the assessment of genotypic stability of amplified CHO cells is a prerequisite for understanding their production stability during long-term culture in the absence of selection pressure.

Secretory immunoglobulin A heavy chain presents Galbeta1-3GalNAc binding structures for Actinomyces naeslundii genospecies 1.

Adherence of Actinomyces naeslundii ATCC 12104 to hydroxyapatite beads coated with protein fractions of parotid saliva, obtained by gel filtration on S-200 HR columns, showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to high-molecular-weight proteins (Strömberg et al., 1992). The present study investigates the nature of these high-molecular-weight binding proteins and determines their specific ability to mediate adherence to representative strains of Actinomyces species. Strain ATCC 12104 bound specifically in a lactose-inhibitable manner to the heavy chain of secretory immunoglobulin A (S-IgA), contained within a high-molecular-weight parotid protein fraction separated on SDS-PAGE and transferred to a solid membrane support. Lactose-inhibitable binding to the heavy chain of S-IgA from human colostrum was also demonstrated. Peanut agglutinin bound to the heavy chain of parotid and colostrum S-IgAs contained on solid support membranes, confirming the presence of Galbeta1-3GalNAc residues on these molecules. Both salivary and colostrum S-IgA aggregated with strain ATCC 12104 in a GalNAcbeta1-3Galalpha-O-ethyl-inhibitable fashion. Further separation of high-molecular-weight salivary proteins on S-500 HR columns showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to both mucin- and S-IgA-containing fractions. The presence of S-IgA in salivary pellicles formed in vivo on teeth was demonstrated by Western blot analysis of pellicle extracts with anti-IgA antibodies. Among strains representing A. naeslundii genospecies 1 and 2 and A. odontolyticus, only those of genospecies 1 with a particular adherence profile showed efficient GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to S-IgA. Thus, oligosaccharides on S-IgA may promote bacterial aggregation (or adherence) and provide a mechanism by which S-IgA can interact with bacteria without prior immunological challenge.

Expression of IL-4, Cepsilon RNA, and Iepsilon RNA in the nasal mucosa of patients with seasonal rhinitis: effect of topical corticosteroids.

BACKGROUND: Nasal allergen provocation has demonstrated that allergen-induced rhinitis is associated with an increase in local IL-4 mRNA and IgE heavy chain (Cepsilon) and IgE heavy chain promoter (Iepsilon) RNA and that pretreatment with topical glucocorticosteroids inhibits the increase in these transcripts. OBJECTIVE: This study was undertaken to determine whether observations made after acute allergen provocation can be extended to the case of chronic exposure experienced during the pollen season. METHODS: Biopsy specimens were obtained from the inferior turbinate of 33 pollen-sensitive subjects with allergic rhinitis before and during pollen season. Patients were randomized in a double-blind fashion and treated with either topical steroids (200 microg fluticasone propionate twice daily; n = 16) or matched placebo nasal spray (n = 17) before the pollen season. Alkaline phosphatase anti-alkaline phosphatase immunocytochemistry was used to identify B cells (CD20+), and in situ hybridization was used to detect IL-4, Cepsilon, and Iepsilon RNA+ cells. RESULTS: Baseline examination revealed IL-4 and Cepsilon RNA but virtually no Iepsilon RNA+ cells in the nasal mucosa. Analysis revealed a significant difference in the expression of Cepsilon and Iepsilon RNA+ cells (p < 0.001). Biopsy specimens taken after antigen exposure exhibited highly significant increases in placebo-treated (p < 0.001) but not steroid-treated patients. In both groups, the number of CD20+ cells was unchanged when preexposure and postexposure biopsy specimens were compared. CONCLUSIONS: These results show strong support for the hypothesis that IgE class switching occurs locally within the nasal mucosa of subjects with seasonal allergic rhinitis and that this response can be inhibited through strategies directed against local IgE production.

Isolation and characterization of a cDNA encoding a Xenopus immunoglobulin binding protein, BiP (grp78).

We have isolated a full-length cDNA clone encoding a Xenopus laevis immunoglobulin binding protein (BiP; also called glucose-regulated protein or grp78). The Bip cDNA sequence includes an open reading frame of 1,965 bp encoding a 655 amino acid protein with an N-terminal hydrophobic leader sequence and a C-terminal KDEL tetrapeptide which has been found in other lumenal proteins of the endoplasmic reticulum. The 3' untranslated region contains a polyadenylation and an adenylation control element (ACE) as well as a putative mRNA instability sequence. The Xenopus BiP amino acid sequence displayed high identity with BiP from other vertebrates including chicken (91.3%), rat (90.7%), and human (89.9%). Northern hybridization analysis demonstrated that BiP mRNA was present constitutively in the Xenopus A6 kidney epithelial cell line and that BiP mRNA levels could be enhanced by treatment of the cells with galactose-free media, 2-deoxyglucose, 2-deoxygalactose, glucosamine, tunicamycin, heat shock, dithiothreitol, and the calcium ionophore, A23187. Finally, while BiP mRNA was detected in all of the adult tissues examined, the relative level of BiP mRNA differed dramatically between organs. For example, relatively high levels of BiP mRNA were detected in liver with moderate levels in testis, ovary and heart and reduced levels in eye and muscle tissue.

Cleavage of immunoglobulin A1, A2 and G by proteases from clinical isolates of Pasteurella multocida.

Several Pasteurella multocida strains were examined for their ability to produce extracellular enzymes that cleave immunoglobulin A and G (Ig A and Ig G) molecules. Two strains isolated from human pulmonary and genital infections produced proteases that cleaved human IgA and IgG, colostral IgA and human myeloma IgA1 and IgA2. Human IgM was not degraded by these enzymes. Examination of cleavage digests showed two main fragments with different electrophoretic mobilities. The two P. multocida strains produced a protease that cleaved IgA and IgG heavy chains outside the hinge region, and differed in this respect from the hinge-cutting proteases of other bacteria. Protease production may be a virulence mechanism for P. multocida strains.