Search

Chronic lymphocytic leukaemia (CLL) is characterised by the expansion of a neoplastic mature B cell clone. CLL clinical outcome is very heterogeneous, with some subjects never requiring therapy and some showing an aggressive disease. Genetic and epigenetic alterations and pro-inflammatory microenvironment influence CLL progression and prognosis. The involvement of immune-mediated mechanisms in CLL control needs to be investigated. We analyse the activation profile of innate and adaptive cytotoxic immune effectors in a cohort of 26 CLL patients with stable disease, as key elements for immune-mediated control of cancer progression. We observed an increase in CD54 expression and interferon (IFN)-γ production by cytotoxic T cells (CTL). CTL ability to recognise tumour-targets depends on human leukocyte antigens (HLA)-class I expression. We observed a decreased expression of HLA-A and HLA-BC on B cells of CLL subjects, associated with a significant reduction in intracellular calnexin that is relevant for HLA surface expression. Natural killer (NK) cells and CTL from CLL subjects show an increased expression of the activating receptor KIR2DS2 and a reduction of 3DL1 and NKG2A inhibiting molecules. Therefore, an activation profile characterises CTL and NK cells of CLL subjects with stable disease. This profile is conceivable with the functional involvement of cytotoxic effectors in CLL control.

Objective: To report for the first time an Italian epidemiological analysis of the prevalence of multiple sclerosis (MS) in patients with endometriosis (EMS), through the study of the endometriosis population of our referral center; to analyze the clinical profile and perform a laboratory analysis to examine the immune profile and the possible correlation to other autoimmune diseases of the enrolled patients. Methods: We evaluated 1652 women registered with EMS in the University of Naples Federico II and retrospectively searched patients with a co-diagnosis of MS. Clinical features of both conditions were recorded. Serum autoantibody and immune profiles were analyzed. Results: 9 out of 1652 patients presented a co-diagnosis of EMS and MS (9/1652 = 0.005%). Clinically, EMS and MS presented in mild forms. Hashimoto’s thyroiditis was found in two patients (2/9). Even if not statistically significant, a trend of variation in CD4- CD8 T lymphocytes and of B cells were found. Conclusion: Our findings suggest an increased risk of MS in women with EMS. However, large-scale prospective studies are needed.

Ethnopharmacological relevance: Chamomile (M. chamomilla L.) is an herbaceous plant from family Astereaceae, that has a long history of use in traditional medicine. It has been used as herbal remedies for thousands of years to treat several diseases, including infections, neuropsychiatric, respiratory, gastrointestinal, and liver disorders. Chronic inflammation is involved in the pathogenesis of most infectious and non-infectious diseases and macrophages are considered the major cellular players that drive disease initiation and maintenance. Aim of the study: The aim of this study was to evaluate the variation in the chemical profile of the essential oil of M. chamomilla plants collected in three experimental field sites in the Molise region. Additionally, we evaluated the pharmacological mechanism behind the anti-inflammatory effect of M. chamomilla essential oils. Material and methods: Three essential oils (called GC1, GC2 and GC3) were extracted from aerial parts of M. chamomilla by hydrodistillation and chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS). The essential oils were tested for their ability to modulate pro-inflammatory murine macrophages and human peripheral blood mononuclear cells (PBMCs) functions. Results: The chemical analysis of the samples revealed the presence of a high content of the oxygenated sesquiterpenes that represented more than the half of the entire oils. GC1, GC2 and GC3 essential oils significantly attenuated LPS/IFN-γ-induced inflammation by reducing M1 polarization. In details, they showed significant anti-inflammatory property by inhibiting NO, TNF-α and IL-6 production. These effects were correlated to a suppression of LPS-mediated p65 activation, the critical transactivation subunit for NF-κB transcription factor. Oxidative stress may trigger macrophages activation and elicit strong immune responses. Our study demonstrated that GC1, GC2 and GC3 were highly effective at increasing GCL and HMOX-1 anti-oxidant enzymes expression leading to the rapid scavenging of ROS. The antioxidant activity of these oils was explained throughout the activation of NRF2 signaling pathway. Next, we demonstrated that essential oils were able to reduce CD4+ T cell activation which are also involved in inflammatory processes. Conclusions: Our data describe for the first time that chamomile essential oils exerted their anti-inflammatory and antioxidant activity by modulating macrophages and CD4+ T cells-mediate immune response.

Emergence of dysplastic haematopoietic precursor/s, cytopenia and variable leukaemia risk characterise myelodysplastic syndromes (MDS). Impaired immune-regulation, preferentially affecting cytotoxic T cells (CTL), has been largely observed in MDS. Recently, we described the TTo analyse the relationship between TPeripheral blood and BM specimens, obtained at disease onset in a cohort of 58 subjects, were analysed by immune-fluorescence and flow cytometry, to preserve the complexity of the biological sample.We observed that a trend-increase of BM TThese data add T

An unresolved issue in autoimmunity is the lack of surrogate biomarkers of immunological self-tolerance for disease monitoring. Here, we show that peripheral frequency of a regulatory T cell population, characterized by the coexpression of CD3 and CD56 molecules (TR3-56), is reduced in individuals with new-onset type 1 diabetes (T1D). In three independent T1D cohorts, we find that low frequency of circulating TR3-56 cells is associated with reduced beta-cell function and with the presence of diabetic ketoacidosis. Since autoreactive CD8+ T cells mediate disruption of insulin-producing beta cells1–3, we demonstrate that TR3-56 cells can suppress CD8+ T cell functions in vitro by reducing the levels of intracellular reactive oxygen species. The suppressive function, phenotype and transcriptional signature of TR3-56 cells are also altered in children with T1D. Together, our findings indicate that TR3-56 cells constitute a regulatory cell population that controls CD8+ effector functions, whose peripheral frequency may represent a traceable biomarker for monitoring immunological self-tolerance in T1D. Low numbers of circulating CD3+CD56+ regulatory T cells are associated with reduced beta-cell function and diabetic ketoacidosis in three separate cohorts of children with type 1 diabetes.

Natural killer (NK) cells are indicated as favorite candidates for innovative therapeutic treatment and are divided into two subclasses: immature regulatory NK CD56bright and mature cytotoxic NK CD56dim. Therefore, the ability to discriminate CD56dim from CD56bright could be very useful because of their higher cytotoxicity. Nowadays, NK cell classification is routinely performed by cytometric analysis based on surface receptor expression. Here, we present an in-flow, label-free and non-invasive biophysical analysis of NK cells through a combination of light scattering and machine learning (ML) for NK cell subclass classification. In this respect, to identify relevant biophysical cell features, we stimulated NK cells with interleukine-15 inducing a subclass transition from CD56bright to CD56dim. We trained our ML algorithm with sorted NK cell subclasses (≥86% accuracy). Next, we applied our NK cell classification algorithm to cells stimulated over time, to investigate the transition of CD56bright to CD56dim and their biophysical feature changes. Finally, we tested our approach on several proband samples, highlighting the potential of our measurement approach. We show a label-free way for the robust identification of NK cell subclasses based on biophysical features, which can be applied in both cell biology and cell therapy.

An unresolved issue in autoimmunity is the lack of surrogate biomarkers of immunological self-tolerance for disease monitoring. Here, we show that peripheral frequency of a regulatory T cell population, characterized by the co-expression of CD3 and CD56 molecules (TR3-56), is reduced in subjects with new-onset type 1 diabetes (T1D). In three independent T1D cohorts, we find that low frequency of circulating TR3-56 cells is associated with reduced β-cell function and with the presence of diabetic ketoacidosis. As autoreactive CD8+ T cells mediate disruption of insulin-producing β-cells1–3, we demonstrate that TR3-56 cells can suppress CD8+ T cell functions in vitro by reducing levels of intracellular reactive oxygen species. The suppressive function, phenotype and transcriptional signature of TR3-56 cells are also altered in T1D children. Together, our findings indicate that TR3-56 cells constitute a regulatory cell population that controls CD8+ effector functions, whose peripheral frequency may represent a traceable biomarker for monitoring immunological self-tolerance in T1D.

This study aimed to investigate the effects of a weight loss program (WLP) on biochemical and immunological profile, and cardiovascular parameters in a cohort of dogs with naturally occurring obesity. Eleven obese dogs [body condition scoring (BCS), ≥7/9] were enrolled into the study and underwent clinical and cardiovascular examination, and blood testing before (T0) and after 6 months (T1) of WLP. Eleven normal weight (BCS, 4/5) healthy dogs were used as a control (CTR) group. Compared to the CTR group, at T0 obese dogs expressed higher serum leptin concentrations (p < 0.0005) that significantly decreased after weight loss (p < 0.005) but remained higher than the CTR group. Furthermore, obese dogs showed considerably lower levels (p < 0.0005) of regulatory T cell (Treg) compared to the CTR group, but they did not change after weight loss at T1. In obese dogs, tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentrations were substantially reduced at T1 (p < 0.0001 and p < 0.005). Regarding the cardiovascular parameters, only one obese dog was hypertensive at T0, and systolic blood pressure values showed no significant differences at the end of the WLP. The ratio of interventricular septal thickness in diastole to left ventricle internal diameter in diastole (IVSd/LVIDd) was significantly greater in obese dogs at T0 than in the CTR group (p < 0.005). It decreased after weight loss (p < 0.05). In obese dogs, troponin I level significantly reduced with weight loss (p < 0.05), while endothelin-1 level did not differ statistically. The results suggest that the immune dysregulation in the presence of high leptin levels and reduced number of Treg could affect obese dogs as well as humans. Based on our findings, we may speculate that a more complete immune-regulation restore could be obtained by a greater reduction in fat mass and a longer-term WLP. Finally, left ventricular remodeling may occur in some obese dogs. However, in canine species, further studies are needed to investigate the impact of obesity and related WLP on cardiovascular system.

CRISPR/Cas9 has become a powerful method to engineer genomes and to activate or to repress genes expression. As such, in cancer research CRISPR/Cas9 technology represents an efficient tool to dissect mechanisms of tumorigenesis and to discover novel targets for drug development. Here, we employed the CRISPR/Cas9 technology for studying the role of prostaglandin-endoperoxide synthase 2 (PTGS2) in melanoma development and progression. Melanoma is the most aggressive form of skin cancer with a median survival of less than 1 year. Although oncogene-targeted drugs and immune checkpoint inhibitors have demonstrated a significant success in improving overall survival in patients, related toxicity and emerging resistance are ongoing challenges. Gene therapy appears to be an appealing option to enhance the efficacy of currently available melanoma therapeutics leading to better patient prognosis. Several gene therapy targets have been identified and have proven to be effective against melanoma cells. Particularly, PTGS2 is frequently expressed in malignant melanomas and its expression significantly correlates with poor survival in patients. In this study we investigated on the effect of ptgs2 knockdown in B16F10 murine melanoma cells. Our results show that reduced expression of ptgs2 in melanoma cells: i) inhibits cell proliferation, migration, and invasiveness; ii) modulates immune response by impairing myeloid derived suppressor cell differentiation; iii) reduces tumor development and metastasis in vivo. Collectively, these findings indicate that ptgs2 could represent an ideal gene to be targeted to improve success rates in the development of new and highly selective drugs for melanoma treatment.

Over-nutrition and obesity have been associated with impaired immunity and low-grade inflammation in humans and mouse models. In this context, a causal role for unbalanced T regulatory cell (Treg)-dependent mechanisms has been largely suggested. Obesity is the most common nutritional disorder in dogs. However, it is not defined whether canine obesity may influence circulating Treg as well as if their number variation might be associated with the occurrence of systemic inflammation. The present study investigated the immune profile of healthy adult obese dogs belonging to the Labrador Retriever breed, in comparison with the normal weight counterpart. Indeed, obesity has been described as particularly evident in this dogs. With this purpose, 26 healthy dogs were enrolled and divided into two groups based on body condition score (BCS): controls (CTR: BCS 4–5) and obeses (OB: BCS ≥ 7). Our data indicate that adult obese Labrador Retrievers are characterised by the inverse correlation between leptin serum concentration and circulating Treg (CD4+CD25highFoxp3+) levels. In addition, an increased number of cytotoxic T cell effectors (CD3+CD8+) and a higher IFN-γ production by cytotoxic T lymphocytes were observed in OB group. These results may provide new insights into the immunological dysregulation frequently associated to obesity in humans and still undefined in dogs.

BACKGROUND AND PURPOSE: Myeloid‐derived suppressor cells (MDSCs) represent a major obstacle to cancer treatment, as they negatively regulate anti‐tumour immunity through the suppression of tumour‐specific T lymphocytes. Thus, the efficacy of immunotherapies may be improved by targeting MDSCs. In this study, we assessed the ability of hydrogen sulfide (H(2)S), a gasotransmitter whose anti‐cancer effects are well known, to inhibit the accumulation and immunosuppressive functions of MDSCs in melanoma. EXPERIMENTAL APPROACH: Effects of H(2)S on the host immune response to cancer were evaluated using an in vivo syngeneic model of murine melanoma. B16F10‐melanoma‐bearing mice were treated with the H(2)S donor, diallyl trisulfide (DATS) and analysed for content of MDSCs, dendritic cells (DCs) and T cells. Effects of H(2)S on expression of immunosuppressive genes in MDSCs and on T cell proliferation were evaluated. KEY RESULTS: In melanoma‐bearing mice, DATS inhibited tumour growth, and this effect was associated with a reduction in the frequency of MDSCs in the spleen, in the blood as well as in the tumour micro‐environment. In addition, we found that CD8(+) T cells and DCs were increased. Furthermore, DATS reduced the immuno‐suppressive activity of MDSCs, restoring T cell proliferation. CONCLUSIONS AND IMPLICATIONS: The H(2)S donor compound, DATS, inhibited the expansion and the suppressive functions of MDSCs, suggesting a novel role for H(2)S as a modulator of MDSCs in cancer. Therefore, H(2)S donors may provide a novel approach for enhancing the efficacy of melanoma immunotherapy. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc

The Tyrosine Kinase Receptor ErbB2 (HER2) when overexpressed in breast cancer (BC) is associated with poor prognosis. The monoclonal antibody Trastuzumab has become a standard treatment of ErbB2+BC. The antibody treatment has limited efficacy, often meets resistance and induces cardiotoxicity. T-cell recruiting bispecific antibody derivatives (TRBA) offer a more effective alternative to standard antibody therapy. We evaluated a panel of TRBAs targeting 3 different epitopes on the ErbB2 receptor either in a bivalent targeting tribody structure or as a monovalent scFv-fusion (BiTE format) for binding, cytotoxicity on Trastuzumab-resistant cell lines, and induction of cardiotoxicity. All three TRBAs bind with high affinity to the ErbB2 extracellular domain and a large panel of ErbB2-positive tumor cells. Tribodies had an increased in vitro cytotoxic potency as compared to BiTEs. It is interesting to note that, Tribodies targeting the epitopes on ErbB2 receptor domains I and II bind and activate lysis of mammary and gastric tumor cells more efficiently than a Tribody targeting the Trastuzumab epitope on domain IV. The first 2 are also active on Trastuzumab-resistant cancer cells lacking or masking the epitope recognized by Trastuzumab. None of the Tribodies studied showed significant toxicity on human cardiomyocytes. Altogether these results make these novel anti-ErbB2 bispecific Tribodies candidates for therapeutic development for treating ErbB2-positive Trastuzumab-resistant cancer patients.

Mechanical properties of living cells can be used as reliable markers of their state, such as the presence of a pathological state or their differentiation phase. The mechanical behavior of cells depends on the organization of their cytoskeletal network and the main contribution typically comes from the actomyosin contractile system, in both suspended and adherent cells. In the present study, we investigated the effect of a pharmaceutical formulation (OTC – Ossitetraciclina liquida 20%) used as antibiotic, on the mechanical properties of K562 cells by using the Micropipette Aspiration Technique (MAT). This formulation has been shown to increase in a time dependent way the inflammation and toxicity in terms of apoptosis in in vitro experiments on K562 and other types of cells. Here we show that by measuring the mechanical properties of cells exposed to OTC for different incubation times, it is possible to infer modifications induced by the formulation to the actomyosin contractile system. We emphasize that this system is involved in the first stages of the apoptotic process where an increase of the cortical tension leads to the formation of blebs. We discuss the possible relation between the observed mechanical behavior of cells aspirated inside a micropipette and apoptosis.

Antibiotics are widely used in zoo technical and veterinary practices as feed supplementation to ensure wellness of farmed animals and livestock. Several evidences have been suggesting both the toxic role for tetracyclines, particularly for oxytetracycline (OTC). This potential toxicity appears of great relevance for human nutrition and for domestic animals. This study aimed to extend the evaluation of such toxicity. The biologic impact of the drug was assessed by evaluating the proinflammatory effect of OTC and their bone residues on cytokine secretion by in vitro human peripheral blood lymphocytes. Our results showed that both OTC and OTC‐bone residues significantly induced the T lymphocyte and non‐T cell secretion of interferon (IFN)‐γ, as cytokine involved in inflammatory responses in humans as well as in animals. These results may suggest a possible implication for new potential human and animal health risks depending on the entry of tetracyclines in the food‐processing chain.

Background Oxytetracycline (OTC), which is largely employed in zootechnical and veterinary practices to ensure wellness of farmed animals, is partially absorbed within the gastrointestinal tract depositing in several tissues. Therefore, the potential OTC toxicity is relevant when considering the putative risk derived by the entry and accumulation of such drug in human and pet food chain supply. Despite scientific literature highlights several OTC-dependent toxic effects on human and animal health, the molecular mechanisms of such toxicity are still poorly understood. Methods Here, we evaluated DNA damages and epigenetic alterations by quantitative reverse transcription polymerase chain reaction, quantitative polymerase chain reaction, chromatin immuno-precipitation and Western blot analysis. Results We observed that human peripheral blood mononuclear cells (PBMCs) expressed DNA damage features (activation of ATM and p53, phosphorylation of H2AX and modifications of histone H3 methylation of lysine K4 in the chromatin) after the in vitro exposure to OTC. These changes are linked to a robust inflammatory response indicated by an increased expression of Interferon (IFN)-γ and type 1 superoxide dismutase (SOD1). Discussion Our data reveal an unexpected biological in vitro activity of OTC able to modify DNA and chromatin in cultured human PBMC. In this regard, OTC presence in foods of animal origin could represent a potential risk for both the human and animal health.

Simple Summary We evaluated the effects of a commercial nutraceutical supplementation in dogs naturally affected by Leishmania infantum. This nutraceutical supplement is proposed to be added to dog diet to foster physiological immune-response during chronic leishmaniasis. Parasite clearance is infrequently obtained by immune response in both the human and canine leishmaniasis. Pharmacological therapies frequently fail in the elimination of L. infantum that could have settled in deep organs, escaping immune responses and therapy. The conventional drug therapy improves clinical signs, reduces parasitemia as well as relapse of infection. This nutraceutical supplementation can improve the impaired immune response of the infected dogs to fight the disease. The use of nutraceuticals as immunomodulators in the treatment of visceral leishmaniasis has generated interest in the current approaches to treat the disease. In this clinical and immunological study, we investigated whether the administration of a nutritional supplement mediates the immune-modulatory response in canine leishmaniosis (CL) and improves the clinical outcome of the disease. With this purpose, we analysed T lymphocyte subsets in peripheral blood (PB) of 12 dogs naturally infected by Leishmania infantum, following treatment with a nutritional supplement. The regulatory T (Treg) cells and the T helper (Th) 1 population were specifically evaluated. The animals underwent complete clinical examination and blood sample collection for haematological, biochemical, serological and immunological analysis before treatment (T0), one month (T30) and 3 months (T90) after the onset of the nutraceutical supplementation. We observed that nutraceutical supplementation was associated with immunomodulation of Th1 response and significant clinical improvement of the animals. No side effects were observed. Therefore, a potential supportive role for the nutraceutical supplement during canine leishmaniasis is proposed.

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and possible evolution to acute leukemia. Occurrence of stem cell defects and of immune-mediated mechanisms was evidenced as relevant for pathophysiology of MDS. Here, we described one case of MDS patient carrying CD14(+) CD56(+) monocytes in bone marrow (BM), in the presence of a defective human leukocyte antigen (HLA)-E expression on peripheral blood (PB) cells and of natural killer (NK) cell expansion in PB and BM. The defective HLA-E expression and the NK expansion are proposed to be relevant for the pathogenesis of myelodysplasia in those patients showing CD14(+) CD56(+) monocytes in BM.

Chronic mucocutaneous candidiasis (CMCC) is a heterogeneous group of disorders characterized by non-invasive persistent Candida species infections of the skin, nails, and mucous membranes. Heterozygous dominant gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) have been described as causing impaired STAT1 dephosphorylation, diminished IL-17- producing T-cell numbers, and CMCC (1, 2). Here, we report on the case of a 17-year-old boy who presented to our Department for CMCC. He was born preterm (36 weeks) to healthy non-consanguineous parents from Italy, by a pregnancy complicated by threatened miscarriage and gestosis. This article is protected by copyright. All rights reserved.

Several extrinsic factors, like drugs and chemicals, can foster autoimmunity. Tetracyclines, in particular oxytetracycline (OTC), appear to correlate with the emergence of immune-mediated diseases. Accumulation of OTC, the elective drug for gastrointestinal and respiratory infectious disease treatment in broiler chickens, was reported in chicken edible tissues and could represent a potential risk for pets and humans that could assume this antibiotic as residue in meat or in meat-derived byproducts. We investigated thein vitroanti-inflammatory properties of a pool of thirteen botanicals as a part of a nutraceutical diet, with proven immunomodulatory activity.In addition, we evaluated the effect of such botanicals in contrasting thein vitroproinflammatory toxicity of OTC. Our results showed a significant reduction in interferon- (INF-)γproduction by human and canine lymphocytes in presence of botanicals (p⁎<0.05). Increased INF-γproduction, dependent on 24-hour OTC-incubation of T lymphocytes, was significantly reduced by the coincubation withHaematococcus pluvialis, withGlycine max, and with the mix of all botanicals (p⁎<0.05). In conclusion, the use of these botanicals was shown to be able to contrast OTC-toxicity and could represent a new approach for the development of functional foods useful to enhance the standard pharmacological treatment in infections as well as in preventing or reducing the emergence of inflammatory diseases.

Tetracyclines, which represent one of the most commonly used antibiotics for poultry, are known to be deposited in bones, where they can remain, despite the observation of appropriate withdrawal times. The aim of the study was to determine the concentration of oxytretracycline (OTC) residues in the bone and muscle of chickens, following the oral administration of a commercially available liquid formulation, and to test their cytotoxic effects on an in vitro cell culture model. Seventy-two 1-day-old broiler chickens were randomly allotted into 2 groups (control and treated animals). OTC (40 mg/kg BW) was administered via drinking water during the 1 to 5 and 20 to 25 days of life periods. At the end of the trial, the birds were slaughtered and the OTC residues in the target tissues were measured by means of liquid chromatography (LC) - tandem mass spectrometry (MS/MS). Cytotoxicity was assessed by evaluating the pro-apoptotic effect of the bone residues on the K562 erythroleukemic line and on the peripheral blood mononuclear cells (PBMC). In all the animals, the OTC residues in the muscle were far below the established MRL of 100 μg/kg. The OTC levels in the bones of the treated animals were instead found in the parts per million (ppm) range. Cell cytotoxicity was assessed by evaluating the pro-apoptotic effect of OTC bone residues on the haematopoietic cell system. This in vitro system has revealed a significant pro-apoptotic effect on both the K562 cell line and PBMC cultures. This result suggests potential human and animal health risks due to the entry of tetracycline residues contained in the bones of treated livestock into the food-chain. This could be of concern, particularly for canine and feline diets, as meat, bone meal, and poultry by-products represent some of the main ingredients of pet foods, especially in the case of dry pet food. Further studies are needed to define the underlying mechanisms of cytotoxicity and to evaluate the in vivo toxicological implications due to the observed in vitro effects.

Summary Intracellular metabolic pathways dependent upon the mammalian target of rapamycin (mTOR) play a key role in immune-tolerance control. In this study, we focused on long-term mTOR-dependent immune-modulating effects in kidney transplant recipients undergoing conversion from calcineurin inhibitors (CNI) to mTOR inhibitors (everolimus) in a 1-year follow-up. The conversion to everolimus is associated with a decrease of neutrophils and of CD8+ T cells. In addition, we observed a reduced production of interferon (IFN)-γ by CD8+ T cells and of interleukin (IL)-17 by CD4+ T lymphocytes. An increase in CD4+CD25+ forkhead box protein 3 (FoxP3)+ [regulatory T cell [(Treg)] numbers was also seen. Treg increase correlated with a higher proliferation rate of this regulatory subpopulation when compared with the CD4+FoxP3− effector counterpart. Basal phosphorylation level of S6 kinase, a major mTOR-dependent molecular target, was substantially maintained in patients treated with everolimus. Moreover, oscillations in serum concentration of everolimus were associated with changes in basal and activation-dependent S6 kinase phosphorylation of CD4+ and CD8+ T cells. Indeed, T cell receptor (TCR) triggering was observed to induce significantly higher S6 kinase phosphorylation in the presence of lower everolimus serum concentrations. These results unveil the complex mTOR-dependent immune-metabolic network leading to long-term immune-modulation and might have relevance for novel therapeutic settings in kidney transplants.

Background Clinical appearance and evolution of Canine Leishmaniosis (CL) are the consequence of complex interactions between the parasite and the genetic and immunological backgrounds. We investigated the effect of an immune-modulating diet in CL. Dogs were treated with anti- Leishmania pharmacological therapy combined with standard diet (SD Group) or with the immune-modulating diet (IMMD Group). CD3+ CD4+ Foxp3+ Regulatory T cells (Treg) and CD3+ CD4+ IFN-γ + T helper 1 (Th1) were analyzed by flow cytometry. Results All sick dogs showed low platelet number at diagnosis (T0). A platelet increase was observed after six months (T6) SD Group, with still remaining in the normal range at twelve months (T12). IMMD Group showed an increase in platelet number becoming similar to healthy dogs at T6 and T12. An increase of CD4/CD8 ratio was revealed in SD Group after three months (T3), while at T6 and at T12 the values resembled to T0. The increase in CD4/CD8 ratio at T3 was maintained at T6 and T12 in IMMD Group. A reduction in the percentage of Treg of all sick dogs was observed at T0. A recovery of Treg percentage was observed only at T3 in SD Group, while this effect disappeared at T6 and T12. In contrast, Treg percentage became similar to healthy animals in IMDD Group at T3, T6 and T12. Sick dogs showed an increase of Th1 cells at T0 as compared with healthy dogs. We observed the occurrence of a decrease of Th1 cells from T3 to T12 in SD Group, although a trend of increase was observed at T6 and T12. At variance, IMMD Group dogs showed a progressive decrease of Th1 cells, whose levels became similar to healthy controls at T6 and T12. Conclusion The immune-modulating diet appears to regulate the immune response in CL during the standard pharmacological treatment. The presence of nutraceuticals in the diet correlates with the decrease of Th1 cells and with the increase of Treg in sick dogs. Therefore, the administration of the specific dietary supplement improved the clinical response to the standard treatment in a model of CL.

Background: Helicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection. Results: In the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD50 lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae Always correlated with the induction of apoptosis in hemocytes. Conclusions: G. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients’ disease status.

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. Copper-zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the "non-T" cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors. © 2013 Elsevier B.V.

Canine leishmaniasis caused by the protozoan parasite Leishmania infantum is a chronic systemic disease endemic in Mediterranean basin. The aim of the study is to investigate the immune profile of dogs naturally infected by Leishmania infantum. In order to address such issue, CD4(+) and CD8(+) lymphocyte T cell subsets, peripheral CD4(+)CD3(+)Foxp3(+) (Treg) levels and the presence of pro-inflammatory T cells have been assessed, in 45 infected dogs and in 30 healthy animals, by using immunofluorescence and flow cytometry detection. Animals were categorised according to their clinical-pathological status and their antibody titer at diagnosis. Results showing a significant increase of CD8(+)CD3(+) T lymphocytes, a reduced percentage of the T regulatory CD4(+)CD3(+)Foxp3(+) subset and a significant increase of T(H)1 cells, characterise the infected dogs, regardless of their antibody titer or the occurrence of clinical symptomatic disease. These data may provide new insights into the pathogenesis of immune-mediated alterations associated with canine leishmaniasis.

Relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones in myelodysplastic syndromes (MDS) was suggested, but valuable or predictive criteria on this involvement are lacking. We previously reported that reduced T-regulatory cells (Treg) and high CD54 expression on T cell identify a sub-group of patients in whom an immune-pathogenesis might be inferred. Here, we suggest the occurrence of immune-selection of dysplastic clones in a subgroup of MDS patients, with reduced HLA-I and HLA-E on PMN, and propose that an altered immune profile might represent a valuable criterion to classify Low/Int-1 patients on the basis of immune-pathogenesis of MDS.

Paroxysmal Nocturnal Haemoglobinuria (PNH) is due to pathological expansion of a stem progenitor bearing a somatic mutation of PIG-A gene involved in the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor. Numerous data suggest a role for immune-mediated mechanisms in the selection/expansion of GPI-defective clone. Haemolytic anaemia in PNH is dependent on the effect of complement against GPI-defective red cells. Eculizumab, an anti-C5 monoclonal antibody, is dramatically effective in controlling haemolysis and thrombosis, in reducing fatigue and in improving quality of life of patients. However, this therapy presents new challenges that need to be properly faced. Here, we report the decrease in B, Natural Killer (NK) and regulatory T cells (Treg), an altered cytokine profile of invariant-NKT cells (NKTi) and the increasing of C-X-C chemokine receptor type 4 (CXCR4) receptor in PNH patients before the Eculizumab therapy. Treatment significantly affects some of these alterations: after Eculizumab, the number of B lymphocytes, the cytokine secretion of NKTi and CXCR4 expression on CD8 T cells became similar to healthy donors. No effects were observed on NK and Treg. The amplitude of the GPI-defective compartment remained unchanged.