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Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with an increased intestinal permeability. Several studies have shown that microRNAs (miRNAs) are involved in the IBD pathogenesis. Here, we aimed to functionally characterize the role of miRNAs in the regulation of intestinal permeability and barrier function. We identified 18 dysregulated miRNAs in intestinal epithelial cells (IECs) from the ulcerative colitis (UC) mice model and control mice. Among them, down-regulated miR-195-5p targeted claudin-2 (CLDN2) and was involved in impaired barrier function. CLDN2 expression levels were increased in UC mice models and negatively correlated with miR-195-5p expression. We demonstrated that gain-of-function of miR-195-5p in colonic epithelial cell lines decreased the CLDN2 levels. This modulation, in turn, downregulated claudin-1 (CLDN1) expression at protein level but not that of occludin. Our data support a previously unreported role of miR-195-5p in intestinal tight junctions' regulation and suggest a potential pharmacological target for new therapeutic approaches in IBD.

Background & Aims: Inflammatory bowel diseases are multifactorial diseases commonly treated with either immunomodulatory drugs or anti-tumor necrosis factor (TNF). Currently, failure to respond to anti-TNF therapy (assessed no earlier than 8-12 weeks after starting treatment) occurs in 20%-40% of patients enrolled in clinical trials and in 10%-20% in clinical practice. Murine models of inflammatory bowel disease provide important tools to better understand disease mechanism(s). In this context and among the numerous models available, Winnie-TNF-knockout (KO) mice recently were reported to show characteristics of ulcerative colitis (UC) that are independent of TNF, and with increased interleukin (IL)1β production., Methods: Herein, the efficacy of recombinant IL1-receptor antagonist (anakinra) administration was evaluated in Winnie-TNF-KO mice, used as a UC model of primary anti-TNF nonresponders., Results: We analyzed gut mucosal biopsy specimens and circulating cytokine profiles of a cohort of 30 UC patients; approximately 75% of primary nonresponders were characterized by abundant IL1β in both the serum and local intestinal tissues. In Winnie-TNF-KO mice, administration of anakinra efficiently reduced the histologic score of the distal colon, which represents the most common site of inflammation in Winnie mice. Furthermore, among lamina propria and mesenteric lymph node-derived T cells, interferon γ-expressing CD8 + T cells were reduced significantly after anakinra administration., Conclusions: Our study provides new insight and alternative approaches to treat UC patients, and points to anti-IL1 strategies (ie, anakinra) that may be a more effective therapeutic option for primary nonresponders to anti-TNF therapy., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

(1) Background: Colorectal cancer (CRC) is among the best examples of the relationship between inflammation and increased cancer risk. (2) Methods: To examine the effects of spontaneous low-grade chronic inflammation on the pathogenesis of CRC, we developed a new murine model of colitis-associated cancer (CAC) by crossing Mucin 2 mutated mice (Winnie) with Apc Min/+ mice. (3) Results: The resulting Winnie- Apc Min/+ model combines an inflammatory background with a genetic predisposition to small intestinal polyposis. Winnie- Apc Min/+ mice show an early occurrence of inflammatory signs and dysplastic lesions in the distal colon with a specific molecular signature. (4) Conclusion: The Winnie- Apc Min/+ model is a perfect model to demonstrate that chronic inflammation represents a crucial risk factor for the onset and progression of tumoral lesions in individuals genetically predisposed to CRC.

Celiac disease (CD) is a chronic immune-mediated disorder, characterized by enhanced paracellular permeability across the intestinal epithelium. The complex system of intercellular junctions, including tight junctions (TJs) and adherens junctions (AJs), seals together the epithelial cells to form a continuous layer. The improvements in barrier integrity have been related to modifications in intercellular junction protein expression. Polyamines (spermidine, spermine, and putrescine) actively participate in the modulation of the AJ expression. Both in vitro and in vivo studies have demonstrated that also probiotics can promote the integrity and the function of the intestinal barrier. On these bases, the present work investigated the protective effects exerted by Lactobacillus rhamnosus GG (L.GG) against the pepsin-trypsin-digested gliadin (PTG)-induced enteropathy in jejunal tissue samples of Wistar rats. In particular, the probiotic effects have been evaluated on the intestinal mucosal architecture, polyamine metabolism and intercellular junction protein expression (ZO-1, Occludin, Claudin-1, β-catenin and E-cadherin). The results from this study indicate that L.GG protects the intestinal mucosa of rats from PTG-induced damage, by preventing the reduction of the expression of the intercellular junction proteins. Consequently, a role for L.GG in the therapeutic management of the gluten-related disorders in humans could be hypothesized.

Background/aim: The expression of cannabinoid receptor-1 (CB1-R) seems to be modulated by bioactive natural components such as the flavonoid quercetin. The aim of this study was to determine in an animal model of induced-colon cancer, whether quercetin inhibits colon carcinogenesis through changes in the expression of CB1-R., Materials and Methods: C57BL/6J male mice were randomly assigned to standard diet or experimental diet supplemented with 0.5% quercetin. Azoxymethane (AOM) (10 mg/kg body weight) or saline solution (PBS) was intraperitoneally injected, once weekly for 6 weeks., Results: The diet supplemented with quercetin induced CB1-R gene expression and protein, inhibiting the protein levels of STAT3 and p-STAT3 (both mediators of cell proliferation). Dietary quercetin also caused a significant increase in Bax/Bcl2 ratio protein expression., Conclusion: The anti-proliferative and pro-apoptotic effects of quercetin in AOM-treated mice are mediated by induction of the protein and gene expression levels of CB1-R., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

BACKGROUND: Cannabinoid- 2 (CB2) receptor is known for its anti-obesity effects silencing the activated immune cells that are key drivers of metabolic syndrome and inflammation. Nutritional interventions in experimental models of carcinogenesis have been demonstrated to modulate tissue inflammation state and proliferation. OBJECTIVE: Aim of this study was to test, in Apc Min/+ mice, whether a diet enriched with olive oil, omega- 3 and omega-6- PUFAs affects the adipose tissue inflammation status. METHODS: Four groups of animal were studied: ST group, receiving a standard diet; OO group, receiving the standard diet in which soybean oil (source of fats) was replaced with olive oil; OM-3 group, receiving the standard diet in which soybean oil was replaced with salmon oil; OM-6 group, receiving the standard diet in which soybean oil was replaced with oenothera oil. Gene and protein expression, in adipose tissue, were evaluated by RT-PCR and Western Blotting, respectively. Enzymatic activities were assayed by fluorescent and radiometric method, where appropriated. RESULTS: The diet enriched with olive oil significantly induced CB2 receptor expression and it was able to control inflammatory and proliferative activity of mice adipose tissue. CONCLUSIONS: The present findings open opportunities for developing novel nutritional strategies considering olive oil a key ingredient of a healthy dietary pattern.

The statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and orlistat, an inhibitor of fatty acid synthase (FAS), inhibit tumor cell growth by restricting cholesterol and fatty acid synthesis, respectively. We previously demonstrated that an omega (ω)-3 polyunsaturated fatty acid (PUFA)- or olive oil-enriched diet reduced the polyp number and volume in ApcMin/+ mice. This phenomenon was associated with a significant inhibition of FAS and HMGCoAR, as well as an increase in the estrogen receptor (ER)β/α ratio. Herein, we evaluated the effect of lovastatin and orlistat on polyp development and ER expression in ApcMin/+ mice, in order to confirm previous data obtained with ω‑3-PUFAs and olive oil. As expected, the use of lovastatin and orlistat significantly reduced HMGCoAR and FAS enzymatic activities and gene expression in colonic tissues, but did not affect the number of intestinal polyps, while there was a statistically significant reduction in polyp volume only in the mouse group treated with lovastatin. In the mice receiving orlistat, we observed a significant increase in cell proliferation in the polyp tissue, as well as enhanced expression of ERα. Moreover, the overexpression of ERα was associated with a statistically significant increase in PES1, Shh and Gli1 protein levels, considered ERα-related molecular targets.

Background and Aims: Augmenter of Liver Regeneration is a protein encoded by the Growth Factor Erv1-Like gene. Its biological properties are crucial for cell survival since knock-out mice for Growth Factor Erv1-Like gene do not survive. In this study, we injected hepatotropic adenoviral particles harboring oligonucleotide sequences against Growth Factor Erv1-Like gene into 70% partially hepatectomized rats and studied the effect of gene silencing on the progression liver regeneration., Methods: Partially hepatectomized rats were divided into three groups of animals and, before surgery, received either phosphate buffer saline, or adenoviral particles alone or adenoviral particles harboring the oligonucleotide silencing sequence. In each group, rats were sacrificed at 12, 24 and 48 h after surgery. Liver tissues were collected to analyze the expression of Augmenter of Liver Regeneration, Bax, Bcl-2 and activated Caspase-9 and -3, as well as hepatocyte proliferation and apoptosis, polyamines levels and histological and ultrastructural features., Results: Growth Factor Erv1-Like gene silencing reduced the compensatory hepatocellular proliferation triggered by surgery through (i) the reduction of polyamines synthesis, hepatocyte proliferation and anti-apoptotic gene expression and (ii) the increase of pro-apoptotic gene expression and caspase activation., Conclusions: For the first time, using a technique of gene silencing in vivo, our results demonstrate that Growth Factor Erv1-Like gene knock-down, i.e., the lack of Augmenter of Liver Regeneration, modifies the expression of genes involved in cell apoptosis and inhibits early phase of DNA synthesis. As a consequence, a promotion of cell death and a reduction of cell proliferation occurs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aim: To study, in intact male transgenic mice, the effects of three diets based on olive oil and olive oil diet supplemented with lovastatin and orlistat on hepatic lipogenic enzymes expression, considered markers of cell proliferation., Methods: Forty Apc(Min/+) mice were randomly divided into 4 groups and fed for 10 wk: olive oil (OO) group, n = 10 animals received a diet with olive oil 12%; olive oil plus lovastatin (LOVA) group, n = 10 animals received the same diet with olive oil supplemented with lovastatin 5 mg/kg; olive oil plus orlistat (OR) group, n = 10 animals fed the diet with olive oil supplemented with orlistat 50 mg/kg and SD group, n = 10 animals fed a standard diet. The activity of lipogenic enzymes and their gene expression were evaluated by radiometric and real-time reverse transcription-polymerase chain reaction assay, respectively., Results: After 10 wk of dietary treatment, the body weight was no different among animal groups (21.3 ± 3.1 g for standard group, 22.1 ± 3.6 g for OO group, 22.0 ± 3.2 g for LOVA group and 20.7 ± 3.4 g for OR group, data expressed as mean ± SD), observing a generalized well-being in all animals. All the dietary managed treated groups presented significantly reduced hepatic levels of fatty acid synthase, farnesyl pyrophosphate synthase and 3-hydroxyl-3-methyl-glutaryl CoA reductase activity and gene expression when compared with the mice fed the standard diet. To evaluate cell proliferation in the liver of treated mice, the levels of cyclin E mRNA have been measured, demonstrating a significant reduction of cyclin E gene expression in all treated groups. Evidence of reduced hepatic cell proliferation was present overall in OO group mice., Conclusion: We confirm the role of lipogenic enzymes as markers of cell proliferation, suggesting that appropriate dietary management alone or with drugs can be a feasible approach to counteract hepatic cell proliferation in mice.

In many cases, it is difficult or even impossible to distinguish parathyroid lesions from thyroid ones at ultrasound as well as at scintiscan and even at cytology, because they often share common features. The aim of this study was to evaluate the role of Parathyroid Hormone (PTH) determination in the aspirates in the differential diagnosis of parathyroid from thyroid lesions in an area of mild iodine deficiency and high prevalence of thyroid nodules. Forty-six consecutive patients were suspected to have one or more nodule(s) of parathyroid origin because of their position in the posterior aspect of thyroid lobes and/or their shape and echo-pattern at ultrasound examination. In 13 cases, there were also laboratory findings suggestive for primary hyperparathyroidism, with clinical evidence in 6 of these patients. A total of 55 lesions suspected to be of parathyroid origin were selected. After obtaining cytological preparations, the needle used to perform the fine-needle aspirate (FNA) was washed using 1 ml of normal saline. Intact PTH determination in the washout was done whereas the evaluation was performed directly in the aspirated fluid in case of cystic lesions. The values of PTH in the aspirates ranged from 6.7 to 16640 pg/ml. Sixteen patients underwent surgical intervention and the histological examination of the 23 operated lesions previously submitted to FNA-PTH showed 11 parathyroid adenomas, 5 hyperplasic parathyroid lesions and 7 benign thyroid nodules. A strong positive correlation between high levels of PTH in the aspirate and the histological findings of parathyroid lesions was found. A value over 245 pg/ml was constantly associated to the parathyroid lesions. Our results confirmed the high accuracy of FNA-PTH determination in differentiating parathyroid lesions from thyroid nodules and this is of special value in an area of mild iodine deficiency with a high prevalence of thyroid nodules.

Aim: The aim of the present study was to evaluate the effects of a diet supplementation with either olive oil, or n-3 or n-6-polyunsaturatedFatty acids (PUFAs) on tumour development and gene expression for lipogenic enzymes in Apc(Min/+) mice., Materials and Methods: In the control group, the mice received a standard diet, the OO group was fed on a diet with 12% olive oil, the OM-3 group with 12% salmon fish rich in n-3 PUFAs, the OM-6 group with 12% oenothera oil rich in n-6 PUFAs. Gene expression of lipogenic enzymes was evaluated by real-time reverse transcription polymerase chain reaction., Results: All mice in the treated groups presented a reduction in total intestinal polyp number and load, which was particularly marked in the OM-3 group. Treated mice showed an induction of low density lipoprotein receptor gene expression and a significant reduction of expression of lipogenic gene., Conclusion: Our data provide new insights into the mechanism of cell growth inhibition and apoptotic regulation by dietary olive oil and PUFAs in Apc(Min/+) mice.

To evaluate the growth-inhibitory properties of the potent multi-kinase antagonist Regorafenib (Fluoro-Sorafenib), which was synthesized as a more potent Sorafenib, a Raf inhibitor and to determine whether similar mechanisms were involved, human hepatoma cell lines were grown in the presence or absence of Regorafanib and examined for growth inhibition. Western blots were performed for Raf targets, apoptosis, and autophagy. Regorafenib inhibited growth of human Hep3B, PLC/PRF/5, and HepG2 cells in a concentration- and time-dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho-ERK and phospho-JNK and its target phospho-c-Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin-1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications., (Copyright © 2012 Wiley Periodicals, Inc.)

Familial Adenomatous Polyposis, Cowden's Syndrome, and Peutz-Jeghers Syndrome are well known as Intestinal Polyposis Syndromes, inherited conditions characterized by the development of polyps of the gastro-intestinal tract in association with extra-intestinal manifestations, in particular malignant tumors at different sites. Thyroid carcinoma is sometimes a part of the clinical picture of these syndromes. The aim of this paper is to review the literature dealing with the association between differentiated thyroid carcinomas and Intestinal Polyposis Syndromes in order to point out peculiar aspects, providing suggestions for the screening and the management of thyroid tumors in these patients.

In a group of 14 healthy aged subjects, donkey and goat milk was administered respectively, for a period of one month. Cytokine profile [interleukin (IL)-12, IL-10, IL-1beta, IL-8, IL-6 and Tumor Necrosis Factor (TNF)-alpha] was assessed before and after milk intake by means of a cytometric bead array test. Data demonstrated that IL-12 was undetectable, while IL-10, IL-1beta and TNF-alpha were released in very low amounts. Quite interestingly, IL-8 was increased by donkey milk administration, while same cytokine was dramatically decreased following goat milk intake. Same pattern of response was noted with IL-6 even if levels of these cytokine were lower than those detectable in the case of IL-8. Taken together, these findings indicate that administration of donkey milk in the aged host is able to upregulate the immune response, while goat milk seems to reduce the exaggerated acute phase response in elderly.

Pro-inflammatory mediators hold important functions in human body in response to infection, trauma and vascular disease. However, their action is down regulated by the release of anti-inflammatory cytokines, thus restoring a balance which reflects the immune status of a given individual. Recent studies have stressed out the importance of circulating levels of cytokines for forensic purposes even if there is a lack of studies regarding the role of post-mortem mucosa-associated lymphoid tissue. In this respect, Peyer's patches (PP), represent one of the most important immunological site of the body and the major component of the gut -associated lymphoid tissue. The aim of this study was to evaluate post-mortem PP immune response in 40 serial autopsy cases of people who died from natural and traumatic death. The study examined spontaneous release of the following cytokines by fresh isolated PP cells: interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, IL-10, IL-6, IL-1 beta, and IL-8. Results will show that higher levels of TNF-alpha, IL-6, IL-1 beta, and IL-8 are statistically correlated with the traumatic death group. From a forensic point of view these data demonstrate that fundamental lymphoid organs, such as PP, may have a potential in diagnosing the cause of death.

The intestinal mucosa contains a highly specialized immune system which plays a central role in the induction of immune reactions. In the small bowel, Gut-Associated Lymphoid Tissue (GALT) is organized in lymphoid aggregates which are known as Peyer's Patches (PP). Even though human PP involvement in systemic immunity has been described, little is known about their anatomy and morphology and viability. The aim of this study was to examine PP according to their macroscopic anatomy, distribution and cell viability after death. Specimens from the distal ileum were obtained from 72 serial autopsy cases: PP were identified and, parts of them were analyzed for histological examination. Moreover, viability of recovered PP cells was assessed by the trypan blue exclusion test. Most of the PP (90%) were situated on the antimesenteric border of ileum, and the greatest density of PP occurred in the most distal segment. The number of PP varied with age, with the maximum number observed in 21- to 30-years old cadavers. Histological examination showed their remarkable architectural preservation at different post-mortem intervals (PMI), while the mucosal surface underwent autolysis. In 56% of cases PP cells were still viable, especially at PMI < 24 hours after death. These data confirm that human PP are still well preserved in a remarkable percentage of cadavers also several hours after death, and their availability may be helpful in various fields of research.

Polyphenols contained in red wine possess a broad array of properties which seem to be beneficial to human and animal health. We have investigated the ability of red wine polyphenols to promote the in vitro release of both proinflammatory and antiinflammatory cytokines from human healthy mononuclear cells, as well as of immunoglobulins from B cells. Following red wine (Negroamaro) pretreatment of lymphomonocytes, results will show a production of regulatory [Interleukin(IL)-12], proinflammatory (IL-1 beta and IL-6), and anti-inflammatory (IL-10) cytokines, as well as of IgA and IgG. The fine balance between inflammation and antiinflammation, as well as the role of humoral immune response either systemic or mucosal will be discussed as a consequence of red wine intake. Finally, since ageing is characterized by a decline of many immune functions, our results suggest that moderate use of red wine may be beneficial in age-related disorders where the host immune response is very often not effective against a variety of antigens.

Donkey's milk is the best substitute of human milk for its content in lactose, proteins, minerals, and omega-3 fatty acids. Here, we have evaluated the effects of colostrum and milk from donkeys (Martina Franca breed) on the function of human peripheral blood mononuclear cells (PBMCs) at different intervals from lactation. Colostrum induced more IgA responses, while milk induced predominantly more IgG responses. Both milk and colostrum induced expression of CD25 and CD69 on PBMCs. The ability to induce release of interleukins (IL) (IL-12, IL-1 beta and IL-10) and tumor necrosis factor-alpha was confined only to milk, while colostrum was devoid of this capacity. Finally, both colostrum and milk induced nitric oxide (NO) release from PBMCs but milk exhibited a greater capacity than colostrum in NO generation. Taken together, these immunological activities exerted by both colostrum and milk from donkeys may be useful in the treatment of human immune-related diseases. In particular, NO induction by donkey's milk may be very useful in the prevention of atherosclerosis, being a strong vasodilator and an effective antimicrobial agent since pathogens and/or their products may play a proatherogenic role.

Evidence has been provided that red wine possesses antiatherogenic activities in virtue of its content in polyphenols (flavonoids and non-flavonoids substances). Here, some red wines (Negroamaro, Primitivo and Lambrusco) were tested for their ability to trigger nitric oxide (NO) production from human healthy peripheral blood mononuclear cells (PBMC). Negroamaro was the strongest inducer of NO from PBMC and deprivation of polyphenols did not influence its NO generation capacity. This fact supports the involvement of polyphenols in the NO production even in the absence of alcohol, which also per se does not exert any significant activity. These results are also corroborated by the evidence that PBMC inducible-nitric oxide synthase expression occurred by the effect of samples containing polyphenols but this expression was very weak when polyphenols were removed from the whole Negroamaro. In synthesis, flavonoids and resveratrol, major constituents of red wine, once absorbed at intestinal level, enter circulation and trigger monocytes for NO production. To the best of our knowledge, this is the first demonstration of a direct effect of red wine on monocytes for NO release to occur. On the other hand, also the macrophage contingent from gut-associated lymphoid tissue can contribute to NO generation, besides the aliquot produced by endothelial cells, as previously demonstrated by various authors. Taken together, these results support the concept that moderate intake of red wine can prevent atherosclerosis via production of NO, a potent vasodilator of terminal vessels.

Anthrax edema toxin (EdTx) is an AB-type toxin that binds to anthrax toxin receptors on target cells via the binding subunit, protective Ag (PA). Edema factor, the enzymatic A subunit of EdTx, is an adenylate cyclase. We found that nasal delivery of EdTx enhanced systemic immunity to nasally coadministered OVA and resulted in high OVA-specific plasma IgA and IgG (mainly IgG1 and IgG2b). The edema factor also enhanced immunity to the binding PA subunit itself and promoted high levels of plasma IgG and IgA responses as well as neutralizing PA Abs. Mice given OVA and EdTx also exhibited both PA- and OVA-specific IgA and IgG Ab responses in saliva as well as IgA Ab responses in vaginal washes. EdTx as adjuvant triggered OVA- and PA-specific + T cells which secreted IFN-gamma and selected Th2-type cytokines. The EdTx up-regulated costimulatory molecule expression by APCs but was less effective than cholera toxin for inducing IL-6 responses either by APCs in vitro or in nasal washes in vivo. Finally, nasally administered EdTx did not target CNS tissues and did not induce IL-1 mRNA responses in the nasopharyngeal-associated lymphoepithelial tissue or in the olfactory bulb epithelium. Thus, EdTx derivatives could represent an alternative to the ganglioside-binding enterotoxin adjuvants and provide new tools for inducing protective immunity to PA-based anthrax vaccines.

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.

In humans, monocytes and macrophages (Mphi) play a central role in immune regulation, tissue maintenance and pathogen control. In lower vertebrates, a few studies have been conducted on Mphi like cells. In acute monocytic leukemia monocytic cells, as immature cells restrained in one of the phases of their ontogenesis, would offer the opportunity to rebuild an archaic condition helpful to understand the phylogenesis. Therefore, aim of this work was to characterize in the Rainbow trout (Salmo Gairdneri Richardson) Mphi and compare them with acute leukemia monocytic cells. In the trout, Mphi's morphology is similar to that of mammals. In particular, Mphi possess an irregular embryoshaped nucleus occupying 2/3 of the cell, while the peripheral cytoplasmic profile is irregular with extroflexed plasmalemma and pseudopods. A morphological transition towards Mphi is featured by a wavy hyaline classical membrane and an irregular and extroflexed surface. Some aspects of erythrophagocytosis represented a finding of great interest indicating that the hemocatheretic function could take place directly in circulation. This condition, also observed in human acute monocytic leukemia, suggests that the information to the erythrophagocytosis is restrained under physiological conditions. Non-specific esterases, which are positive in human Mphi smear and Mphi from human lymph node tissue, were also positive in the teleost studied but with a dysomogeneous pattern. Consequently non-specific esterase system is phylogenetically conserved. A lack of immune-reactivity with the anti-CD68 monoclonal antibody (MoAb) on smear and trout tissue sections was observed. On the contrary, strong positivity was detected on human lymph node sections. In trout, the presence of Mphi and circulating Mphi like cells exhibiting an erythrocatheretic function in the circulation would indicate a primordial function that has later been replaced by the liver and the spleen.

The mucosal immune system has evolved alongside, but separate, from the general systemic immune system. As a major consequence of this dichotomy, only immune responses initiated in mucosal inductive sites can result in effective immunity in mucosal tissues themselves. Oral tolerance, as usually assessed as orally-induced systemic unresponsiveness, contributes to mucosal homoeostasis by preventing unwanted immune reactions to food or environmental antigens. It is now established that tolerance can also be induced by the nasal route and mucosally-induced tolerance is being actively investigated for immune therapy against a number of diseases. Nontoxic derivatives of cholera toxin and the heat labile toxin of Escherichia coli as well as chimeric enterotoxins have been developed. These molecules retain the mucosal adjuvant activity of native enterotoxins and are effective at inducing targeted Th1 or Th2- type immune responses. Mucosal delivery of cytokines as adjuvants represents a safer alternative to parenteral cytokine injection. Nasally administered cytokines such as IL-1 and IL-12 or chemokines including RANTES, lymphotactin, MIP-1 beta, all act as mucosal adjuvants for co-administered antigens. Each of these cytokines promote specific pattern of CD4(+) T helper cell cytokine responses that could be exploited for targeted immune therapy. Although GALT and NALT are both parts of the Common Mucosal Immune System, there are major differences between orally and nasally induced immune responses. Nasal vaccines more effectively promote protective immunity in the genitourinary tract than do oral vaccines. In addition, aging affects mucosal tolerance or immunity in GALT more than is seen in NALT. Therapeutic manipulation of mucosal immunity involves regulation of CD4(+) T cell cytokine responses and thus, should require a careful examination of the host status, including the occurrence of inflammatory bowel diseases.

In Salmo gairdneri Richardson trouts, a comparison was made between macrophages (MO) derived from head kidney and peripheral blood monocytes. Morphologically and cytochemically no differences were observed between these two types of mononucleated cells. On the other hand, in parallel studies the ability of trout erythrocytes to engulf Candida albicans (CA) was evaluated and compared to the MO phagocytosis. In erythrocytes, engulfment is preceded by binding to CA and cell membrane invagination, while this was not the case for MO. Finally, MO stimulated with lipopolysaccharides (LPS) did not modify their phagocytic capacities, thus suggesting a lack of LPS receptors or a tolerance to LPS.

The role of fish erythrocytes (FE) as phagocytic cells has poorly been investigated, until now. Here, we have focussed our attention on the interplay between rainbow trout (Salmo gairdneri Richardson) erythrocytes and Candida albicans (CA). At the same time, the intervention of autologous head kidney macrophages (MO) in the CA processing by FE has been studied. Data show that CA particles bind to FE, which, in turn, are able to engulf but not kill them. In the presence of MO, a decrease of FE with bound CA occurs and, in some microscopic images, FE form rosettes with MO. Phagocytosis of CA is higher in rosetting MO than in non-rosetting ones. According to our findings, it appears that FE represent a reservoir of engulfed CA and rosetting is an efficacious phenomenon of presentation of pathogens to MO, where an effective clearance of them can take place.

In two pups (A and B) naturally infected with canine parvovirus type 1 (CPV1) phagocytic responses were evaluated over a period of two weeks (day 0 = T0; day 3 = T1; day 7 = T2; day 14 = T3). CPV1 infection led to a marked reduction of monocyte (MO) phagocytosis in both pups. Also MO killing was impaired and in pup B this function was totally absent. Polymorphonuclear (PMN) phagocytosis values of both pups fluctuated within normal ranges, as well as PMN killing of pup A. In pup B, killing exerted by PMN was absent at T0, then increased but again dropped below normal ranges at T3. The described alterations of phagocytic functions may be regarded as possible viral mechanisms of immune evasion.

Over the past few years, many reports have pointed out that open, but not minimally invasive, cholecystectomy was associated with reduced immune functions. Also, after laparoscopic surgery, a reduced impairment of T cell functions and lower levels of proinflammatory cytokines, epinephrine, and norepinephrine were found in comparison with those detected in patients who underwent conventional cholecystectomy. We investigated polymorphonuclear cell- and monocyte-mediated phagocytosis and killing and T-cell-mediated antibacterial activity in 12 patients who underwent open cholecystectomy versus another group of 12 patients who underwent laparoscopic cholecystectomy. Our data show that polymorphonuclear and monocyte killing activities are preserved or are less affected in patients who undergo laparoscopy when compared with patients who undergo conventional operation. On the other hand, in both groups of patients, T-cell-mediated antibacterial activity was significantly reduced in the preoperative period, and, therefore, we could not draw conclusions on the effects of the surgical techniques used on the above immune parameter. The overall data suggest that laparoscopic cholecystectomy is a valid alternative to open surgery because of the moderate postoperative immune suppression and decreased risk of postsurgical infections.

Host responses to fungi result from a coordinate interplay between innate and adaptative immune system. Neutrophils and monocytes are involved in the non specific clearance of yeasts (e.g. Candida albicans and Cryptococcus neoformans), while T helper 1 type responses are protective via release of interferon gamma. By contrast, T helper 2 responses (IL-4 and IL 10 release) correlate with disease exacerbation and pathology. IL-12 production which enhances T helper 1 type responses seem to exert a beneficial role in the course of Candida infection. In particular, its production from neutrophilis may support memory T helper 1 cell responses of the fungus. With respect to anti-Candida vaccines several approaches are in progress, such as use of heat-killed Candida albicans in combination with adjuvants, purified peptides and proteins and immunogenic peptide-lipid conjugates. Furthermore, exogenous IL-12 may play an important role in inducing a T helper 1 anticandidal response, also replacing neutrophils in neutropenic patients. At the same time, granulocyte-colony stimulating factor has exhibited therapeutic efficacy in experimental and human models of fungal infection.

Three pups 2-4 months old were vaccinated subcutaneously with the modified live canine parvovirus, CPV-2b/29-97 strain. During an observation period of two weeks pups remained clinically health, exhibiting a vigorous post-vaccinal active serological response (haemoagglutinating inhibiting antibody titers for CPV-2 ranging from 1:2560 to 1:5120 at 21 days post inoculation). Phagocytosis and killing of Candida albicans exerted by polymorphonuclear cells and monocytes did not undergo significant modifications 3-6 days post vaccination up to 30 days. Antibacterial activity mediated by peripheral blood lymphocytes (Salmonella typhi was used as a target) was slightly, but not significantly decreased 3 days post vaccination. Conclusively, in pups the CPV type 2b vaccine seems to be safe as far as natural immune responses are concerned, while its immunogenicity is preserved.