Predicting the immunomodulatory activity of probiotic lactic acid bacteria using supervised machine learning in a Cornu aspersum snail model.

In the process of screening for probiotic strains, there are no clearly established bacterial phenotypic markers which could be used for the prediction of their in vivo mechanism of action. In this work, we demonstrate for the first time that Machine Learning (ML) methods can be used for accurately predicting the in vivo immunomodulatory activity of probiotic strains based on their cell surface phenotypic features using a snail host-microbe interaction model. A broad range of snail gut presumptive probiotics, including 240 new lactic acid bacterial strains (Lactobacillus, Leuconostoc, Lactococcus, and Enterococcus), were isolated and characterized based on their capacity to withstand snails' gastrointestinal defense barriers, such as the pedal mucus, gastric mucus, gastric juices, and acidic pH, in association with their cell surface hydrophobicity, autoaggregation, and biofilm formation ability. The implemented ML pipeline predicted with high accuracy (88 %) strains with a strong capacity to enhance chemotaxis and phagocytic activity of snails' hemolymph cells, while also revealed bacterial autoaggregation and cell surface hydrophobicity as the most important parameters that significantly affect host immune responses. The results show that ML approaches may be useful to derive a predictive understanding of host-probiotic interactions, while also highlighted the use of snails as an efficient animal model for screening presumptive probiotic strains in the light of their interaction with cellular innate immune responses.

Effect of silver nanoparticles on gene transcription of land snail Helix aspersa.

Silver nanoparticles (Ag-NPs) are extremely useful in a diverse range of consumer goods. However, their impact on the environment is still under research, especially regarding the mechanisms involved in their effect. Aiming to provide some insight, the present work analyzes the transcriptional activity of six genes (Hsp83, Hsp17.2, Hsp19.8, SOD Cu-Zn, Mn-SOD, and BPI) in the terrestrial snail Helix aspersa in the presence of different concentrations of Ag-NPs. The animals were exposed for seven days to Lactuca sativa soaked for one hour in different concentrations of Ag-NPs (20, 50, 100 mg/L). The results revealed that the highest concentration tested of Ag-NPs (100 mg/L) led to a statistically significant induction of the Hsp83 and BPI expression in the digestive gland compared to the control group. However, a trend to upregulation with no statistical significance was observed for all the genes in the digestive gland and the foot, while in the hemolymph, the trend was to downregulation. Ag-NPs affected the stress response and immunity under the tested conditions, although the impact was weak. It is necessary to explore longer exposure times to confirm that the effect can be maintained and impact on health. Our results highlight the usefulness of the terrestrial snail Helix aspersa as a bioindicator organism for silver nanoparticle pollution biomonitoring and, in particular, the use of molecular biomarkers of pollutant effect as candidates to be included in a multi-biomarker strategy.

Phenoloxidase is involved in the immune reaction of Helix lucorum to parasitic infestation by dicrocoeliid trematode.

INTRODUCTION: Phenoloxidases are known to play a role in the immune defences of arthropods and molluscs. In the invertebrates, phenoloxidases mediate three major physiologically important processes: sclerotization, wound healing, and defence reactions. Helix lucorum serve as the first intermediate host for the larval stages of dicrocoeliid trematodes which infects animals as well as human beings. OBJECTIVE: The aim of the study is to investigate the effect of larval forms of dicrocoeliid trematodes to phenoloxidase acitivity in H. lucorum, Linneaus, 1758, in Bitlis, Turkey. The effect of the snail's shell colour to phenoloxidase activity was also investigated. MATERIAL AND METHODS: Land snails (n=200) were collected by hand from their natural habitats during the period May - June 2019 in Bitlis, Turkey. Evaluation of the process was performed by measuring immune reaction of the snails against larval forms of dicrocoeliid trematodes. Phenoloxidase activity assay was carried out using a spectrophotometer device based on 3,4-Dihydroxy-L-phenylalanine (L-dopa) hydrolysis. RESULTS: The natural infection rate of the land snails with the developmental stages of dicrocoeliid trematodes was 20%. Phenoloxidase activity was found to be significantly higher (*p<0.05) in larval forms of dicrocoeliid trematodes infected snails when compared with non-infected snails. No effect of shell colours to phenoloxidase activity was observed. CONCLUSIONS: To the best of the authors' knowledge, this study is the first to report that the phenoloxidase system is involved in the immune reaction of Helix lucorum to parasitic infestation by larval forms of dicrocoeliid trematodes.

Helix pomatia hemocyanin - a novel bio-adjuvant for viral and bacterial antigens.

BACKGROUND: New generated subunit vaccines are characterized by increased safety and lack of side effects, however they suffer from weak immunogenicity. The adjuvants are substances that have the ability to enhance the magnitude and duration of the immune response and to increase vaccine efficacy, but the different vaccines may require diverse adjuvants. The urgent need of novel adjuvant formulations occurs, thus ensuring protective cellular and humoral responses against infectious pathogens. The hemocyanins, oxygen binding copper proteins in the hemolymph of molluscs and arthropods, are widely used as peptide carriers and vaccine adjuvants. RESULTS: In the present study we promote the hemocyanin isolated from the terrestrial gastropod Helix pomatia (HPH) as bio-adjuvant, combined with standard antigens. The purified HPH combined with influenza virus hemagglutinin intersubunit peptide (IP) or with tetanus toxoid (TT) were used for immunization. Administration of tetanus toxoid combined with HPH in mice resulted in an increased number of anti-TT IgG producing plasmocytes and induced a significant increase of B and T cell proliferation. The level of the anti-TT IgG antibodies in mice sera was comparable to the group administered with TT+Al(OH)3. An immunization of experimental animals with IP combined with H. pomatia hemocyanin led to generation of strong anti-influenza cytotoxic response. CONCLUSION: The vaccination of mice demonstrates that the HPH is acceptable as a potential bio-adjuvant for subunit vaccines and it could be used as a natural adjuvant or protein carrier.

Antioxidant and molecular chaperone defences during estivation and arousal in the South American apple snail Pomacea canaliculata.

The invasive Pomacea canaliculata estivates during periods of drought and should cope with harmful effects of reoxygenation during arousal. We studied thiobarbituric acid reactive substances (TBARS), enzymatic (superoxide dismutase, SOD and catalase, CAT) and non-enzymatic antioxidants (uric acid and reduced glutathione), and heat shock protein expression (Hsc70, Hsp70 and Hsp90) in (1) active control snails, (2) snails after 45 days of estivation, and (3) aroused snails 20 min and (4) 24 h after water exposure, in midgut gland, kidney and foot. Both kidney and foot (but not the midgut gland) showed a TBARS increase during estivation and a decrease after arousal. Tissue SOD and CAT did not change in any experimental groups. Uric acid increased during estivation in all tissues, and it decreased after arousal in the kidney. Allantoin, the oxidation product of uric acid, remained constant in the midgut gland but it decreased in the kidney until 20 min after arousal; however, allantoin levels rose in both kidney and foot 24 h after arousal. Reduced glutathione decreased during estivation and arousal, in both midgut gland and kidney, and it remained constant in the foot. Hsc70 and Hsp70 kidney levels were stable during the activity-estivation cycle and Hsp90 expression decreases during estivation and recovers in the early arousal. In foot, the expression of Hsp70 and Hsp90 was high during activity and estivation periods and disminished after arousal. Results indicate that a panoply of antioxidant and molecular chaperone defences may be involved during the activity-estivation cycle in this freshwater gastropod.

Identification and characterization of major proteins carrying ABO blood group antigens in the human kidney.

BACKGROUND: It is generally admitted that ABO(H) blood group antigens are linked to lipids and proteins. Although glycolipids carrying ABO antigens have been well characterized in human kidneys, glycoproteins carrying ABO antigens are largely unknown, and their molecular properties remain to be elucidated. METHODS: All the blood group A antigen-linked proteins in human kidney could be solubilized and captured on immobilized Helix pomatia lectin that recognizes A antigens. These proteins were separated on SDS-PAGE gels. The gel pieces containing protein bands immunoreactive with anti-A antibody were excised, in-gel digested with trypsin, and analyzed by nanoLC tandem mass spectrometer. Protein candidates that carry ABO antigens were confirmed by immunoprecipitation and double-labeled immunofluorescense microscopy. RESULTS: All the glycoproteins carrying ABO antigens were found to be Asn-linked glycoproteins, and presented as multiple bands on SDS-PAGE with molecular masses ranging from 60 to 270 kDa. The protein bands were subjected for mass spectrometric analysis, which identified 121 distinct proteins with high confidence. Of the identified proteins, 55 N-glycosylated, membrane proteins were selected as glycoprotein candidates that carry ABO antigens. Among them, most abundantly expressed proteins as estimated by the number of peptide matches in the MS spectrometric analysis, such as platelet endothelial cell adhesion molecule 1, plasmalemmal vesicle-associated protein, and von Willebrand factor, were further characterized. CONCLUSIONS: Several glycoproteins were identified that represented major glycoproteins carrying ABO antigens in the human kidney, which exhibited distinct features in localization to most of vascular endothelial cells.

Involvement of glycans in the immunological cross-reaction between alpha-macroglobulin and hemocyanin of the gastropod Helix pomatia.

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between alpha-macroglobulin (alphaM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: alphaM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: alpha(D)-HpH, alpha(N)-HpH and beta-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With beta-HpH, which is richer in carbohydrates than alpha(D)-HpH and alpha(N)-HpH, mainly due to a higher 3-O-methyl-d-galactose content, the cross-reaction with HpalphaM was highest. (ii) From the 8 functional units, designated a-h, isolated from beta-HpH, two that lack carbohydrates (c and f) were not recognized by antibodies against HpalphaM, while the six glycosylated ones were strongly cross-reacting. The nearly complete loss of the cross-reactivity upon deglycosylation of functional units d and g and the inhibition in competitive ELISA experiments by glycopeptides isolated from both beta-HpH and HpalphaM are further evidence that glycans are involved in the immunological relationship between HpH and HpalphaM. Carbohydrate analyses indicated that the glycan structures present on HpalphaM are very similar (or identical) to those found on HpH, suggesting that glycans are common epitopes on both proteins. Especially d-xylose and 3-O-methyl-d-galactose seem to be responsible for the cross-reactivity since the alpha-macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react.

The Helix aspersa (brown garden snail) allergen repertoire.

BACKGROUND: Ingestion of snails can induce strong asthmatic or anaphylactic responses, mainly in house-dust-mite-sensitized patients. The aim of this study was to identify the Helix aspersa (Hel a), Theba pisana (The p) and Otala lactea (Ota l) allergens and the extent of their cross-reactivity with the Dermatophagoides pteronyssinus (Der p) mite. PATIENTS AND METHODS: In 60 atopic patients, skin prick tests (SPT) to snail and D. pteronyssinus, total and specific IgE, specific IgE immunoblots, RAST and immunoblot inhibition assays were performed. RESULTS: Mean total IgE was >1,000 kU/l. Mean specific IgE (class 6 for Der p and class 2 for Hel a) SPT were positive in 44 patients for snail and in 56 for mite. Isoelectric focusing (IEF) and SDS-PAGE followed by immunoblotting of H. aspersa extract enabled the identification of 27 and 20 allergens, respectively. Myosin heavy chains from snails (molecular weight >208 kDa) disclosed two major allergens. Hel a and Der p RAST were strongly inhibited by their homologous extracts, with Hel a RAST being inhibited by the Der p extract to a much greater extent (72.6%) than the inverse (5.6%). A complete inhibition of the immunoblots by their homologous extract was obtained. However, Hel a extract did not inhibit Der p IEF separated recognition. On the other hand, mite extract extensively inhibited snail immunoblots from both IEF and SDS-PAGE separations. Immune detection on chicken, pig, rabbit, cow and horse myosins did not reveal any IgE cross recognition with snail. CONCLUSIONS: In most cases of snail allergy, mite appeared to be the sensitizing agent. Nevertheless, snails may also be able to induce sensitization by themselves. This hypothesis is supported by the finding of specific IgE to Hel a in 2 patients who did not show specific IgE to Der p, and one of them was suffering from asthma after snail ingestion.

Cloning, isolation, and IgE-binding properties of Helix aspersa (brown garden snail) tropomyosin.

BACKGROUND: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail. This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates. METHODS: A tropomyosin-specific cDNA encoding H. aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein. IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients. RESULTS: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84-69% identity) as well as with those from arthropods (65-62%), and less homology with vertebrate ones (56% identity). Tropomyosin reacted with 18% of the sera from patients with snail allergy. Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins. Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts. CONCLUSIONS: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity.

Cross-reactivity between terrestrial snails (Helix species) and house-dust mite (Dermatophagoides pteronyssinus). I. In vivo study.

Clinical reports have suggested an unusual frequency in the number of patients with food allergy to snails who are also allergic to the house-dust mite (HDM). As allergy to HDM is one of the most frequent sensitizations in atopic patients of Western countries, evaluation of the relevance of the concomitant sensitization to Dermatophagoides pteronyssinus and to snails is an important consideration. To evaluate the responsibility of different snail components and of snail mites for inducing in vivo hypersensitivity in patients allergic to HDM, the in vivo reactivity of patients with clinical symptoms after ingestion of snails was assessed by skin prick tests with extracts and hemolymph from four different Helix species snails, and extracts from the snail parasitic mite, Riccardoella limacum. In addition, to obtain epidemiologic data on cosensitization to HDM and snails in allergic patients, the frequency of snail sensitization and its relationship to HDM sensitization were determined in a population of 169 allergic children. All patients allergic to snails had positive skin prick tests to the snail extracts and none to R. limacum extract. The number of positive skin reactions did not significantly differ whatever the species, snail part, or heating procedure used. The strongest reactions were obtained with Helix pomatia (Burgundy snail). Among the 169 prospectively tested children, 38 had a positive prick test to snail extracts; 79% of the snail-sensitized children had sensitization to HDM; and 31% of the children allergic to HDM were found to be sensitized to snails. These results show that snail components, and not the mite R. limacum, were responsible for the in vivo hypersensitivity. These snail components reacting in vivo are present in different parts of snails, including the hemolymph. One-third of the children allergic to HDM were sensitized to snails without any previous ingestion of snails: this observation suggests that HDM was the sensitizing agent and that the cross-reaction could be clinically relevant in countries where eating snails is common.

Cross-reactivity between terrestrial snails (Helix species) and house-dust mite (Dermatophagoides pteronyssinus). II. In vitro study.

Epidemiologic and in vitro data have shown that the association of house-dust mite (HDM) allergy and snail allergy in the same patients was due to cross-reactivity between HDM and snail allergenic components. However, the cross-reacting allergen(s) have not yet been identified. In vitro reactivity of seven patients' sera to the various extracts and hemolymph of four different Helix snail species was analyzed by IgE detection and immunodots and Western blots. Cross-reactivity between snails and Dermatophagoides pteronyssinus was assessed by immunodot and ELISA inhibition in two patients. Heterologous inhibition of the snail immunodot and ELISA was observed in one serum. Western blotting showed a specific binding on all four snail species extracts; molecular weights of snail allergens ranged from < 21 to 200 kDa. Marked individual differences were observed in the seven sera under study; most sera demonstrated IgE recognition of multiple bands, illustrating that no single allergen is responsible for cross-reactivity between snail and mite. These results confirm that cross-reactivity exists between snails of the Helix genus and HDM. This cross-reactivity, involving more than a single allergen, may be of clinical significance in atopic patients allergic to D. pteronyssinus. The identity of the cross-reacting allergens remains to be determined. Potential candidates include the thermostable minor allergens of D. pteronyssinus, tropomyosin and hemocyanin.

Allogeneic and xenogeneic grafts in pulmonate gastropod molluscs: fates of neural transplants.

Neural intracerebral allo- and xenografts in pulmonate gastropods demonstrated a variation in the tolerance of neural xenogeneic grafts that was dependent on the phylogenetic distance between the donor and the host. Like allografts, neural congeneric xenografts (Hp/Haa and H1/Haa) of cerebral ganglia (CG) were tolerated and restored growth in juvenile mesocerebrum-deprived (Haa) snails. However, CG neural xenografts between different genera of stylommatophorans (Achatina fulica/Haa) or between genera of different orders (Lymnaea stagnalis: Basommatophora/Haa: Stylommatophora) revealed an interspecific histoincompatibility. These results, compared with those described by other authors, suggest that gastropods possess mechanisms for the recognition of non-self that depend on the organ considered and the phylogenetic distance separating host and donor. Research should now attempt to identify the factors responsible for graft destruction.

Isolation of IgA1 from human serum by affinity chromatography using an immobilized extract of the albumin gland of Helix pomatia.

An extract of the albumin gland of Helix pomatia was linked to Sepharose-4B and used to prepare IgA from group O human serum; immunoelectrophoresis showed that the preparation was free of IgG and IgM. From studies with specific IgA subclass antisera and by comparison with the activity of jacalin-produced material the Helix pomatia extract was found to be IgA1 specific. The preparation had red cell anti-A, B specificity and was suitable for standardizing and controlling anti-human IgA reagents. Preparations using six different carbohydrates as eluants inhibited the agglutination reaction between anti-human IgA and IgA-coated red cells to varying degrees. The pattern of reactions suggested that N-acetyl glucosamine was the IgA binding site for Helix pomatia; this differed from its blood group A determinant (N-acetyl galactosamine) which was the same as that for the IgA1 reactive component of jacalin.

HLA-DR expression is induced on keratinocytes in delayed hypersensitivity but not in allergen induced late-phase reactions.

In view of increasing evidence suggesting an active immunoregulatory role of the skin keratinocytes and the observation that the differentiation of allergen specific T lymphocytes is critical in the development of allergy, we evaluated epidermal expression of HLA-DR antigen in skin reactions induced with an atopen (house dust mite) and with an non-atopic antigen (Hemocyanin). Two groups of patients with house dust mite (Dermatophagoides pteronyssinus [Der p]) allergy were compared, one group was skin tested with Der p, the other group was immunized and subsequently skin tested with Helix pomatia Hemocyanin (HPH). Biopsy specimens taken at 48 h after the HPH (n = 11) and Der p (n = 11) tests were analysed immunohistologically. Reactions in both groups were comparable in size. Immunohistological analysis showed domination by CD4+ lymphocytes. Expression of HLA-DR antigen by epidermal keratinocytes was observed in six out of 11 of the HPH induced reactions, but in none of the Der p induced reactions. Eosinophils were spotted only throughout the Der p induced reactions, showing a good correlation with the number of CD4 positive lymphocytes. The lack of HLA-DR expression by keratinocytes during the allergen-induced reaction, compared with the Hemocyanin induced reaction can be the result of a difference in cytokine profile of the lymphocytes dominating the dermal infiltrate. On the other hand evidence exists that defective HLA-DR expression by keratinocytes enhances antigen induced lymphocyte activation, and may thus contribute to the development of allergen-specific T-lymphocytes.

Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: evidence for donor origin of the antibody producing cells.

A rapid recovery of specific humoral immunity in the recipient of an allogeneic bone marrow transplantation (BMT) can be observed after immunization of the donor before graft sampling. This has been attributed to transfer of specific immunity from donor to recipient. However, to maintain the concept of transfer the origin of the antibody producing cells in the recipient after BMT must be demonstrated. To this end, donor-recipient pairs with differences in Gm-allotypes were selected and immunized before BMT with the neo-antigen Helix pomatia haemocyanin (HPH) according to three immunization protocols. Additionally, the recipients were immunized at day 42 after BMT. Serum samples were weekly obtained from the recipients in the first 100 d after BMT. The origin of HPH-specific antibody producing cells was assessed by two approaches: (1) determination of the Gm-allotypes of anti-HPH antibodies within a distinct IgG subclass, (2) analysis of anti-HPH antibody spectrotypes by isoelectric focusing combined with immunoblotting. The results obtained with these two approaches show concordance in most instances and led to the conclusion that the antibody producing cells are of donor origin.

Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: effect of conditioning on the specific immune response of the graft recipient.

Transfer of specific immunity was investigated in a group of 28 paediatric and adult leukaemia patients during the first 100 d after allogeneic bone marrow transplantation (BMT). These patients and/or their donors were immunized 7-13 d before transplantation with the recall antigen tetanus toxoid (TT) and the neo-antigen Helix pomatia haemocyanin (HPH). The recipients were booster immunized with both antigens at day 42 after transplantation. Transfer of a primary IgM and IgG response to HPH was successful in most paediatric and adult patients, but transfer of a secondary response to TT was established in only a few paediatric recipients. After booster immunization at day 42 most paediatric recipients responded with a rise in serum antibody titre to HPH as opposed to only two of 18 adult recipients. This incapability of the adult recipients to mount a secondary immune response may be related to their conditioning regimen which included Campath-IG in vivo. The results from this study indicate that transfer of immunity against recall- and neo-antigens is possible. However, the establishment of long-term memory may be affected by the regimen used to condition the graft recipient.

Experimental epilepsy in vitro: neuromodulating activity of anti-brain autoantibodies from rats exposed to electroconvulsive shock.

Neuromodulating activity of anti-brain autoantibodies obtained from electroshocked (ECS) rats was tested on the neurons of isolated suboesophageal ganglion of the snail Helix pomatia. In 16 out of 18 spontaneously active (pacemaker) neurons, ECS IgG containing anti-brain autoantibodies induced short-lasting epileptiform discharges and membrane depolarization. Membrane input resistance and time constant decreased, while membrane capacitance increased after addition of ECS IgG. Amplitude of evoked action potential (AP) decreased, whereas AP duration, rise time and fall time slightly increased. Thus, anti-neural autoantibody-positive IgG from rats with experimental epilepsy, but not autoantibody-negative IgG from control rats, significantly affected the bioelectrical properties of the isolated snail neurons. These results suggest that anti-neural autoantibodies present in epileptic animals are capable of influencing in vivo the function of the brain neurons.

Food allergy to Helix terrestre (snail).

Among the rare foods capable of producing food allergies is the snail (Helix terrestre). The snail is a delicacy eaten in Spain, France and Portugal. This study presents the findings of an allergic study of 10 patients with this infrequent food allergy during the past 10 years. The shock organ in the majority (80%) of these patients was the bronchial tree. Six of them did not have any digestive or skin symptoms which are usually seen in cases of food allergy. All patients manifested the symptomatology after ingestion of Helix terrestre. Two also had reactions after eating Patella vulgata (limpet). The snail and the limpet are within the phylogenetic line of molluscs, i.e. of gastropods. All patients tolerated the ingestion of cephalopods and bivalves which belong to two other phylogenetic lines. Skin tests to seafoods (squids, prawns, lobsters and clams) were negative for all patients. This suggests that the sensitizing antigen is probably a protein found only in gastropod molluscs. Skin tests along with the histamine release test were valid diagnostic methods for this food allergy. The limited bibliography on this subject is probably due to the fact that the consumption of snails as well as limpets is limited to specific geographical areas.