Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants.

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68-81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68-61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

IgA Plasma Cells Are Long-Lived Residents of Gut and Bone Marrow That Express Isotype- and Tissue-Specific Gene Expression Patterns.

Antibody secreting plasma cells are made in response to a variety of pathogenic and commensal microbes. While all plasma cells express a core gene transcription program that allows them to secrete large quantities of immunoglobulin, unique transcriptional profiles are linked to plasma cells expressing different antibody isotypes. IgA expressing plasma cells are generally thought of as short-lived in mucosal tissues and they have been understudied in systemic sites like the bone marrow. We find that IgA+ plasma cells in both the small intestine lamina propria and the bone marrow are long-lived and transcriptionally related compared to IgG and IgM expressing bone marrow plasma cells. IgA+ plasma cells show signs of shared clonality between the gut and bone marrow, but they do not recirculate at a significant rate and are found within bone marrow plasma cells niches. These data suggest that systemic and mucosal IgA+ plasma cells are from a common source, but they do not migrate between tissues. However, comparison of the plasma cells from the small intestine lamina propria to the bone marrow demonstrate a tissue specific gene transcription program. Understanding how these tissue specific gene networks are regulated in plasma cells could lead to increased understanding of the induction of mucosal versus systemic antibody responses and improve vaccine design.

High microbiota reactivity of adult human intestinal IgA requires somatic mutations.

The gut is home to the body's largest population of plasma cells. In healthy individuals, IgA is the dominating isotype, whereas patients with inflammatory bowel disease also produce high concentrations of IgG. In the gut lumen, secretory IgA binds pathogens and toxins but also the microbiota. However, the antigen specificity of IgA and IgG for the microbiota and underlying mechanisms of antibody binding to bacteria are largely unknown. Here we show that microbiota binding is a defining property of human intestinal antibodies in both healthy and inflamed gut. Some bacterial taxa were commonly targeted by different monoclonal antibodies, whereas others selectively bound single antibodies. Interestingly, individual human monoclonal antibodies from both healthy and inflamed intestines bound phylogenetically unrelated bacterial species. This microbiota cross-species reactivity did not correlate with antibody polyreactivity but was crucially dependent on the accumulation of somatic mutations. Therefore, our data suggest that a system of affinity-matured, microbiota cross-species-reactive IgA is a common aspect of SIgA-microbiota interactions in the gut.

Deoxycholic acid disrupts the intestinal mucosal barrier and promotes intestinal tumorigenesis.

High-fat diet, which leads to an increased level of deoxycholic acid (DCA) in the intestine, is a major environmental factor in the development of colorectal cancer (CRC). However, evidence relating to bile acids and intestinal tumorigenesis remains unclear. In this study, we investigated the effects of DCA on the intestinal mucosal barrier and its impact on the development of CRC. Here we showed that DCA disrupted cell monolayer integrity and increased proinflammatory cytokine production in intestinal cancer and precancerous cell lines (Caco-2 and IMCE). Apcmin/+ mice receiving DCA increased the number and size of intestinal adenomas and promoted the adenoma-adenocarcinoma sequence. Importantly, DCA induced the activation of the NLRP3 inflammasome, increased the production of inflammatory cytokines, and led to intestinal low grade inflammation. A reduction of tight junction protein zonula occludens 1 (ZO-1) and the number of intestinal cells including goblet cells and Paneth cells was also observed after DCA treatment. Moreover, DCA significantly reduced the level of secretory immunoglobulin A (sIgA), and promoted the polarization of M2 macrophages in the intestine of Apcmin/+ mice. In conclusion, these data suggested that DCA induced intestinal low grade inflammation and disrupted the mucosal physical and functional barriers, aggravating intestinal tumorigenesis.

Insulin-like growth factor I reduces the occurrence of necrotizing enterocolitis by reducing inflammatory response and protecting intestinal mucosal barrier in neonatal rats model.

OBJECTIVE: To observe the effect of enteral supplement of insulin-like growth factor I (IGF-1) on dynamic changes of TLR4, NF-κB, IL-6, SIgA and MUC2 in intestinal tissues of neonatal rats, and to investigate the protective effects and possible mechanisms of IGF-1 on necrotizing enterocolitis (NEC). MATERIALS AND METHODS: Specific pathogen free (SPF) neonatal Sprague Dawley (SD) rats aged 3 days old were randomly divided into 3 groups, namely, normal control group, NEC model group and IGF-1 intervention group. In NEC group, the neonatal NEC rat models were established using artificial feeding, hypoxia and cold stimulation. In IGF-1 intervention group, the models were established by means of artificial feeding plus hypoxia and cold stimulation, and IGF-1 (22 ug/L) at a physiological concentration similar to the breast milk was added to milk replacer for intervention. The rats in the three groups were killed after the blood was collected from the heart at 24, 48 and 72 h, respectively, following the establishment of models; then, 3 cm of the terminal ilea were dissected and used for histopathological examination, RT-PCR and ELISA studies after hematoxylin and eosin (HE) staining. RESULTS: Symptoms in IGF-1 intervention group were significantly relieved, and the incidence rate of NEC was lowered remarkably. In NEC model group, the peak expression of TLR4 mRNA occurred later than that of NF-κB mRNA and IL-6, and the expressions of TLR4 mRNA, NF-κB mRNA and IL-6 were decreased at 72 h after IGF-1 intervention. In NEC model group, the expression of MUC2 showed a transient decrease, the expression of SIgA was on the decline, but the expressions of MUC2 and SIgA were increased after IGF-1 intervention. CONCLUSIONS: The enteral administration of IGF-1 at a physiological concentration can ameliorate the clinical symptoms in neonatal NEC rat models and decrease the occurrence rate. The possible mechanism is that IGF-1 down-regulates the TLR4 mRNA expression to inhibit the production of inflammatory mediators, and it up-regulates the expressions of MUC2 and SIgA to protect the mechanical and immuno-barrier functions of the intestinal mucous.

IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches.

Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-ß receptor (LTßR)-dependent maintenance of DCs. PP DCs augment IgA production by integrin αvß8-mediated activation of transforming growth factor-ß (TGFß). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGFß activation and induction of mucosal IgA responses.

Effects of dietary supplementation of synbiotics on growth performance, intestinal morphology, sIgA content and antioxidant capacities of broilers.

Today, several strategies are being used to decrease the serious effects of antibiotics abuse on broilers industry and public health, among which synbiotics are one of the most promising antibiotic alternative. This study was undertaken to assess the effects of synbiotics, which composed of probiotics (Bacillus subtilis) and prebiotics (xylooligosaccharide and mannanoligosaccharide), on growth performance, intestinal morphology, sIgA content and antioxidant parameters of broilers. Four hundred and fifty one-day-old commercial Cobb48 broilers were assigned to five treatments consisting of six replicates of 15 birds each pen. Five dietary treatments include basal diets (control), basal diets plus antibiotics (4 mg/kg Xanthomycin), basal diets plus 1 g of probiotics B. subtilis product/kg of diets (4 × 108  cfu/kg), basal diets plus 150 mg/kg xylooligosaccharide (35%) and 1 g/kg mannanoligosaccharide (75%), and basal diets plus synbiotics (1 g of probiotics B. subtilis product/kg of diets (4 × 108  cfu/kg), 150 mg/kg xylooligosaccharide (35%) and 1 g/kg mannanoligosaccharide (75%). The results demonstrated that on 21 and 42 days, dietary supplementation of the synbiotics significantly increased daily weight gain (p < 0.05), feed efficiency (p < 0.05), the villus height and villus:crypt ratio in the duodenum, jejunum and ileum (p < 0.05), as well as intestinal mucosa sIgA content (p < 0.05), serum T-SOD activity (p < 0.05) and lysozyme content (p < 0.05), comparing with control group. In conclusion, synbiotics (B. subtilis and xylooligosaccharide and mannanoligosaccharide) is one of the safe and ideal dietary supplementations to increase broilers' growth performance by improving small intestinal morphology, sIgA content and antioxidant capabilities.

Enhanced differentiation of intraepithelial lymphocytes in the intestine of polymeric immunoglobulin receptor-deficient mice.

To clarify the effect of secretory IgA (sIgA) deficiency on gut homeostasis, we examined intraepithelial lymphocytes (IELs) in the small intestine (SI) of polymeric immunoglobulin receptor-deficient (pIgR(-/-) ) mice. The pIgR(-/-) mice exhibited the accumulation of CD8αß(+) T-cell receptor (TCR)-αß(+) IELs (CD8αß(+) αß-IELs) after weaning, but no increase of CD8αß(+) γδ-IELs was detected in pIgR(-/-) TCR-ß(-/-) mice compared with pIgR(+/+) TCR-ß(-/-) mice. When 5-bromo-2'-deoxyuridine (BrdU) was given for 14 days, the proportion of BrdU-labelled cells in SI-IELs was not different between pIgR(+/+) mice and pIgR(-/-) mice. However, the proportion of BrdU-labelled CD8αß(+) -IELs became higher in pIgR(-/-) mice than pIgR(+/+) mice 10 days after discontinuing BrdU-labelling. Intravenously transferred splenic T cells migrated into the intraepithelial compartments of pIgR(+/+) TCR-ß(-/-) mice and pIgR(-/-) TCR-ß(-/-) mice to a similar extent. In contrast, in the case of injection of immature bone marrow cells, CD8αß(+) αß-IELs increased much more in the SI of pIgR(-/-) TCR-ß(-/-) mice than pIgR(+/+) TCR-ß(-/-) mice 8 weeks after the transfer. αß-IELs from pIgR(-/-) mice could produce more interferon-γ and interleukin-17 than those of pIgR(+/+) mice, and intestinal permeability tended to increase in the SI of pIgR(-/-) mice with aging. Taken together, these results indicate that activated CD8αß(+) αß-IELs preferentially accumulate in pIgR(-/-) mice through the enhanced differentiation of immature haematopoietic precursor cells, which may subsequently result in the disruption of epithelial integrity.

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV.

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 µg/g leaf fresh mass (LFM) in transgenic tobacco and 25 µg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.

Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis.

The DevR/DosR regulator is believed to play a key role in dormancy adaptation mechanisms of Mycobacterium tuberculosis in response to a multitude of gaseous stresses, including hypoxia, which prevails within granulomas. DevR activates transcription by binding to target promoters containing a minimum of two binding sites. The proximal site overlaps with the SigA -35 element, suggesting that DevR-SigA interaction is required for activating transcription. We evaluated the roles of 14 charged residues of DevR in transcriptional activation under hypoxic stress. Seven of the 14 alanine substitution mutants were defective in regulon activation, of which K191A, R197A, and K179A+K168A (designated K179A*) mutants were significantly or completely compromised in DNA binding. Four mutants, namely, E154A, R155A, E178A, and K208A, were activation defective in spite of binding to DNA and were classified as positive-control (pc) mutants. The SigA interaction defect of the E154A and E178A proteins was established by in vitro and in vivo assays and implies that these substitutions lead to an activation defect because they disrupt an interaction(s) with SigA. The relevance of DevR interaction to the transcriptional machinery was further established by the hypoxia survival phenotype displayed by SigA interaction-defective mutants. Our findings demonstrate the role of DevR-SigA interaction in the activation mechanism and in bacterial survival under hypoxia and establish the housekeeping sigma factor SigA as a molecular target of DevR. The interaction of DevR and RNA polymerase suggests a new and novel interceptable molecular interface for future antidormancy strategies for Mycobacterium tuberculosis.

Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction.

Although both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) are present in the gut-associated lymphoid tissues (GALT), the roles of pDCs in the gut remain largely unknown. Here we show a critical role for pDCs in T cell-independent (TI) IgA production by B cells in the GALT. When pDCs of the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) (which are representative GALT) were cultured with naive B cells to induce TI IgA class switch recombination (CSR), IgA production was substantially higher than in cocultures of these cells with cDCs. IgA production was dependent on APRIL and BAFF production by pDCs. Importantly, pDC expression of APRIL and BAFF was dependent on stromal cell-derived type I IFN signaling under steady-state conditions. Our findings provide insight into the molecular basis of pDC conditioning to induce mucosal TI IgA production, which may lead to improvements in vaccination strategies and treatment for mucosal-related disorders.

From the fetal liver to spleen and gut: the highway to natural antibody.

The film of sIgA lining the intestinal epithelium plays a role in the regulation of the commensal microflora and prevention of pathogen invasion. We show that, in the absence of intentional immunization, all sIgA in the gut is produced by B-1a B cells. We also show that B-1a B cells and sIgA derive from lineage-negative precursors found in the fetal liver and located in the spleen after birth. The splenic precursors do not generate B cells of the adaptive immune system in bone marrow, spleen, and lymph nodes, but efficiently replenish the cells producing the natural antibodies. Therefore, B-1a B cells with their splenic progenitors and their progeny of plasma cells fill the same function of the primordial immune system of lower vertebrates. The natural antibodies in the serum and on the intestinal epithelium may be an evolutionary ancient tool for the immediate protection against commensal and pathogenic bacteria.

Local production of secretory IgA in the eye-associated lymphoid tissue (EALT) of the normal human ocular surface.

PURPOSE: Secretory IgA (SIgA) is a critical local defense mechanism of mucosal immunity. Although the conjunctiva, as part of the ocular surface, has a mucosa-associated lymphoid tissue, the production of SIgA by local plasma cells and its transport is unequivocally accepted to occur only in the upstream lacrimal gland (LG). The molecular components were therefore investigated by immunohistochemistry (IHC) and their local production verified by RT-PCR. METHODS: Tissues from 18 conjunctivas and 9 LGs of human donor eyes with normal ocular surfaces were analyzed by histology and IHC. Different zones of 12 further conjunctivas and LG tissues were analyzed by RT-PCR for the presence of the respective mRNA. RESULTS: Plasma cells were present in the diffuse lymphoid tissue of all investigated specimens and showed an intense immunoreactivity for IgA. This immunoreactivity was absent when the antiserum was preadsorbed with the protein. The luminal epithelium, with the exception of goblet and basal cells, was strongly positive for the epithelial transporter molecule secretory component (SC) in the conjunctiva and interconnecting excretory duct similar to the LG. PCR products for IgA, the monomeric IgA-joining molecule (J-chain) and SC were regularly found in all conjunctival zones and in the LG in gel electrophoresis and were sequenced. CONCLUSIONS: The local production of SIgA is for the first time verified by RT-PCR in the human conjunctiva and in the LG. This finding points to an active role of the conjunctiva in secretory immune protection of the ocular surface and supports the presence and importance of EALT at the normal ocular surface.

Human scFv SIgA expressed on Lactococcus lactis as a vector for the treatment of mucosal disease.

The gastrointestinal tract is a complex niche and the main port of entry of many pathogens that trigger a wide range of diseases like inflammatory bowel disease (IBD) and colon cancer. Antibodies are effective for treating such diseases, but a system capable of local delivery at the site of the pathology, thus avoiding systemic side effects, is not yet available. Here we report a novel recombinant scFvSIgA1 protein produced by Lactococcus lactis, anchored to the bacterial membrane, which retains its full immuno-recognizing potential. This scFv fragment employed was specific for a colon cancer epitope, epithelial glycoprotein protein-2 (EGP-2). Accordingly L. lactis expressing this chimeric protein was capable of binding cells expressing this epitope. Expression of specific antibodies on bacteria may allow local delivery of anticancer agents produced by such bacteria in conjunction with the antibody and provides a new avenue in the quest for targeted drug delivery.

Dynamic interactions between bacteria and immune cells leading to intestinal IgA synthesis.

The gastrointestinal tract is colonized by an immense number of bacteria that are in a constant dialog with our immune cells. One obvious question is how the mucosal immune system maintains a state of hypo-responsiveness toward the commensal bacteria and a state of readiness that allows efficient and prompt responses against pathogens. The answers have important implications for immunologists who seek to understand the fundamental aspects of bacteria-immune cell interactions in the steady-state condition and wish to elucidate the patho-physiologic mechanisms in immune disregulations, such as inflammatory bowel diseases. An important adjustment of the immune system to bacterial colonization of the gut is the "constitutive" production of IgA by the gut-associated lymphoid tissues (GALT). In this review, we summarize the sites and mechanisms for IgA synthesis in mice. We emphasize the important role played by secretory IgAs in maintenance of an appropriate intestinal microbiota, which is required for local and systemic immune homeostasis.

Inhibition of HIV-1 infectivity and epithelial cell transfer by human monoclonal IgG and IgA antibodies carrying the b12 V region.

Both IgG and secretory IgA Abs in mucosal secretions have been implicated in blocking the earliest events in HIV-1 transit across epithelial barriers, although the mechanisms by which this occurs remain largely unknown. In this study, we report the production and characterization of a human rIgA(2) mAb that carries the V regions of IgG1 b12, a potent and broadly neutralizing anti-gp120 Ab which has been shown to protect macaques against vaginal simian/HIV challenge. Monomeric, dimeric, polymeric, and secretory IgA(2) derivatives of b12 reacted with gp120 and neutralized CCR5- and CXCR4-tropic strains of HIV-1 in vitro. With respect to the protective effects of these Abs at mucosal surfaces, we demonstrated that IgG1 b12 and IgA(2) b12 inhibited the transfer of cell-free HIV-1 from ME-180 cells, a human cervical epithelial cell line, as well as Caco-2 cells, a human colonic epithelial cell line, to human PBMCs. Inhibition of viral transfer was due to the ability of b12 to block both viral attachment to and uptake by epithelial cells. These data demonstrate that IgG and IgA MAbs directed against a highly conserved epitope on gp120 can interfere with the earliest steps in HIV-1 transmission across mucosal surfaces, and reveal a possible mechanism by which b12 protects the vaginal mucosal against viral challenge in vivo.

Secretory antibody formation: conserved binding interactions between J chain and polymeric Ig receptor from humans and amphibians.

Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.

Expression and secretion of immunoglobulin alpha heavy chain with diverse VDJ recombinations by human epithelial cancer cells.

Generally, only B lymphocytes express immunoglobulin. Recently, we found the expression of Ig alpha heavy chain in human epithelial cancer cells unexpectedly. We first detected Ig VDJ-Calpha and Ialpha-Calpha transcripts in multiple cancer cell lines. Further, the configuration of the Ig heavy chain genomic locus was analyzed in human cancer cells. We found that cancer cells have the recombination VDJ region, but bear Ig Salpha region in germline configuration, which is different from Ig expression pattern in B cells. And human epithelial cancers possess the essential effectors including RAG-1 and RAG-2, but not activation induced cytidine deaminase (AID) protein. These provide further proofs for Ig alpha expression. In addition, we found that human cancer cells not only express the protein of Ig alpha chain, but also secrete the protein in secretory IgA (SIgA) pattern. Importantly, diverse CDR3 recombinations were found in human cancer cells of different epithelial origin. Since IgA is the key immunoglobulin which contributes to local immunity of mucous membrane, the aberrant expression of Ig alpha heavy chain might increase our further comprehension to development and immunity of cancers.

Plant expression of chicken secretory antibodies derived from combinatorial libraries.

Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA immunoglobulin heavy and light chains, J-chain and secretory component) has been completed, allowing recombinant, up scaled production of chicken sIgA and extension of passive immune strategies to poultry. To test the suitability of the plant cell factory for bulk production of chicken sIgA, we studied the expression of chicken IgA, dIgA and sIgA in planta. To that end, new cassettes were designed that allowed the grafting of immunoglobulin variable regions derived from combinatorial libraries into full-size chicken IgA frames ready for plant expression. Using this system, 10 individual phage display clones, which had previously been selected against Eimeria acervulina antigens, were transferred "from phage to plant". Plant-made chicken antibodies showed strong differences in expression levels, which seemed governed mainly by the stability of their respective light chains. Finally, with the co-expression of chicken IgA heavy and light chains, J-chain and secretory component in N. benthamiana leaves we showed that plant cells are suitable biofactories for the production of assembled chicken sIgA complexes.

Measurement of messenger RNA encoding the alpha-chain, polymeric immunoglobulin receptor, and J-chain in duodenal mucosa from dogs with and without chronic diarrhea by use of quantitative real-time reverse transcription-polymerase chain reaction assays.

OBJECTIVE: To examine the difference in expression of messenger RNA (mRNA) transcripts for polymeric immunoglobulin receptor (plgR), alpha-chain, and J-chain determined by use of quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) assays in duodenal biopsy specimens obtained from dogs with and without chronic diarrhea. SAMPLE POPULATION: Biopsy specimens of the proximal portion of the duodenum were obtained endoscopically from 39 dogs evaluated because of chronic diarrhea (12 German Shepherd Dogs and 27 non-German Shepherd Dog breeds); specimens were also obtained from a control group of 7 dogs evaluated because of other gastrointestinal tract diseases and 2 dogs that were euthanatized as a result of nongastrointestinal tract disease. PROCEDURE: Dogs were anesthetized, and multiple mucosal biopsy specimens were obtained endoscopically at the level of the caudal duodenal flexure by use of biopsy forceps; in 2 control dogs, samples were obtained from the descending duodenum within 5 minutes of euthanasia. One-step QRT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase, plgR, alpha-chain, and J-chain in duodenal mucosal tissue. RESULTS: There was no significant difference in the level of expression of any transcript among non-German Shepherd Dog breeds without diarrhea (control group), non-German Shepherd Dog breeds with chronic diarrhea, and German Shepherd Dogs with chronic diarrhea. Conclusions and Clinical Relevance-Results indicated that the susceptibility of German Shepherd Dogs to chronic diarrhea is not a result of simple failure of transcription of the key genes that encode molecules involved in mucosal IgA secretion.