Poly Ig Research Articles

The Distribution of IgT mRNA+ Cells in the Gut of the Atlantic Salmon (Salmo salar L.).

The newly discovered IgT+ B cell is thought to play a dominant role in mucosal immunity, but limited studies have examined its distribution in fish species, hindering our understanding of its function. This study investigated IgT and poly Ig receptor (pIgR) mRNA+ cell distribution in Atlantic salmon (Salmo salar) gut using RNAscope in situ hybridization (ISH) and assessed the effects of vaccination. The pyloric caeca, mid-intestine (first and second parts), and posterior segment in two weight stages (Group 1: avg. 153 g, Group 2: avg. 1717 g) were examined in both vaccinated and unvaccinated fish. ISH revealed more IgT mRNA+ cells in the second part of the midgut compared to other intestinal segments, as well as a higher number of positive cells in Group 2 (older fish). In line with previous findings, intraperitoneal vaccination had no significant impact on the number of IgT+ transcripts. IgT mRNA+ cells were found mostly in the lamina propria and near capillaries, while pIgR was registered in both the lamina propria and mucosa. Interestingly, vaccinated fish presented adhesions and granulomatous tissue in the peritoneum, with both IgT and pIgR mRNA+ cells. Taken together, these results suggest that the distribution of IgT mRNA+ cells in the intestine of Atlantic salmon is region-specific and is not affected by intraperitoneal vaccination but varies with fish age.

DOP51 Loss of PTPN23 in the intestinal epithelium results in epithelial hyperproliferation and lethal diarrhea in a microbiota dependent manner

Abstract Background Protein tyrosine phosphtase non-receptor type 23 (PTPN23) plays a critical role in regulating epidermal growth factor (EGF) receptor signaling and its loss has been associated with aberrant cell proliferation and promotes onset of epithelial cancers. However, the role of PTPN23 in the intestinal epithelium has not been investigated yet. Here, we investigated how PTPN23 deletion in intestinal epithelial cells (IEC) affects intestinal homeostasis. Methods To study the role of PTPN23 in the intestinal epithelium, we crossed mice with a LoxP flanked PTPN23 gene (PTPN23fl/fl mice) to mice expressing the CreERT construct under the villin promoter (PTPN23-VilCreERT mice). PTPN23 was deleted in IEC in these mice by tamoxifen-injections on five consecutive days. PTPN23 fl/fl mice injected with tamoxifen served as control. Results PTPN23 deletion in IEC resulted in drastic loss of weight starting around 10-14days after the first tamoxifen injection, eventually leading to death within 3–5 weeks due to severe wasting disease with severe diarrhea. Histology revealed massive hyper-proliferation of the colonic epithelium accompanied with elevated immune cell infiltration. In line with previous reports on the function of PTPN23, we observed highly elevated levels of EGF receptor, possibly accounting for the massive epithelial hyper-proliferation and aberrant water secretion/defective water resorption in these mice. Furthermore, we observed bacterial translocation to the spleen and the liver, indicating defective anti-bacterial defense in the intestinal epithelium of PTPN23-VilCreERT mice. In line with this, 16S sequencing revealed significant differences in the intestinal microbiome with an overgrowth of bacteria that have been reported to be heavily coated with IgA. PTPN23-VilCreERT mice showed reduced apical presence of the poly Ig receptor, a receptor that transports IgA from the lamina propria through IEC into the gut lumen. Notably, while serum IgA levels were normal, its levels in the stool were reduced, confirming defective transport of IgA into the gut lumen. In addition, autophagy was significantly reduced in PTPN23-VilCreERT mice when compared to their littermates. Of interest, antibiotic treatment was sufficient to prevent epithelial hyper-proliferation, diarrhea and death in PTPN23VilCreERT mice, indicating that this drastic phenotype was microbiota-dependent and likely the result of defects in bacterial handling. Conclusion Our results demonstrate that PTPN23 is indispensible for normal IEC function and its loss renders the intestinal epithelium unable to cope with invading bacteria. This uncovers a novel, so far unknown role of PTPN23 for regulating pathways involved in bacterial handling.

T-cell cytokines affect mucosal immunoglobulin A transport

Background Secretory immunoglobulin A (sIgA) is the principle immune defense against bacteria and other pathogens in the gut and other mucosal surfaces. sIgA is produced locally by plasma cells and transported (transcytosis) across epithelial cells into luminal secretions. sIgA production and transcytosis are governed by multiple signals, which may be affected by T cells. The purpose of this study was to elucidate the role of the Th1-type cytokine, interferon gamma (IFN-γ), and the Th2-type cytokine interleukin-4 (IL-4) on IgA transcytosis across intestinal epithelial cells in vitro. Methods HT-29 cell monolayers were established in a 2-chamber cell culture system. Cell monolayers were exposed to either IFN-γ, IL-4, or both on the apical or basal side of the culture system systems for 48 hours. Dimeric IgA then was added to the basolateral chamber and transcytosis into the apical chamber was quantified by enzyme-linked immunosorbent assay at timed intervals. IgA transcytosis across HT-29 cells without prior addition of either cytokine served as a control. Results Poly Ig–receptor up-regulation was shown by both IFN and IL-4. A combination of these 2 cytokines caused the greatest increase in receptor expression. A total of 3.7% of HT-29 cells expressed the polymeric Ig receptor in the control group. This increased to 23.7% in IL-4–treated cells, 66.3% in IFN-γ–treated cells, and 71.5% of cells treated with both cytokines. These findings re-show previously published findings. The effects of IFN-γ added to the basal chamber of HT-29 cell cultures shows a 7- to 10-fold increase in sIgA transcytosis at the 1-, 3-, and 12-hour time intervals. The effect of the addition of IL-4 to the basal chamber was similar to the results noted with IFN-γ alone. The greatest IgA transcytosis was noted when IFN-γ and IL-4 both were added to the basal chamber. However, these same results did not occur with cytokine exposure to the apical side of cell monolayers. Conclusions IFN-γ and IL-4 increased IgA transport across HT-29 cells in a polar fashion. An increased effect on IgA transport was noted with the combination of IFN-γ and IL-4. Delivery of T-cell cytokines to mucosal surfaces may support local antibody defense of mucosal surfaces and prevent infection.

The CMRF-35 mAb recognizes a second leukocyte membrane molecule with a domain similar to the poly Ig receptor.

The CMRF-35 mAb recognizes an antigen found on most leukocytes including monocytes, neutrophils, macrophages, dendritic cells, and subpopulations of lymphocytes and bone marrow cells. A cDNA expressing the CMRF-35 epitope was isolated by expression cloning and this predicts for a type I cell surface glycoprotein belonging to the Ig superfamily. Here we demonstrate that the CMRF-35 mAb recognizes an epitope more widely distributed on hemopoietic cells and cell lines than suggested by expression analysis of the CMRF-35 mRNA. Furthermore, we have isolated a novel cDNA (CMRF-35-H9) that encodes a protein product also recognized by the CMRF-35 mAb. This cDNA product is a type I cell surface glycoprotein with a single Ig V-like domain. Although the sequences of the extracellular V-like domains of the two molecules are very similar, there is little similarity between the remainder of their sequences. The two transcripts are expressed independently of each other, and their presence accounts for the discrepancy between CMRF-35 mAb binding and mRNA analysis. The cytoplasmic tail of CMRF-35-H9 contains motifs similar to the inhibitory motifs found in some leukocyte surface receptors. Their expression in hemopoietic cells suggests that these two molecules may play distinct but related roles in the regulation of leukocyte function.

Dimeric recombinant IgA directed against carcino-embryonic antigen, a novel tool for carcinoma localization

Carcinoembryonic antigen (CEA) has been shown to be one of the best markers for in vivo tumor targeting of radiolabeled antibodies, despite the fact that it is localized predominantly at the apical side of human colon carcinoma cells within the fairly closed pseudolumen structures formed by these tumors. Due to this particular histological localization, a large proportion of the CEA molecules may remain inaccessible to the intravenously injected radiolabeled anti-CEA antibodies of IgG isotype, which are widely used in the clinic. In order to improve targeting, we made a recombinant dimeric IgA, which should have the capacity to translocate from the basolateral to the apical side of the pseudolumen formed by colon carcinoma cells after binding to the poly Ig receptor (plgR). A genomic chimeric mouse-human IgA 2 construct was made using one of our most specific anti-CEA hybridomas, CE-25. The chimeric IgA (chlgA) was expressed in the Sp2/0 myeloma cell line. The secreted recombinant antibody was found to consist mostly of a dimeric form of IgA with a molecular weight of about 350 kDa. The dimeric chlgA was shown to translocate efficiently in vitro across a monolayer of epithelial cells expressing the plgR and to retain full CEA binding activity.

Rat secretory component binds poorly to rodent IgM.

Our previous studies and those of others indicated that human secretory component (SC), the five domain extracellular portion of the poly Ig receptor, binds avidly to both pIgA and IgM. In this study we report that in rodents, SC binds primarily to pIgA. Rat secretory component was isolated from bile and radiolabeled to known specific activity with 125I. Radiolabeled rat SC was incubated with rat and mouse monoclonal proteins for 1 h at room temperature and overnight at 4 degrees D. Binding of 125I-rat SC to Ig was determined in two ways: 1) immunoprecipitation of putative 125I-rat SC-Ig complexes with anti-L chain antibodies; 2) HPLC gel filtration on an analytical TSK 4000 column that separated free 125I-rat SC from 125I-rat SC bound to Ig. Both methods of analysis yielded similar results. Rat and mouse polymeric (p) IgA bound rat SC with high avidity, although the binding activity of the IgM from either species was virtually nil. The number of SC-binding sites on rat polymeric Ig was determined by immunoprecipitation of mixtures of rat pIg with saturating concentrations of 125I-rat SC and yielded values of 1.0 and 0.05 for rat pIgA and IgM, respectively. The significance of these findings with respect to the biologic function of the pIg R in rodents and the nature of the pIg R-binding site on pIg is discussed.

Molecular cloning of a novel member of the immunoglobulin gene superfamily homologous to the polymeric immunoglobulin receptor.

The CMRF35 monoclonal antibody recognizes a cell membrane antigen present on the surface of monocytes, neutrophils, a proportion of peripheral blood T and B lymphocytes and lymphocytic cell lines. Initial studies with CMRF35 suggested an unusual pattern of serological reactivity which did not correspond to any of the known leukocyte differentiation antigen clusters. We describe here the cloning and sequencing of a cDNA encoding the CMRF35 antigen by means of expression in COS cells and immunoselection with the CMRF35 monoclonal antibody. The cDNA encodes a novel integral membrane glycoprotein of 224 amino acids that represents a new member of the immunoglobulin (Ig) gene superfamily. The molecule comprises (a) a single extracellular Ig variable domain remarkably similar to the Fc receptor for polymeric IgA and IgM, (b) a membrane-proximal domain containing a high proportion of proline, serine and threonine residues that was predicted to be heavily O-glycosylated, (c) an unusual transmembrane anchor that contained a glutamic acid and a proline residue and (d) a short cytoplasmic tail. Transcripts encoding the CMRF35 protein were detected in early monocytic cell lines, in peripheral blood T cells and in some B lymphoblastoid cell lines, confirming the results of immunocytological staining. However, the level of CMRF35 expression on peripheral blood T cells was shown to decrease in response to mitogenic stimulation. The likelihood that the CMRF35 antigen shares a common evolutionary ancestor with the poly Ig Fc receptor is discussed.

Binding of secretory component to protein 511, a pIgA mouse protein lacking 36 amino acid residues of the Cα3 domain

Protein 511, a murine IgA protein described previously by Robinson and Appella [ Proc. natn. Acad. Sci. U.S.A. 77, 4909–4913 (1980)] which lacks 36 amino acids in the Cα3 domain, was tested for its ability to bind to radiolabelled secretory component ( 125I-rat SC) and to be transported from blood to bile in the rat, a function described previously to be mediated by the poly Ig receptor (pIg R). When compared to other mouse pIgA proteins, the naturally occurring mutant protein 511 bound 125I-rat SC and was transported from blood to bile in a manner indistinguishable from wild-type pIgA protein. We conclude that the region of Fcα which is missing in protein 511, is not involved in mediating the binding of pIgA to the pIg R.

Expression du récepteur des immunoglobulines polymères (R. poly Ig ou composante sécrétoire) par les cellules conjonctivales dans la maladie de Gougerot-Sjögren

We report for the first time the expression of the poly Ig Receptor at the conjonctival epithelial cell surface in normal subjects and in patients with Sjögren disease. Expression of the poly IgR was shown to disappear on conjonctival areas with metaplasia, suggesting an impairement of IgA transport into the lacrimal fluid.

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA-II adaptors (found in plasma membrane coated pits) and the HA-I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6-phosphate (M6P) receptor-a receptor which is concentrated in coated pits on both the plasma membrane and in the trans-Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA-II plasma membrane adaptor, which recognizes a 'tyrosine' signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA-I adaptors. This suggests that there is an HA-I recognition signal in another region of the M6P receptor tail, C-terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans-Golgi network, thus mediating efficient transfer of these enzymes to lysosomes.

Effects of sonication on physicochemical and biological properties of synthetic double-stranded polyribonucleotides as interferon inducer.

Two kinds of synthetic double-stranded polyribonucleotides, poly IG∶C1 and poly I∶:C and their constituent single-stranded polymers were subjected to sonication. Sonication of both poly IG∶C and poly I∶C resulted in decreases in viscosity, molecular size and heterogeneity in the size distribution. In poly IG∶C, whose average sedimentation constant was larger than or around 11 S, these changes were accompanied with enhancements of interferon inducing activity in rabbits and mice and antiviral activity in mice, and moreover with a decrease in the systemic toxicity in mice. In poly I∶C, however, such an enhancement in the interferon inducing activity was observed only when its molecular size corresponded to that of poly IG∶C. Previous sonication of poly C of the relatively large molecular size (> 10 S) has also been shown effective, to a certain extent, in obtaining double-stranded RNA of smaller size distribution with increased interferon inducing activity and lowered toxicity. It has been shown that these changes induced by sonication were based on the breakage of phosphodiester bonds of both double and single-stranded polyribonucleotides. On the basis of the analyses of the correlations between molecular sizes and the biological activities, it has been suggested that, while toxicity decreases always when the molecular size becomes smaller, the optimal size of the double-stranded polyribonucleotide complexes for interferon production ranges roughly from 10 S (9.1×105 daltons) to 5 S (1.2 ×105 daltons).

Influence of pH and of certain anions on DNA strand separation by poly IG

Influence of pH and of certain anions on DNA strand separation by poly IG

Melting of Complexes of Polyribocytidylic Acid Plus Copolymers of Inosinic and Guanylic Acids in Solution

Abstract Three copolymers of riboinosinic acid and riboguanylic acid (poly IG), with guanylic acid contents of 21.6%, 33%, and 45%, have been prepared, as well as three homopolymers of ribocytidylic acid (poly C), riboinosinic acid (poly I), and riboguanylic acid (poly G). On mixing both the poly IG and the poly G with poly C in various mole ratios in an aqueous solution, a marked hypochromicity was observed in the 250 mμ region, and it was shown that there are double helical complexes similar to the well-known poly(I+C) formed in the solutions. The optical melting profile of each complex into random coils was examined, and it was found that the guanine-cytosine pair is 20% (in enthalpy) more stable than the hypoxanthine-cytosine pair. It was also found that actinomycin D, which has no effect on the melting of poly (I+C), stabilizes the helical structure of poly(IG+C).

Regions containing repeating base-pairs in DNA from some oncogenic and nononcogenic animal viruses.

It is evident that DNA's from oncogenic animal viruses contain information necessary for malignant transformation of the host cell. However, the molecular mechanism by which these viruses transform cells is not well understood. There is a correlation between DNA base composition and the oncogenicity of hunmain adenovirus types.' In addition, viral genes apparently persist in cells tranisformed by polyoma virus, adenovirus type 12, and simian virus 40 (SV40).2-5 Further knowledge of the viral oncogenic process might be gained if more were known about the structural properties of oncogenic and nononcogenic virus DNA's. Recent studies indicate that DNA's from natural sources contain regions in which a given base-pair predominates.6' Pyrimidineand purine-rich sequences thus occur in one DNA strand, while the complementary and antiparallel sequences occur in the other. These sequences are detected in denatured DNA on the basis of their ability to bind certain ribopolymers. Sequences rich in deoxycytosi.ne (dO) form hydrogen-bonded complexes with poly IG, and sequences rich in deoxyadenosine (dA) form similar complexes with poly U. The DNA: RNA complexes can, then be identified in CsCl density gradients because their buoyant densities are greater than that of the denatured DNA. The density increments are related to the degree of DNA reactivity with poly IG or poly U. The presence of arn asymmetrical distribution of dC-rich sequences between the two DNA strands results in more poly IG binding by one strand. The same is true for poly U binding to DNA strands with differernt contents of dA-rich sequences. In these cases a bimodal pattern, of DNA: ribopolymer complexes is observed in the density gradient. The mode anld degree of reactivity with the ribopolymers is highly reproducible and specific for a given DNA.' 7 This technique thus permits an assessment of the content and distribution of dCand dA-rich sequences in the DNA molecule. A comparison of the relative ribopolymer reactivities of DNA's from oncogenic and nononcogenic viruses might then offer a clue as to genetic differences whi,ch could relate to viral oncogenicity. Several human adenoviruses were selected for investigation because they contain DNA of about the same molecular weight and have a spectrum of oncogenic capability.8-11 A highly oncogenic an.d unrelated virus, SV40, was also studied.12 13 It was found that DNA's from the oncogenic adenoviruses have fewer poly IG binding sites and more poly U binding sites than nononcogenic adenovirus DNA's. Furthermore, it was observed that SV40 virus DNA has few (if any) poly IG binding sites and is relatively more reactive with poly U than the DNA's from highly oncogenic adenoviruses. From these findings it is concluded that the oncogenic adenovirus DNA's contain less dC-rich sequences and more dA-rich sequences than DNA's from the nononcogenic adenoviruses. Materials and Methods.-Reagents: Poly IG and poly IJ were purchased from

Patterns of interaction between polyribonucleotides and individual DNA strands derived from several vertebrates, bacteria and bacteriophages

Poly G, poly IG, ribosomal RNA and poly U exhibit species-characteristic interactions with neither, one, or both strands of denatured DNA. Only one of the two complementary strands of DNA extracted from Bacillus and Clostridium species or T-odd coliphages forms complexes with ribosomal RNA, poly G or poly IG. Using CsCl density-gradient centrifugation, preparative separation of the lighter strand (dN G′) from the denser poly IG complex (DE G) containing the complementary strand is possible with the DNA of these micro-organisms. In the case of E. coli and other Enterobacteriaceae, band separation is less pronounced, since both strands complex with poly IG, albeit to various extents. Poly G is more efficient than poly IG or ribosomal RNA in effecting fractionation of the DNA strands into two widely separated bands in the CsCl gradient. The intact complementary strands of T1 and T7 coliphage DNA are separated in the presence of poly IG, indicating that cytosine-rich sequences are prevalent on only one of the two DNA strands. With the DNA of some species there is no strand separation into two distinct bands, since poly IG either reacts with both strands to similar extents (e.g. Sarcina lutea or Micrococcus lysodeikticus DNA), or reacts hardly at all (e.g. DNA from Cytophaga johnsonii or from T-even coliphage DNA). Poly U reacts with both strands of denatured DNA extracted from most of the organisms tested, with the exception of Micrococcus lysodeikticus, Sarcina lutea and coliphages T3 and T7. Every DNA tested exhibited affinity for at least one of the ribopolymers. These results seem to indicate that the mRNA-transcribing DNA strand contains widely spaced pyrimidine-rich sequences which act as the initiation points for the polycistronic RNA messages. At neutral pH, poly C, poly A and poly I do not interact with DNA. Poly C, however, interferes with the formation of complexes between denatured DNA and poly G, poly IG or ribosomal RNA, whereas poly A prevents similar DNA interaction with poly U. Complexes between denatured DNA and polynucleotides exhibit various degrees of resistance to pancreatic and T 1 RNases.