Volume 1

Pablo Roman, ... Diana Cardona, in Comprehensive Gut Microbiota, 2022

1.29.3.3 Gut-Microbiota and Toxic Dietary Components

Lectins and phytohemagglutinins (PHA) are natural toxicants present in many foods, especially in beans and other dietary pulses, which can have toxic effects when consumed without adequate cooking, occasionally leading to an acute gastroenteritis (Kumar et al., 2013). Toxic GIT effects may be secondary to increased gut bacteria and intestinal permeability. Indeed, rats exposed to crude red kidney beans (Phaseoulus vulgaris) for 24 h showed weight loss and increased intestinal permeability, as well as increased bacterial load and increased bacterial translocation to the liver (Ramadass et al., 2010). Lectins seem to increase the levels of facultative aerobes, without an increase in obligate anaerobes (Rhodes et al., 2003). Furthermore, in vitro and ex vivo studies led to the conclusion that some lectins (at least concanavalin A) may act by promoting bacterial adherence to the small intestine, thereby facilitating colonization and infection and leading to bacterial overgrowth (Abud et al., 1989). Early in vivo studies showed that, after exposure to PHA derived from red kidney beans, the predominant bacteria proliferating in the jejunum were E. coli, Streptococcus spp. and Lactobacillus. Also, E. coli and Streptococcus spp. populations increased within 24 h of PHA exposure and remained high during further exposure to PHA but declined on reversion to a control diet within 24–48 h, with a shift to gram-positive rod and coccus flora through days 1–4 of the reversion (Banwell et al., 1988). Anyhow, adequate cooking (at 100 °C for at least 30 min) avoids the presence of toxic lectins in legumes, which may otherwise produce beneficial effects for the colonic microenvironment (including the microbiota, mucus and epithelial barriers), particularly associated with their contents in fermentable fibers, which are the source of gut-health promoting bioactive microbial metabolites, including SCFAs (acetic acid, propionic acid and butyric acid). In these regards, mice fed with diets supplemented with 20% cooked navy bean or black bean flours increased the abundance of carbohydrate fermenting bacteria, SCFAs and their receptors (Monk et al., 2017). However, a recent opinion review (Panacer and Whorwell, 2019) called for a deeper study of the impact of dietary lectins. FODMAP stands for “fermentable oligosaccharides, disaccharides, monosaccharides, and polyols”; these carbohydrates are poorly absorbed in the gut and are, therefore, fermented by colonic bacteria, leading to gastrointestinal symptoms, especially in patients with IBS. Although the use of a diet low in FODMAPs can dramatically improve the symptoms of many patients, gut microbiota may become affected by such restrictive diet (Staudacher and Whelan, 2017). The authors highlighted that many of the foods excluded in the low FODMAP diet also contain lectins, suggesting that restrictions of more specific dietary components (i.e., lectins) might be more broadly beneficial for IBS patients (Panacer and Whorwell, 2019). Since dysbiosis occurs in IBS patients (Carco et al., 2020), more research is needed to clarify the role of lectins on this (Spiller, 2021).

Dietary AGEs are not fully digested and absorbed in the GI tract, thus reaching the colon, where they may undergo fermentation by the resident microbiota. Colonic protein fermentation leads to the formation of ammonia, amines, phenols and sulfides (Tuohy et al., 2006; Gilbert et al., 2018), that negatively affect the colon. As a consequence, the composition of the microbiota and its production of SCFAs, which are generally considered anti-inflammatory, can change (Vinolo et al., 2011; Li et al., 2018). In animal models, diets high in dietary AGEs were associated with a decrease in Bacteroidetes, Bifidobacteria, Lactobacilli, and alpha-diversity, suggesting a less healthy condition of the GIT (Snelson and Coughlan, 2019). In vitro studies confirmed that the effects of dietary AGEs on gut microbiota are largely dependent on food composition and thermal treatment (Pérez-Burillo et al., 2018). Furthermore, dietary AGEs may contribute to some of the symptoms and possibly the disease course of IBD through microbiota modulation, suggesting that decreasing the levels of foods rich in these compounds may be helpful in IBD patients (van der Lugt et al., 2020).

Acrylamide and its metabolites have been recognized as potential carcinogens or co-carcinogens (including in the colon, see (Raju et al., 2013; Hogervorst et al., 2014; El Asri et al., 2020): and neurotoxic agents (including to the enteric nervous system, see (Lourenssen et al., 2009). However, the direct impact of acrylamide on gut microorganisms is not clear. Different deleterious effects were demonstrated to occur in Bacillus subtilis (Tsuda et al., 1993), E. coli (Starostina et al., 1983) and Saccharomyces cerevisiae (Kwolek-Mirek et al., 2011) cultures, whereas Salmonella typhymurium was not affected (Shipp et al., 2006). As mentioned earlier, fructose-asparagine (F-Asn) is a precursor to acrylamide that is found in human foods, and a nutrient source for Salmonella enterica, a foodborne pathogen, whose serovars Typhimurium and Enteriditis cause inflammatory diarrhea in humans. Interestingly, it was found that some species present in the normal intestinal microbiota, particularly three members of the Clostridium genus, are able to use F-Asn, suggesting that these clostridial species may contribute to competitive exclusion of Salmonella (Sabag-Daigle et al., 2018). Furthermore, some microorganisms present in the colon or delivered with food may be able to degrade acrylamide by amidase production, including Bacillus clausii (Lippolis et al., 2013), Enterococcus faecalis (Mesnage et al., 2008) and Helicobacter pylori (Bury-Moné et al., 2003). Some lactic acid bacteria were shown to remove acrylamide in aqueous solution by simply physically binding the toxin to the bacterial cell wall (Rivas-Jimenez et al., 2016), suggesting a beneficial effect of probiotics to reduce acrylamide absorption. Interestingly, Petka et al. (2020) found that several Lactobacillus species were tolerant to acrylamide even at high concentrations (up to 1 g/mL) in culture. Besides, some of them, like L. plantarum, L. lactis sp. Lactis, and L. brevis, as well as the probiotic strain Lactobacillus acidophilus LA-5 showed an increased growth rate in the presence of high concentrations of the toxin. These results suggest that these bacteria may use this toxin as a source of energy. However, L. plantarum cell division was uncoupled, and these bacteria appeared in the form of chains and diplobacillus. The authors concluded that acrylamide-containing products would be not as harmful as previously thought, provided a properly functioning intestinal microbiota is present (Petka et al., 2020).

The impact of mycotoxins on the gut microbiota has recently been thoroughly reviewed (Liew and Mohd-Redzwan, 2018; Gao et al., 2020; Guerre, 2020). The intestinal barrier is composed of the physical (enterocytes), chemical (mucin, etc), immune and microbial (gut microbiota) barriers and all of them may be affected by mycotoxins (Gao et al., 2020), favoring the development and exacerbation of chronic intestinal inflammatory diseases (IBD, celiac disease, and even IBS). Some in vivo studies reported mycotoxin-induced modifications in gut microbiota of animals by advanced molecular approaches. For example, Coprococcus genus was more abundant in rats fed with the trichothecene deoxynivalenol (DON) (Miró-Abella et al., 2018). In pigs fed with DON and zearalenone (ZEN) Lactobacillus was found to be more abundant in one report (Reddy et al., 2018), whereas increased abundance of Erysipelotrichaceae and decreased abundance of Ruminococcaceae, Streptococcaceae, and Veillonellaceae were reported in another study (Le Sciellour et al., 2020). However, Daud et al. (2020) did not find any impact of mycotoxin (unconjugated or masked forms) on the growth of any bacterial strain tested, suggesting that mycotoxins do not act directly on gut microbes (Daud et al., 2020). Thus, the toxicity exerted by mycotoxins on the large intestine and gut microbiota might be more likely attributable to secondary effects on microbiota composition associated with host-toxicity and consequent barrier impairment, which warrants further investigation. Interestingly, these authors also demonstrated that several strains of gut bacteria are likely to hydrolyze masked mycotoxins in the human gut due to their high activity and high prevalence in the gut, including Eubacterium rectale A1-86, Roseburia intestinalis L1-82, Butyrivibrio fibrisolvens 16/4 and Prevotella copri DSM 18205, as well as Bifidobacterium adolescentis and Lactiplantibacillus plantarum. Masked mycotoxins are sugar conjugates formed by the cereals and are stable toward small intestinal digestion, i.e., they are not absorbed intact (Gratz, 2017), but the parent mycotoxins may be released by the gut microbiota (Daud et al., 2020) and absorbed and excreted in the urine, as shown in humans (Vidal et al., 2018), contributing to overall mycotoxin systemic toxicity. Importantly, this process will vary depending on the individual composition of the gut microbiota. Furthermore, the use of probiotics (Bifidobacterium and Lactobacillus spp.) and prebiotics boosting the numbers of specific gut bacteria may influence the hydrolysis of masked mycotoxins (Daud et al., 2020). It is also important to realize that the co-occurrence of different mycotoxins in contaminated foods is the most likely situation and therefore, the possible synergistic, additive, and antagonistic effects of these compounds need to be also taken into account, but has only been scarcely addressed so far, through complex combinatorial approaches (Gao et al., 2020).

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The Role of Intracellular Mediators in the Immune Response

CHARLES W. PARKER, in Biology of the Lymphokines, 1979

A General Considerations

We have made a number of experimental observations over a period of years bearing on the possible role of cAMP in the activation of purified human peripheral blood lymphocytes by lectins (reviewed in Parker et al., 1974; Wedner and Parker, 1976; Parker, 1976).

(1)

Phytohemagglutinin and Con A were shown to produce small (1.4–3.0-fold) but consistent increases in cAMP. Changes occurred within 60 seconds, preceding other known metabolic alterations in lectin stimulated cells. Maximal responses were observed after 5 to 15 minutes (Parker et al., 1974; Smith et al., 1971a; Lyle and Parker, 1974), followed, as a rule, by a decline to control values by 60–120 minutes. Other investigators have confirmed these observations (Webb et al., 1974; Krishnaraj and Talwar, 1973; Jegasothy et al., 1976; Burleson and Sage, 1976). Increases in cAMP have also been seen with two other mitogens, A23187 and trypsin (Greene et al., 1976a; Shneyour et al., 1976), whereas agents that are not themselves mitogenic but that enhance lectin-induced mitogenesis (such as cysteine and several of the cytochalasins) amplify the early cAMP response to lectin.

(2)

The increase in cAMP in response to Con A was specifically blocked or reversed in a dose-related fashion by low (1–10 mM) concentrations of α-methyl-D-glucoside or α-methyl-D-mannoside, indicating that specific glycoproteins on the cell surface are involved in the stimulation (Lyle and Parker, 1974). This was also suggested by the ability of Con A that had been covalently bound to polylysine-Sepharose beads to produce an increase in cAMP.

(3)

Biphasic dose-response curves to lectin were seen in some experiments with stimulation maxima at high (>30 μg/ml) and low (1–10 μg/ml) PHA or Con A concentrations (Parker et al., 1974). Filtration of the lymphocytes through nylon wool columns to remove the B-cells largely eliminated the high-dose response, whereas removal of most of the T-lymphocytes through rosette formation with sheep erythrocytes decreased the low-dose response. Thus it appeared that both T-cells and B-cells responded to PHA and Con A, but that at the relatively low lectin concentrations optimal for mitogenesis most of the response was in T-cells. Although T- and B-lymphocytes contain approximately equal numbers of binding sites for these lectins, under the usual stimulation conditions most of the mitogenic response involves T-cells. Nonetheless, if lectin is insolubilized or presented in high concentrations in the medium to B-cells, they appear capable of responding (reviewed in Wedner and Parker, 1976). Thus the existence of a cAMP response at high concentrations of lectin does not exclude involvement of cAMP in the mitogenic signal.

(4)

Concanavalin A and PHA stimulated adenylate cyclase activity in crude lymphocyte homogenates, although in purified plasma membrane fractions (Parker et al., 1974; Smith et al., 1971a) the response was small and inconsistent (Snider and Parker, 1977), possibly because of disorganization of the plasma membrane structure or the removal of cofactors essential to the response.

(5)

Cyclic AMP (10–100 nM) and its lipophilic analog N6O2-dibutyryl cAMP stimulated mitogenesis, but the magnitude of changes was quite small (at the most, 2–3-fold), whereas PHA and Con A produced up to 100-fold responses in these same cells (Smith et al., 1971b; Hirschhorn et al., 1970). Higher concentrations of cAMP (>100 μM), as well as of theophylline, isoproterenol, and PGE1 in concentrations producing substantial increases in cAMP concentrations, inhibited the DNA synthetic response both to PHA and Con A. Aminoisobutyric acid (AIB) uptake and phosphatidyl inositol turnover also were inhibited.

(6)

Cells exposed to PHA or Con A appeared to accumulate cAMP in or near the lymphocyte surface, as indicated by cAMP immunofluorescence studies using rabbit anti-cAMP antibody and fluoresceinated goat antirabbit IgG (Wedner et al., 1971a,b; Bloom et al., 1973).

(7)

As will be discussed in more detail below, in lymphocytes stimulated with mitogenic lectins there was a transient increase in protein phosphorylation involving a number of different protein species (Wedner et al., 1975a; Wedner and Parker, 1975).

In addition to lectin-stimulated human lymphocytes, there are a number of other systems in which there is clear or suggestive evidence that an early rise in cAMP may be correlated with some form of lymphocyte stimulation; first of all, in the action of one of the thymic hormones on immature T-cells (Kook and Trainin, 1974); and, second, in the stimulation of antibody responses to red blood cells and to a lesser extent to other antigens in vivo or in vitro (possibly through a direct action on immature T-helper cells or B-lymphocyte precursor cells at some early stage of the response) (Braun and Ishizaka, 1971; Watson et al., 1973; Teh and Paetkau, 1974; Kishimoto and Ishizaka, 1976; Naylor et al., 1976).

As was already indicated, there are also inhibitory effects that appear to be associated with rises in cAMP in lymphocytes. (1) Epinephrine, prostaglandins, theophylline, and histamine all decrease MIF production, T-cell proliferation, B-cell proliferation, and T-cell effector function (Parker et al., 1974). (2) The inhibitor of DNA synthesis (IDS) described by Waksman and his colleagues (Jegasothy et al., 1976) produces a late rise in cAMP right before the onset of the S phase. (3) Wheat germ agglutinin—which markedly inhibits mitogenic responses to PHA, Con A, antithymocyte globulins, and specific antigens—produces a rise in cAMP much like that seen with PHA and Con A (Greene et al., 1976b). (4) Colchicine, another inhibitor of DNA synthesis, produces a delayed rise in cAMP appearing after 30–60 minutes (Greene et al., 1976a). (5) Other substances that produce rapid rises in cAMP and may in some instances inhibit DNA synthesis include latex beads and aliphatic and aromatic alcohols (Atkinson et al., 1976, 1977).

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Antifungal and Antiproliferative Activity of Spotted Bean (Phaseolus vulgaris cv.)

Tzi-Bun Ng, ... Yau-Sang Chan, in Nuts and Seeds in Health and Disease Prevention, 2011

Adverse Effects and Reactions (Allergies and Toxicity)

The toxic lectin phytohemagglutinin is present in many varieties of beans, but is highly concentrated in red kidney beans. Although, using dry beans, it requires 10 minutes at 100°C to degrade the toxin, incidents of poisoning with the use of slow cookers at low temperatures not high enough to degrade the toxin have been reported. The British Public Health Authority, PHLA, has recommended soaking kidney beans for 5 hours before cooking. Sprouts of beans high in hemagglutinins (such as kidney beans) should be avoided.

Consumption of Phaseolus vulgaris brought about a decrement of food intake, body weight, and blood glucose level in rats. This is attributed to the suppression of α-amylase by α-amylase inhibitors, interference with the central mechanism controlling appetite, and stimulation of cholecystokinin secretion from intestinal brushborder cells.

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African Legumes: Nutritional and Health-Promoting Attributes

Kwaku G. Duodu, Franklin B. Apea-Bah, in Gluten-Free Ancient Grains, 2017

3.3.6 Lectins

Lectins, also known as phytohemagglutinins, have been reported in cowpea (Batista et al., 2010b; Carvalho et al., 2012; Marconi et al., 1993, 1997), and African yam bean (Machuka et al., 1999; Machuka and Okeola, 2000; Oboh et al., 1998). Lectins are glycoproteins that are able to bind with carbohydrate membrane receptors due to their affinity for specific sugar molecules (Akande et al., 2010). They can bind to intestinal mucosa and enterocytes and thus interfere with absorption and transportation of nutrients during digestion (Akande et al., 2010; Kumar, 1991) and also agglutinate red blood cells (Kumar, 1991). Generally, lectins are heat labile and are reported to be particularly susceptible to more complete destruction by wet-heat treatment (Ayyagari et al., 1989; Almeida et al., 1991).

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Lectins

Halina Lis, Nathan Sharon, in Encyclopedia of Immunology (Second Edition), 1998

Biological activities

Certain lectins are potent mitogens; PHA and concanavalin A, for example, stimulate T cells, while pokeweed mitogen (PWM) stimulates both T and B cells. The mitogenic lectins are polyclonal activators, in that they activate lymphocytes, including memory-type cells, irrespective of their antigenic specificity. Prior to the advent of monoclonal antibodies to cell surface antigens, lectins were the major tool for studies of the mechanism of mitogenic stimulation of lymphocytes.

In the presence of a mitogenic lectin, a wide variety of antigenically unrelated target cells are lysed by cytotoxic T lymphocytes, a phenomenon known as lectin-dependent cytotoxicity (in analogy to antigen-dependent cytotoxicity). Another form of lectin-dependent cytotoxicity is the killing of tumor cells by macrophages. This activity resembles that of tumor recognizing antibodies that can induce macrophages to lyse tumors.

Several lectins, e.g. concanavalin A and wheat germ agglutinin, are toxic to mammalian cells in culture and to animals, but their toxicity is at least 1000 times lower than that of ricin and abrin. The cytotoxic properties of such lectins serve for the selection of clones of lectin-resistant cell mutants that are widely employed in studies of the genetics, biosynthesis and function of cell surface carbohydrates.

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Functional Glycomics

Junya Mitoma, Minoru Fukuda, in Methods in Enzymology, 2010

2.3 Intravenous injection

When inhibiting lymphocyte homing by lectins, E-PHA (150–400 μg), LEA (150–300 μg), or Con A (150 μg) dissolved in 150 μl of PBS is intravenously injected through a tail vein prior to the injection of labeled lymphocytes. Overdose injection of lectins would result in the death of the recipient mice. Optimal dose should be determined for each lectin used in the experiments. The preinjection of MECA-79 antibody (100–200 μg) can inhibit lymphocyte homing into peripheral lymph nodes and mesenteric lymph nodes. One hour after the injection of the lectin or the antibody, 200 μl of fluorescence-labeled lymphocytes (5 × 106 cells) are injected.

For the short-term homing assay, which evaluates the initial recruitment of lymph nodes into secondary lymphoid organs, the lymph nodes should be collected 1–3 h after the injection of labeled lymphocytes. To analyze long-term homing, which represents steady-state circulation of lymphocyte, the lymph nodes should be removed 24 h after injection. The mice are sacrificed and the peripheral (cervical and lateral axillary) lymph nodes, mesenteric lymph nodes, Peyer's patches, spleen, and thymus are collected into 6-well plates containing 2 ml ice-cold PBS after the removal of adipose tissue. Lymphocytes are squeezed out with two frosted glass slides as described above, and the debris is removed by passing through 70 μm mesh. In the case of spleen, the hypotonic treatment should be performed to remove red blood cells as described above. The cell suspension is then analyzed by flow cytometry. The number of fluorescent cells is compared to the number of total cells. Control experiments are carried out by injecting only PBS. The ratio of control and lectin-inhibited or antibody-inhibited experiments is compared.

Using this approach, we have demonstrated that 6-sulfo sLeX on N-glycans would contribute, at least partially, to lymphocyte homing into peripheral and mesenteric lymph nodes in addition to that on core 2 and extended core 1 O-glycans (Mitoma et al., 2007). In that work, we have established Core2GlcNAcT−/−/Core1-β3GlcNAcT−/− mice to eliminate almost all O-glycans in lymph nodes. Although the mice lack entire extended core 1 and core 2 O-glycans on which the most of L-selectin ligand 6-sulfo sLeX resides, the lymphocyte homing into peripheral lymph nodes remained at 40% of wild-type mice. The preinjection of Con A have a minimum effect on lymphocyte homing since L-selectin ligands are not present on Con A-reactive N-glycans. E-PHA and LEA have a significant inhibitory effect on wild-type mice and almost completely inhibit lymphocyte homing in Core2GlcNAcT−/−/Core1-β3GlcNAcT−/− mice (Fig. 15.3). The inhibitory effect of the lectins is highest in lymphocyte homing to peripheral lymph nodes, moderate to mesenteric lymph nodes and very moderate to Peyer's patches.

Figure 15.3. Lymphocyte homing inhibited by lectins for complex type N-glycans. One hour after intravenous injection of LEA (300 μg), E-PHA (200 μg) or ConA (200 μg) into Core2GlcNAcT−/−/Core-1β3GlcNAcT−/− mice, CMRA-labeled lymphocytes were injected. Peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches were recovered 1 h later and the cells were subjected to flow cytometric analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared to PBS control.

(Reprinted with permission from Mitoma and Fukuda, 2007).
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Production of Colony Stimulating Factors by Lymphoid Tissues

DONALD METCALF, in Biology of the Lymphokines, 1979

C GM-CSF Production in Concanavalin A-Stimulated Cultures

As might be anticipated from the above effects of PHA, the addition of concanavalin A (10μg/ml) to mouse spleen cell suspensions induced the development of colony-stimulating activity in the culture medium within 48 hours with maximum activity demonstrable at 4–7 days (Ruscetti and Chervenick, 1975a). Again, DNA synthesis paralleled and preceded production of GM-CSF. Addition of concanavalin A direct to the bone marrow cultures did not stimulate colony formation.

Addition of the competitive sugar α-methylmannoside did not interfere with the production of active conditioned medium by PHA-stimulated spleen cells, but strongly suppressed concanavalin A-stimulated cultures. The presence of concanavalin A appears to be necessary throughout the incubation period, as removal even after 72 hours led to reduced levels of colony stimulating activity in the medium eventually harvested. Again it was noted that inhibitors of protein synthesis (puromycin and cycloheximide) suppressed production of GM-CSF. In this case, however, the effects did not apepar to have been due to cytotoxicity, as cells washed free of puromycin after 60 minutes were still capable of GM-CSF production.

Blocking of DNA synthesis by the addition of vinblastine, colchicine, or cytosine arabinoside was found not to suppress the development of colony-stimulating activity in the conditioned medium, raising the possibility that neither blast transformation nor DNA synthesis were required in concanavalin-A-stimulated cultures (Ruscetti and Chervenick, 1975a). Other evidence for a dissociation between DNA synthesis or cell division and GM-CSF production was the observation that production of active conditioned medium was not inhibited by in vitro irradiation in doses up to 2000 R. This is a curious observation that is at variance with the results with pokeweed-mitogen-stimulated cultures (see below) and is in need of clarification.

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Neurobiology of Cytokines

Jay M. Weiss, Syam K. Sundar, in Methods in Neurosciences, 1993

Lymphocyte Proliferation to Phytohemagglutinin P

To determine the mitogenic response of T lymphocytes, phytohemagglutinin P (PHA; Difco Laboratories, Detroit, MI) was used. One-tenth of a milliliter of lymphocyte suspension (5 × 106 cells/ml) and an equal volume of PHA are added into the wells of the microculture plates. The dose of PHA used was determined by first conducting studies to establish a dose-response curve (i.e., 1.5, 10, and 20 μg/ml tested) and then an optimal dose (i.e., that which produces a clear but nonasymptomatic response) is selected for repeated use in studies; our experiments have usually used 10 μg/ml. Lymphocyte cultures incubated with RPMI-1640 alone serve as controls. All tests on all measures are carried out in triplicate. After 3 days of incubation, the cultures are pulsed with 1 μCi of [3H]thymidine (New England Nuclear, Boston, MA; 6 Ci/mmol) and harvested 4 hr later. Cells are harvested onto glass filters and incorporated [3H]thymidine is determined in a liquid scintillation counter.

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Amphibian Immune System

Jacques Charlemagne, Annick Tournefier, in Encyclopedia of Immunology (Second Edition), 1998

Immunobiology of T and B cells

Thymectomy in Xenopus either decreases or abolishes allograft rejection, MLR, PHA responsiveness, IgY antibody synthesis and antibody responses to thymus-dependent antigens. It does not abolish in vivo or in vitro responses to thymus-independent antigens or B cell polyclonal activation (LPS). In vitro assays using purified T and B cells from carrier- or hapten-primed Xenopus of various MHC types indicate that T cell help is involved in the differentiation of thymus-dependent antigen-primed B cells and that T–B collaboration is MHC restricted. IgM is produced first following antigenic stimulation, and then is produced in conjunction with IgY. A second injection generates a significantly stronger (×10–100), mainly IgY, secondary response. Although somatic mutation occurs in Xenopus VH segments at almost the same frequency found in mammals, these mutations may not be properly selected, perhaps because of the absence of germinal centers in the lymphoid organs.

Larval thymectomy suppresses allograft rejection in urodeles, but does not abolish in vivo responses to certain thymus-dependent antigens, such as sheep or horse erythrocytes. Larval and adult thymectomy, low dose (50–150 rads; 0.5–1.5 Gy) irradiation and hydrocortisone treatment enhance specific antibody synthesis against erythrocyte antigens. These observations suggest that T cell help is impaired in urodeles, but that some kind of T cell-dependent suppression acts on antibody production. Urodeles can be immunized against particulate but not against soluble antigens, and in normal and thymectomized axolotls IgM is the single antigen-sensitive Ig class, specific IgY is not produced. The kinetics of the antibody response is slow and there is no occurrence of a typical secondary response following hyperimmunization. Thus, although the Xenopus and axolotl IgY molecules are clearly homologous at the molecular level, their respective physiological functions are different: IgY are IgG-like (thymus dependent, sensitive to thymectomy) in anurans, but IgA-like in axolotl, at least in the first 7–8 months of development; most IgY are found associated with the digestive epithelium and are secreted into the gut lumen following transepithelial transport in association with secretory component-like molecules.

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Organism, Pathogen, and Environment

Margaret J. Manning, Teruyuki Nakanishi, in Fish Physiology, 1996

B. Proliferative Responses to Mitogens

In mammals, a number of substances, principally plant lectins such as phytohemagglutinin (PHA) and concanavalin A (Con A), are specific for particular arrays of carbohydrate moieties that occur on T lymphocytes. When present in appropriate doses these T-cell mitogens induce proliferation of T cells but not of B cells. Other mitogens such as bacterial lipopolysaccharides (LPS) activate B cells but not T cells. The majority of studies on mitogen-induced proliferation in fish have been done on teleosts, this being the group for which optimal culture conditions (DeKoning and Kaattari, 1992) and good separation techniques are well established.

In the more primitive lampreys (agnathans), Cooper (1971) showed that lymphocytes from ammocoete larvae respond to PHA in vitro by the production of blast cells, suggesting the existence of T-like cells, while similar studies in cartilaginous fish (the nurse shark, Ginglymostoma cirratum) using Con A and PHA again yielded a T-cell like response (Lopez et al., 1974; Sigel et al., 1978; Pettey and McKinney, 1981). In these experiments on nurse sharks, leucocyte populations were separated on Percoli density gradients and by their adherence to glass beads. Cells cytotoxic to xenogeneic targets were present among the adherent population while cells of the nonadherent component showed suppressor activity. These regulatory suppressor cells are sIg–ve but do not behave in an MHC-restricted manner (Haynes and McKinney, 1991). Thus, although cells activated by T-cell mitogens are present in subpopulations of shark leukocytes, a T-cell/B-cell-like basis for the heterogeneity has yet to be established in chondrichthian fish.

In teleosts, the ability to respond to T-cell and B-cell mitogens was established some twenty years ago (Etlinger et al., 1976), and a number of studies followed (reviewed by Rowley et al., 1988). These include reports on the channel catfish (Sizemore et al., 1984), carp (Caspi and Avtalion, 1984a), rainbow trout (Warr and Simon, 1983; Tillit et al., 1988; Reitan and Thuvander, 1991), and Atlantic salmon, Salmo salar, (Smith and Braun-Nesje, 1982; Reitan and Thuvander, 1991). Documentation of the in vitro culture requirements for each species has been important (Rosenberg-Wiser and Avtalion, 1982; Faulmann et al., 1983; DeKoning and Kaattari, 1991). For example, the addition of fetal bovine serum to the culture medium as the serum supplement may not suffice; in salmonids the cells performed better if homologous plasma was used (DeKoning and Kaattari, 1992).

The requirement for accessory cells for the activation of T lymphocytes by mitogens was confirmed for channel catfish peripheral blood lymphocytes by Sizemore et al. (1984). It now seems likely that these accessory cells (monocytes) were acting by the secretion of cytokines, possibly interleukin 1 (IL-1) (Miller et al., 1985; Clem et al., 1985). Sizemore et al. (1984) separated the sIg+ve cells from sIg–ve cells by panning, and demonstrated that the sIg+ve cells responded to LPS regardless of the presence or absence of monocytes, whereas the sIg–ve population remained unresponsive to either LPS or Con A unless the accessory cells were present, in which case they responded to both mitogens. The reduced but significant response to LPS in the sIg–ve population remains problematic. A similar response of sIg–ve cells to LPS was reported by DeLuca et al. (1983) using pronephric leukocytes of rainbow trout. On the other hand, Marsden et al. (1995) observed a marked dichotomy in the proliferative responses of peripheral blood leukocytes of rainbow trout. The sIg–ve cells were activated by PHA with very little response to LPS and vice versa for the sIg+ve cell population. Whether this reflects the efficiency of the panning techniques or whether some LPS responsive cells are present in genuinely sIg–ve populations remains uncertain. Koumans–Van Diepen et al. (1994) recently noted that, among the peripheral blood lymphocytes of carp, some sIg+ve cells responding to LPS showed only dull immunofluorescent staining. These may have escaped sorting in some experiments.

In rainbow trout, thymocytes proliferate in response to PHA but not to LPS (Reitan and Thuvander, 1991). Essentially similar results were obtained for channel catfish using the thymus from fish about 14 months old and stimulating with Con A (Ellsaesser et al., 1988). Accessory cells (monocytes) were required for the T-cell activation and either autologous or allogeneic cells could be used. Again there was a small response in some cases to LPS. However, this does not detract from the general conclusion that thymocytes are Ig–ve cells that display a T-cell-like response to mitogens. Any discrepancies may be accounted for by the suggestion that the thymus can play a minor role as a residence for a small population of B cells (see Manning, 1994).

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