Release Of IgA Research Articles

Immunoinformatic Identification of Multiple Epitopes of gp120 Protein of HIV-1 to Enhance the Immune Response against HIV-1 Infection.

Acquired Immunodeficiency Syndrome is caused by the Human Immunodeficiency Virus (HIV), and a significant number of fatalities occur annually. There is a dire need to develop an effective vaccine against HIV-1. Understanding the structural proteins of viruses helps in designing a vaccine based on immunogenic peptides. In the current experiment, we identified gp120 epitopes using bioinformatic epitope prediction tools, molecular docking, and MD simulations. The Gb-1 peptide was considered an adjuvant. Consecutive sequences of GTG, GSG, GGTGG, and GGGGS linkers were used to bind the B cell, Cytotoxic T Lymphocytes (CTL), and Helper T Lymphocytes (HTL) epitopes. The final vaccine construct consisted of 315 amino acids and is expected to be a recombinant protein of approximately 35.49 kDa. Based on docking experiments, molecular dynamics simulations, and tertiary structure validation, the analysis of the modeled protein indicates that it possesses a stable structure and can interact with Toll-like receptors. The analysis demonstrates that the proposed vaccine can provoke an immunological response by activating T and B cells, as well as stimulating the release of IgA and IgG antibodies. This vaccine shows potential for HIV-1 prophylaxis. The in-silico design suggests that multiple-epitope constructs can be used as potentially effective immunogens for HIV-1 vaccine development.

Protective Effects of Bacteriocin-Producing Lactiplantibacillus plantarum on Intestinal Barrier of Mice.

Bacteriocins are crucial metabolites of probiotics that display beneficial functions. The intestinal barrier is an important target on which probiotics exert their intestinal health activity. However, the impacts of bacteriocin-producing probiotics on the intestinal barrier are unclear. In this study, the effects of bacteriocin-producing Lactiplantibacillus plantarum Q7 and L. plantarum F3-2 on the intestinal barrier of mice were explored. It was shown that L. plantarum Q7 promoted the expression of mucin MUC2 to enhance the protection provided by the intestinal mucus layer. L. plantarum Q7 up-regulated the gene expression of intestinal tight junction proteins ZO-1 and JAM-1 significantly, and L. plantarum F3-2 up-regulated ZO-1 and Claudin-1 markedly, which exhibited tight junction intestinal barrier function. The two strains promoted the release of IgA and IgG at varying degrees. The antimicrobial peptide gene RegIIIγ was up-regulated markedly, and the gene expression of inflammatory cytokines appeared to exhibit an upward trend with L. plantarum Q7 treatment, so as to enhance intestinal immune regulation function. Furthermore, L. plantarum Q7 and L. plantarum F3-2 increased the abundance of the beneficial bacteria Muribaculaceae, inhibited the growth of the harmful bacteria Parabacteroides, and facilitated the synthesis of total short-chain fatty acids (SCFAs), which seemed to favor the prevention of metabolic diseases. Our results suggested that L. plantarum Q7 and L. plantarum F3-2 showed strain specificity in their protective effects on the intestinal chemical, physical, immunological and biological barriers of mice, which provided theoretical support for the selective utilization of bacteriocin-producing strains to regulate host health.

TFH Cells Induced by Vaccination and Following SIV Challenge Support Env-Specific Humoral Immunity in the Rectal-Genital Tract and Circulation of Female Rhesus Macaques.

T follicular helper (TFH) cells are pivotal in lymph node (LN) germinal center (GC) B cell affinity maturation. Circulating CXCR5+ CD4+ T (cTFH) cells have supported memory B cell activation and broadly neutralizing antibodies in HIV controllers. We investigated the contribution of LN SIV-specific TFH and cTFH cells to Env-specific humoral immunity in female rhesus macaques following a mucosal Ad5hr-SIV recombinant priming and SIV gp120 intramuscular boosting vaccine regimen and following SIV vaginal challenge. TFH and B cells were characterized by flow cytometry. B cell help was evaluated in TFH-B cell co-cultures and by real-time PCR. Vaccination induced Env-specific TFH and Env-specific memory (ESM) B cells in LNs. LN Env-specific TFH cells post-priming and GC ESM B cells post-boosting correlated with rectal Env-specific IgA titers, and GC B cells at the same timepoints correlated with vaginal Env-specific IgG titers. Vaccination expanded cTFH cell responses, including CD25+ Env-specific cTFH cells that correlated negatively with vaginal Env-specific IgG titers but positively with rectal Env-specific IgA titers. Although cTFH cells post-2nd boost positively correlated with viral-loads following SIV challenge, cTFH cells of SIV-infected and protected macaques supported maturation of circulating B cells into plasma cells and IgA release in co-culture. Additionally, cTFH cells of naïve macaques promoted upregulation of genes associated with B cell proliferation, BCR engagement, plasma cell maturation, and antibody production, highlighting the role of cTFH cells in blood B cell maturation. Vaccine-induced LN TFH and GC B cells supported anti-viral mucosal immunity while cTFH cells provided B cell help in the periphery during immunization and after SIV challenge. Induction of TFH responses in blood and secondary lymphoid organs is likely desirable for protective efficacy of HIV vaccines.

Intestinal Dysbiosis, Barrier Dysfunction, and Bacterial Translocation Account for CKD-Related Systemic Inflammation.

CKD associates with systemic inflammation, but the underlying cause is unknown. Here, we investigated the involvement of intestinal microbiota. We report that collagen type 4 α3-deficient mice with Alport syndrome-related progressive CKD displayed systemic inflammation, including increased plasma levels of pentraxin-2 and activated antigen-presenting cells, CD4 and CD8 T cells, and Th17- or IFNγ-producing T cells in the spleen as well as regulatory T cell suppression. CKD-related systemic inflammation in these mice associated with intestinal dysbiosis of proteobacterial blooms, translocation of living bacteria across the intestinal barrier into the liver, and increased serum levels of bacterial endotoxin. Uremia did not affect secretory IgA release into the ileum lumen or mucosal leukocyte subsets. To test for causation between dysbiosis and systemic inflammation in CKD, we eradicated facultative anaerobic microbiota with antibiotics. This eradication prevented bacterial translocation, significantly reduced serum endotoxin levels, and fully reversed all markers of systemic inflammation to the level of nonuremic controls. Therefore, we conclude that uremia associates with intestinal dysbiosis, intestinal barrier dysfunction, and bacterial translocation, which trigger the state of persistent systemic inflammation in CKD. Uremic dysbiosis and intestinal barrier dysfunction may be novel therapeutic targets for intervention to suppress CKD-related systemic inflammation and its consequences.

A pig tonsil cell culture model for evaluating oral, low-dose IFN-α treatments

Oral, low-dose IFN-α treatments proved effective in several models of viral infections and immunopathological conditions. Also, they do not give rise to the serious side effects observed after parenteral inoculation of high doses (105U/kg b.w. and higher). There is convincing evidence that such treatments work through an early, effective interaction with oral lymphoid tissues before the IFN-α molecules are rapidly destroyed by gut enzymes. Yet, the paucity of detailed information about these crucial interactions and the lack of recognized in vitro models hamper the development of proper administration protocols. On the basis of a previous study, we developed an in vitro model of interaction between different types of human and porcine IFNs-α at low/moderate concentrations and pig tonsil cells. The IFNs-α under study showed different properties with respect to three fundamental control actions: (1) IgA release in culture, (2) release of natural antimicrobial compounds, and (3) homeostatic regulation of the inflammatory response. This was checked in pig intestinal epithelial cells (IPEC-J2 cell line) treated with supernatants of control and IFN α-treated tonsil cell cultures, respectively, in terms of inflammatory cytokine and chemokine responses. Some IFNs-α caused a significant inhibition of IL-8 (protein release and gene expression) and beta-defensin 1 (gene expression) probably through second messengers released by IFN α-treated tonsil cells. Interestingly, a human lymphoblastoid IFN-α under study caused the decrease of polyclonal IgA release by pig tonsil cells and significantly stimulated the in vitro recall antibody response of swine PBMC to Foot-and-Mouth Disease virus. The modulation of IgA and antibacterial compounds was accompanied by an anti-inflammatory control action at the same, low to moderate IFN-α concentrations (1–100U/ml). This highlights the very foundation of the homeostatic control actions performed by Type I IFNs: to promote an effective host response to infectious and non-infectious stressors and to turn off noxious inflammatory responses associated with tissue damage and waste of metabolic energy. The described tonsil cell model in vitro can be conducive to a further development of oral cytokine treatments in humans and animals in the “one health” conceptual framework.

The Role of Platelet Factor 4 in Local and Remote Tissue Damage in a Mouse Model of Mesenteric Ischemia/Reperfusion Injury

The robust inflammatory response that occurs during ischemia reperfusion (IR) injury recruits factors from both the innate and adaptive immune systems. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR injury has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration were decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissues. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after IR at both the mRNA and protein levels. In conclusion, these findings demonstrate that may PF4 represent an important mediator of local and remote tissue damage.

The role of Platelet Factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury (120.18)

Abstract The robust inflammatory response that takes place during ischemia reperfusion injury (IR/I) recruits factors from both the innate and adaptive immune system. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR/I has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration was decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissue. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after ischemia/reperfusion at both the mRNA and protein levels. In conclusion, these findings demonstrate that PF4 represents an important mediator of local and remote tissue damage.

The influence of lentinus edodes (shiitake mushroom) preparations on bacteriological and morphological aspects of the small intestine in piglets1

Among substances intended to replace growth promoting antibiotics in pig nutrition, non-digestible oligosaccharides or polysaccharides could be potential alternative compounds. Therefore, the influence of β-1,3-1,6 glucans on bacteriological, biochemical and morphological aspects of the small intestine in weaned piglets was investigated. As sources of β-glucans, Lentinan (extract of Lentinus edodes mycelium) or dried L. edodes mycelium were added to the diet. Four homogenous groups of 5 newly weaned piglets (4 weeks of age) received one of four diets: control diet (C), C supplemented with Avilamycin (50 mg/kg, positive control), C supplemented with 0.1% of Lentinan and C supplemented with 5% of dried L. edodes mycelium powder. A first group of 10 piglets was euthanized after 11 days and the remaining 10 on day 12 of the experiment. The gastrointestinal tract was divided in segments and samples taken from digesta (stomach, proximal and distal jejunum, caecum), mucosal scrapings (jejunum) and ring shaped tissue samples (1 cm) of proximal and distal jejunum. Bacterial counts were made with digesta and mucosal samples, and short-chain fatty acids (SCFA), lactic acid and ammonia concentrations were determined. Tissue samples of both jejunal sites were embedded in paraffin wax for morphometrical (villus length, crypt depth) and histological observations (numbers of intraepithelial lymphocytes (IEL), goblet cells, apoptotic enterocytes on villi, mitotic cells in crypts). Only the diet containing 5% of dried L. edodes consistently resulted in lower viable counts (ca. 1 – 2 log10 CFU) of total bacteria, E. coli, streptococci and lactic acid bacteria, and luminal and mucosal effects agreed very well. With this diet, acetate and butyrate concentrations in the distal jejunum were doubled, which is favourable in view of the trophic effect on enterocytes and colonocytes. Villus length (V) was increased with both diets containing β-glucans while crypt depth (C) was not altered, but V/C was higher. IEL counts were decreased by both diets although bacterial numbers, which is only one parameter of bacterial load, were only diminished with the L. edodes feed. The three supplemented feeds lowered the number of apoptotic enterocytes on the villi, but these numbers were very low (control diet : 44 cells per 100 villi), making clear interpretation difficult. The mitotic index was slightly lower with the L. edodes feed, although not statistically significant. Decreased viable counts observed with the latter diet is a favourable effect as it is accepted that a lower bacterial load causes lower turnover rates of the intestinal epithelial cells, while there is also less competition for specific substrates. A higher V/C ratio, a smaller number of IEL in the epithelium and a lower apoptotic index also indicate slower turnover rate of the mucosa when Lentinan and L. edodes diets were fed. The inconsistent effects observed with Lentinan were probably due to the low amount added to the diet. It should be taken into account that the influence of L. edodes mycelium powder was more likely due to the presence of antibacterial compounds (eg. lenthionine, lentinamycin, terpenoids, polyphenols), rather than to an immunostimulating action of β-glucans with increased release of IgA onto the mucosa surface.

Gastric IgA in cystic fibrosis in relation to the migrating motor complex.

Gastrointestinal symptoms in cystic fibrosis are frequent, but little is known about the underlying pathophysiology. Mucosal secretion of IgA is important for the immunologic function in the human gastrointestinal tract but has not been studied in cystic fibrosis. The aim of this study was to quantify the release of IgA by the gastric mucosa in relation to interdigestive motor activity in patients with cystic fibrosis with different genotypes. The study included 7 healthy adult volunteers and 10 adult patients with cystic fibrosis, all Helicobacter pylori-negative. All patients had pathological sweat tests and clinical symptoms and signs of cystic fibrosis. All but one were colonized with Pseudomonas aeruginosa. Three patients were pancreatic sufficient. The investigation was performed using intragastric perfusion and gastroduodenal manometry. During the investigation, 8 of 10 patients with cystic fibrosis showed the characteristic pattern of interdigestive motility. The patients had significantly lower levels of gastric IgA compared to healthy subjects during phases II and III of migrating motor complex, median (range) 120 (67-442) and 36 (6-299) microg/5 min. 382 (40-1176) and 56 (4-398) (P = 0.03 and P = 0.04), respectively. Only one patient with genotype R668C/unknown showed IgA levels within the normal range. There was no correlation to gastric presence of duodenogastric reflux markers. The interdigestive motility pattern was normal in most patients with cystic fibrosis. The low levels of IgA released from the gastric mucosa in the patients might indicate a defective gastric transmucosal IgA transport in cystic fibrosis.

Gastric IgA in Cystic Fibrosis in Relation to the Migrating Motor Complex

Background: Gastrointestinal symptoms in cystic fibrosis are frequent, but little is known about the underlying pathophysiology. Mucosal secretion of IgA is important for the immunologic function in the human gastrointestinal tract but has not been studied in cystic fibrosis. The aim of this study was to quantify the release of IgA by the gastric mucosa in relation to interdigestive motor activity in patients with cystic fibrosis with different genotypes. Methods: The study included 7 healthy adult volunteers and 10 adult patients with cystic fibrosis, all Helicobacter pylori-negative. All patients had pathological sweat tests and clinical symptoms and signs of cystic fibrosis. All but one were colonized with Pseudomonas aeruginosa. Three patients were pancreatic sufficient. The investigation was performed using intragastric perfusion and gastroduodenal manometry. Results: During the investigation, 8 of 10 patients with cystic fibrosis showed the characteristic pattern of interdigestive motility. The patients had significantly lower levels of gastric IgA compared to healthy subjects during phases II and III of migrating motor complex, median (range) 120 (67-442) and 36 (6-299) µg/5 min, 382 (40-1176) and 56 (4-398) (P = 0.03 and P = 0.04), respectively. Only one patient with genotype R668C/unknown showed IgA levels within the normal range. There was no correlation to gastric presence of duodenogastric reflux markers. Conclusion: The interdigestive motility pattern was normal in most patients with cystic fibrosis. The low levels of IgA released from the gastric mucosa in the patients might indicate a defective gastric transmucosal IgA transport in cystic fibrosis.

Both Microtubules and Actin Filaments Are Required for Efficient Postendocytotic Traffic of the Polymeric Immunoglobulin Receptor in Polarized Madin-Darby Canine Kidney Cells

It has been postulated that membrane traffic in polarized epithelial cells requires both actin filaments and microtubules. We have tested this hypothesis by analyzing the effect of cytochalasin D (cytoD; an actin-disrupting agent), by itself or in combination with nocodazole (a microtubule depolymerizing agent), on postendocytic traffic in Madin-Darby canine kidney cells. CytoD treatment inhibited basolateral to apical transcytosis of IgA in polymeric immunoglobulin receptor-expressing cells by approximately 45%, but had little effect on basolateral recycling of transferrin. Apical recycling of IgA was also inhibited by approximately 20%. Like nocodazole, cytoD acted at an early step in transcytosis, and inhibited translocation of IgA between the basolateral early endosomes and the apical recycling endosome. There was little inhibition of the subsequent release of IgA from the apical recycling endosome of cytoD- or nocodazole-treated cells. Order-of-addition experiments suggest that the cytoD-sensitive step preceded the nocodazole-sensitive step. Treatment with both cytoD and nocodazole inhibited transcytosis 95%. These results suggest that in addition to microtubules, efficient postendocytic traffic in polarized epithelial cells also requires actin filaments.

Influence of fatty acid absorption on bidirectional release of immunoglobulin A into intestinal lumen and intestinal lymph in rats

Effects of absorption of long and middle chain fatty acids on IgA secretion into the intestinal lumen and intestinal lymph and the factors which evoke changes in IgA secretion during the absorptive process were examined in rat small intestine. Bidirectional secretion of IgA from the intestinal mucosa into the intestinal lumen and intestinal lymph was continuously observed in the control condition. Perfusion of oleic acid (a long-chain fatty acid) micelle into the jejunal loop induced a significant increase in IgA output into the intestinal lymph. In contrast, lymphatic output of IgA was significantly decreased when oleic acid micelle was administered intraduodenally. Absorption of octanoic acid, a middle-chain fatty acid, did not produce any significant changes in IgA output into either direction. CR1505, a CCK-receptor antagonist, significantly attenuated the oleic acid-induced increase in IgA secretion into the intestinal lumen, but did not affect the oleic acid-induced decrease in lymphatic IgA secretion. Pluronic L-81, an inhibitor of chylomicron formation and secretion, significantly attenuated the decrease in IgA output into the intestinal lymph during oleic acid absorption without affecting the luminal IgA output. The rate of release of IgA into the intestinal lumen is stimulated by absorption of long-chain fatty acids possibly through the influence of locally released CCK, while the transport process of IgA into lymphatics is controlled by a different mechanism which is closely correlated with the intracellular formation and secretion of chylomicron.

Differential microtubule requirements for transcytosis in MDCK cells.

Given the role of microtubules in directing the transport of many intracellular organelles, we investigated whether intact microtubules were also required for transcytosis across epithelia. Using polarized MDCK cells expressing receptors for the Fc domain of IgG (FcRII-B2) or polymeric immunoglobulin (pIg-R), we examined the involvement of microtubules in apical to basolateral and basolateral to apical transcytosis, respectively. While depolymerization of microtubules with nocodozole had no effect on apical to basolateral transcytosis via FcR, basolateral to apical transcytosis of dimeric IgA via pIg-R was almost completely blocked. Inhibition due to nocodozole was selective for basolateral to apical transcytosis, since neither endocytosis nor receptor recycling was significantly affected at either plasma membrane domain. As shown by confocal microscopy, the block in transcytosis was due to the inability of MDCK cells to translocate IgA-containing vesicles from the basolateral to the apical cytoplasm in the absence of an intact microtubule network. The nocodazole sensitive step could be partially by-passed, however, by allowing cells to internalize IgA at 17 degrees C prior to nocodazole treatment. Although incubation at 17 degrees C blocked release of IgA into the apical medium, it did not prevent translocation of IgA-containing vesicles to the apical cytoplasm. Thus, receptor-mediated transcytosis in opposite directions exhibits distinct requirements for microtubules, a feature which reflects the spatial organization of MDCK cells.

IgA in uterine tissue: effect of estrous cycle, hormone treatment and intrauterine immunization.

In order to determine whether uteri synthesize and store IgA, rats were sacrificed and uterine tissues placed in organ culture for 24 h under the following conditions: (i) at various stages of the estrous cycle, (ii) following ovariectomy and treatment with estradiol, and (iii) after intrauterine immunization with sheep red blood cells (SRBC). When IgA was analyzed in tissues both prior to and following organ culture and in incubation media, no significant increases in total IgA were observed, nor was IgA release into media reduced when cycloheximide, a potent inhibitor of protein synthesis, was present. Analysis of uterine tissues indicated that IgA levels remained relatively constant throughout the estrous cycle and was not markedly increased when ovariectomized rats were treated with estradiol (2 micrograms/day) for 3 days. These results indicate that tissue IgA levels remain relatively constant even during estradiol treatment, when uterine luminal IgA levels are known to increase markedly. Analysis of ovariectomized rats that received intrauterine immunizations with SRBC indicated tenfold greater amounts of IgA in immunized tissues than did uteri from intact or ovariectomized animals. Despite this, no evidence of protein synthesis was obtained, based on measurements of total IgA content before and after organ culture or inhibition of IgA synthesis by cycloheximide. These results indicate that IgA synthesis under the conditions examined is not occurring, but that uterine tissue may serve as a significant storage depot for IgA synthesized either distal to or within uterine tissues at times other than those analyzed in the present study.

29 THE ROLE OF CHOLECYSTOKININ (CCK), FOOD AND SUBSTANCE P (SP) IN THE RELEASE OF INTESTINAL IMMUNOGLOBULIN A (IgA)

The ingestion of food augments the release of IgA in rat intestine. This study is intended to show that this phenomenon is mediated by CCK, and furthermore, that SP another of the gut-brain peptides also promotes a rise in intestinal IgA. Hooded Lister rats weighing 160-180 gm. were immunized with ovalbumin and Freund's complete adjuvant. On day 14 a booster dose was given. On day 21, a 10 cm. long segment of intestine was isolated 10 cm. distal to the pylorus and perfused with saline at a rate of 0.5 ml/2.5 mins. After a 10 mins. equilibration period, the CCK antagonist Proglumide(Milid Laboratories, Milano) 20 mg was injected i.v. to the “food” group. Ten mins. later 1 ml. of the protein hydrolysate Pregestimil (Mead Johnson Co. Evansville) was administered intragastrically. In the SP group, 25 μ m SP were administered i.v. Results: 1. In the “food” group, there was a significant rise of IgA after the administration of food (P < 0.05) and this was inhibited by the prior administration of the CCK antagonist. 2. In the SP group there was a significant rise of IgA at 2.5 mins. (P < 0.001) and this became significant again from, 10-20 mins. Conclusion: 1. Food induced rise of IgA in the intestine is probably mediated by CCK as it is inhibited by a CCK antagonist, and 2. SP is another neuropeptide promoting IgA release in the intestine. The effect of SP is much more prolonged than that of CCK.

Binding of IgA to erythrocytes from patients with IgA nephropathy

Primate erythrocytes (RBCs) may be involved in the transport and processing of C3b-containing immune complexes (IC). Compared to RBCs from healthy controls, increased amounts of IgA were detectable on RBCs from 7 of 17 patients with IgA nephropathy (IgA NP). There was no difference in the amount of IgG or IgM. The highest amount of RBC-bound IgA corresponded to 6 ng IgA 10 8 cells . The mechanisms involved in the binding of IgA to RBCs were investigated in vitro. Isolated IgA1 or IgA2 did not bind to RCBs from a patient with IgA deficiency. In contrast, incubation of RBCs with a polyethyleneglycol (PEG) precipitate of serum from a patient with IgA NP which contained IgA-IC resulted in IgA1 binding. However, this binding was not inhibited by monoclonal anti-CR1 or by an excess of IgG or IgM. Factor I did not cause release of IgA from RBCs from patients with IgA NP. Heat-aggregated IgA1 also bound to RBCs and this binding was not affected by the presence of complement. We conclude that minute amounts of IgA-IC are bound to RBCs by a complement- and Fc receptor-independent mechanism. The quantity of IgA-IC associated with RBC is so small that it is unlikely to represent an important in vivo route of IgA-IC transport or processing.

Raised titres of anti-klebsiella IgA in ankylosing spondylitis, rheumatoid arthritis, and inflammatory bowel disease

Serum titres of IgA are raised in ankylosing spondylitis and increased titres of antibodies to klebsiella have also been reported. The humoral response was investigated in ankylosing spondylitis and other inflammatory disorders. IgA antibodies to klebsiella pneumoniae K43 were measured in patients with ankylosing spondylitis, Crohn's disease, ulcerative colitis, and rheumatoid arthritis and in controls. Significantly raised median titres of anti-klebsiella IgA, measured as optical density at 405 nm with an enzyme linked immunosorbent assay (ELISA), were seen among the patients with ankylosing spondylitis (0.7), Crohn's disease (0.8), rheumatoid arthritis (0.6), and ulcerative colitis (0.8) compared with controls (0.4). Activity of disease in ankylosing spondylitis and titres of anti-klebsiella IgA were not correlated. In contrast, titres of anti-klebsiella IgM were significantly lower in patients with ankylosing spondylitis and ulcerative colitis. The increase in the titres of anti-klebsiella IgA may be due to increased permeability of the gut to bacterial antigens, leading to an increased IgA response in the gut mucosa and permitting the release of IgA into the circulation. As the increased antibody titres were seen in Crohn's disease and rheumatoid arthritis as well as in ankylosing spondylitis the response may be nonspecific, occurring because of possible underlying inflammatory bowel disease in these conditions.

28. IS IMMUNOGLOBULIN ‘A’ (IgA) RELEASE IN THE INTESTINE CONTROLLED BY A CALCIUM DEPENDENT SECRETORY MECHANISM?

It has previously been shown that cholecystokinin octapeptide (CCK-8)as well as pilocarpine can accelerate the release of IgA in the gastrointestinal tract. The purpose of this study was to ascertain if CCK-8-induced IgA release involves calcium-coupled secretory mechanisms in the intestine. Hooded Lister rats were immunized with ovalbumin and Freund's complete adjuvant and a booster injection given 14 days later. On the 21st day a 10 cm. long segment of intestine was isolated 10 cm. distal to the pylorus. This was perfused with saline 0.5 ml/2.5minutes. After a flush out period of 30 mins. aliquots were collected every 2.5 mins. After 10 minutes 1 u CCK was given I.V.(group 1). A second group of animals received a calcium channel blocker Verapramil I.V. at a rate of 2 mcg/min. for 20 minutes. After the first 10 mins. 20 ng CCk-8 were administered I.V. Group 3 was given Verapramil only as above.Results: following CCK-8 there was a significant rise of IgA into the intestinal fluid which began at 2.5 minutes and persisted for 10 minutes. Prior administration of Verapramil completely inhibited the CCK-8-induced release of IgA. Conclusion: it is suggested that CCK-8-induced release of IgA into the gastrointestinal tract is related to calcium dependent secretory mechanisms.

Cholecystokinin-induced release of IgA antibodies in rat intestine.

It has been shown previously that the intravenous (i.v.) injection of cholecystokinin (CCK) into healthy adult volunteers will result in a rise of antibodies belonging to the A, M, E and D classes of immunoglobulins (1,2). It appeared likely that these antibodies were derived largely, if not wholly, from the gall bladder and the pancreas, as CCK is known to cause gall bladder contraction and to promote secretion of pancreatic enzymes. It could, however, not be excluded that CCK had a direct effect on the intestinal release of immunoglobulins. This report, therefore, deals with the effect of CCK on the release of IgA in an isolated perfused loop of rat intestine.

Intracellular Immunoglobulins in Human Milk Macrophages

Immunoglobulins (Ig) were localized by immunofluorescence and immunoperoxidase techniques inside human milk macrophages (HMM phi). Immunoelectron microscopy was employed to study the distribution and localization of Ig within HMM phi. IgA and IgM were detected inside phagocytic vacuoles of different sizes in the vicinity of the cellular membrane and in the periphery of larger vacuoles with a dense and homogeneous content. The intramacrophagic IgA represents 5-10% of the total milk IgA. The in vitro release of IgA into the medium was quantitated using an enzyme-linked immunoabsorbent assay. The amount of IgA released was in most samples around 1,720-5,200 ng/10(6) HMM phi. The concentrations of IgM and IgG released by HMM phi were approximately 50-600 and 20-40 ng/10(6) HMM phi, respectively. The concentrations of Ig determined in the lysated pellets of HMM phi ranged between 2 and 100 micrograms/10(6) HMM phi for IgA and around 500 ng/10(6) HMM phi for IgM. Increased IgA release occurred when HMM phi were cultured in medium with a pH lower than 3. IgA concentrations were not very different between pH 5 and pH 7 and alkalinization of the medium did not produce any significant effect on IgA release. Release of IgA and IgM was not significantly increased when HMM phi were cultured in the presence of phorbol myristate acetate, formylated peptides, latex particles, lymphocyte-derived chemotactic factor or cholate. The kinetic study of IgA release by HMM phi, both with or without adding these stimuli to in vitro culture, indicates that IgA release depends on time and that these substances do not have any effect on it.