Macrophage-Mediated Melanoma Reduction after HP-NAP Treatment in a Zebrafish Xenograft Model.

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.

The lipoprotein HP1454 of Helicobacter pylori regulates T-cell response by shaping T-cell receptor signalling.

Helicobacter pylori (HP) is a Gram-negative bacterium that chronically infects the stomach of more than 50% of human population and represents a major cause of gastric cancer, gastric lymphoma, gastric autoimmunity, and peptic ulcer. It still remains to be elucidated, which HP virulence factors are important in the development of gastric disorders. Here, we analysed the role of the HP protein HP1454 in the host-pathogen interaction. We found that a significant proportion of T cells isolated from HP patients with chronic gastritis and gastric adenocarcinoma proliferated in response to HP1454. Moreover, we demonstrated in vivo that HP1454 protein drives Th1/Th17 inflammatory responses. We further analysed the in vitro response of human T cells exposed either to an HP wild-type strain or to a strain with a deletion of the hp1454 gene, and we revealed that HP1454 triggers the T-cell antigen receptor-dependent signalling and lymphocyte proliferation, as well as the CXCL12-dependent cell adhesion and migration. Our study findings prove that HP1454 is a crucial bacterial factor that exerts its proinflammatory activity by directly modulating the T-cell response. The relevance of these results can be appreciated by considering that compelling evidence suggest that chronic gastric inflammation, a condition that paves the way to HP-associated diseases, is dependent on T cells.

Helicobacter pylori antigenic Lpp20 is a structural homologue of Tipα and promotes epithelial-mesenchymal transition.

BACKGROUND: Helicobacter pylori is a bacterium that affects about 50% of the world population and, despite being often asymptomatic, it is responsible of several gastric diseases, from gastritis to gastric cancer. The protein Lpp20 (HP1456) plays an important role in bacterium survival and host colonization, but the possibility that it might be involved in the etiology of H. pylori-related disorders is an unexplored issue. Lpp20 is a lipoprotein bound to the external membrane of the bacterium, but it is also secreted inside vesicles along with other two proteins of the same operon, i.e. HP1454 and HP1457. RESULTS: In this study we determined the crystal structure of Lpp20 and we found that it has a fold similar to a carcinogenic factor released by H. pylori, namely Tipα. We demonstrate that Lpp20 promotes cell migration and E-cadherin down-regulation in gastric cancer cells, two events recalling the epithelial-mesenchymal transition (EMT) process. Differently from Tipα, Lpp20 also stimulates cell proliferation. CONCLUSIONS: This identifies Lpp20 as a new pathogenic factor produced by H. pylori that promotes EMT and thereby the progression of cancer to the metastatic state.

Toll-like receptors hit calcium.

Mitochondrial Ca(2+) uptake is a multifarious signal that controls both the activity of matrix dehydrogenases and the sensitivity to apoptotic and necrotic challenges. Recent evidence indicates that mitochondria also play a role in triggering inflammation, as mitochondrial DNA, when released by the cell, is an important damage-associated molecular pattern (DAMP). Now, Toll-like receptors (TLRs) are shown to close the loop, by affecting in turn mitochondrial activity. Two studies by Shintani and colleagues, one in this issue of EMBO reports, identify a new TLR transduction mechanism that impinges directly on mitochondrial function. Upon binding of CpG oligodeoxynucleotides, TLR9--which in non-immune cells is retained in the ER--inhibits SERCA2, thus reducing Ca(2+) transfer to the mitochondria and aerobic metabolism.

Cytokine BAFF released by Helicobacter pylori-infected macrophages triggers the Th17 response in human chronic gastritis.

BAFF is a crucial cytokine that affects the activity of both innate and adaptive immune cells. It promotes the expansion of Th17 cells in autoimmune disorders. With this study, we investigated the BAFF/Th17 responses in Helicobacter pylori-induced gastritis in humans. Our results show that the mucosa from Helicobacter(+) patients with chronic gastritis is enriched in IL-17 and BAFF, whereas the two cytokines are weakly expressed in Helicobacter(-) patients with chronic gastritis; moreover, the expression of both BAFF and IL-17 decreases after bacteria eradication. We demonstrate that BAFF accumulates in macrophages in vivo and that it is produced by monocyte-derived macrophages in vitro, after Helicobacter stimulation. Application of BAFF on monocytes triggers the accumulation of reactive oxygen species that are crucial for the release of pro-Th17 cytokines, such as IL-23, IL-1ß, and TGF-ß. Moreover, BAFF directly promotes the differentiation of Th17 cells. In conclusion, our results support the notion that an axis BAFF/Th17 exists in chronic gastritis of Helicobacter(+) patients and that its presence strictly depends on the bacterium. Moreover, we demonstrated that BAFF is able to drive Th17 responses both indirectly, by creating a pro-Th17 cytokine milieu through the involvement of innate immune cells, and directly, via the differentiation of T cells toward the specific profile. The results obtained in this study are of great interest for Helicobacter-related diseases and the development of novel therapeutic strategies based on the inhibition of the BAFF/IL-17 response.

The C2 fragment from Neisseria meningitidis antigen NHBA increases endothelial permeability by destabilizing adherens junctions.

Neisseria meningitidis is a human pathogen that can cause fatal sepsis and meningitis once it reaches the blood stream and the nervous system. Here we demonstrate that a fragment, released upon proteolysis of the surface-exposed protein Neisserial Heparin Binding Antigen (NHBA), by the bacterial protease NalP, alters the endothelial permeability by inducing the internalization of the adherens junction protein VE-cadherin. We found that C2 rapidly accumulates in mitochondria where it induces the production of reactive oxygen species: the latter are required for the phosphorylation of the junctional protein and for its internalization that, in turn, is responsible for the endothelial leakage. Our data support the notion that the NHBA-derived fragment C2 might contribute to the extensive vascular leakage typically associated with meningococcal sepsis.

Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis.

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

Pathogenesis of Helicobacter pylori infection.

Helicobacter pylori relies on multiple colonization and virulence factors to persist in the human stomach for life. In addition, these factors can be modulated and vary to suit the ever-changing environment within the host individual. This article outlines the novel developments in this field of research during the past year, highlighting the cag pathogenicity island, VacA, γ-glutamyl-transpeptidase as well as including recent advances in protein structure, bacteria-host interaction, and the role of stomach microbiota.

Orchestration of inflammation and adaptive immunity in Borrelia burgdorferi-induced arthritis by neutrophil-activating protein A.

OBJECTIVE: Lyme arthritis (LA) is characterized by infiltration of inflammatory cells, mainly neutrophils (polymorphonuclear cells [PMNs]) and T cells, into the joints. This study was undertaken to evaluate the role of the neutrophil-activating protein A (NapA) of Borrelia burgdorferi in eliciting inflammation and in driving the adaptive immune response. METHODS: Levels of NapA, interferon-γ (IFNγ), interleukin-17 (IL-17), and T cell-attracting chemokines were assessed by enzyme-linked immunosorbent assay in synovial fluid from patients with LA. The profile of T cells recruited into the synovia of patients with LA was defined by fluorescence-activated cell sorting analysis. NapA was intraarticularly injected into rat knees, and the cells recruited in synovia were characterized. The role of NapA in recruiting immune cells was confirmed by chemotaxis assays using a Transwell system. RESULTS: NapA, IFNγ, IL-17, CCL2, CCL20, and CXCL10 accumulated in synovial fluid from patients with LA. Accordingly, T cells obtained from these patients produced IFNγ or IL-17, but notably, some produced both cytokines. NapA promoted neutrophil and T lymphocyte recruitment both in vitro and in vivo. Interestingly, the infiltration of T cells not only resulted from the chemotactic activity of NapA but also relied on the chemokines produced by PMNs exposed to NapA. CONCLUSION: We provide evidence that NapA functions as one of the main bacterial products involved in the pathogenesis of LA. Accordingly, we show that, at very early stages of LA, NapA accumulates and, in turn, orchestrates the recruitment of inflammatory cells into the joint cavity. Thereafter, with the contribution of recruited cells, NapA promotes the infiltration of T cells producing IL-17 and/or IFNγ.

Sunflower seed-derived bioactive peptides show antioxidant and anti-inflammatory activity: From in silico simulation to the animal model.

The evolving field of food technology is increasingly dedicated to developing functional foods. This study explored bioactive peptides from sunflower protein isolate (SPI), obtained from defatted flour, a by-product of the oil processing industry. SPI underwent simulated gastrointestinal digestion and the obtained peptide-enriched fraction (PEF) showed antioxidant properties in vivo, in zebrafish. Among the peptides present in PEF identified by mass spectrometry analysis, we selected those with antioxidant properties by in silico evaluation, considering their capability to interact with Keap1, key protein in the regulation of antioxidant response. The selected peptides were synthesized and evaluated in a cellular model. As a result, DVAMPVPK, VETGVIKPG, TTHTNPPPEAE, LTHPQHQQQGPSTG and PADVTPEEKPEV activated Keap1/Nrf2 pathway leading to Antioxidant Response Element-regulated enzymes upregulation. Since the crosstalk between Nrf2 and NF-κB is well known, the potential anti-inflammatory activity of the peptides was assessed and principally PADVTPEEKPEV showed good features both as antioxidant and anti-inflammatory molecule.

Tumor-associated macrophages as major source of APRIL in gastric MALT lymphoma.

Lymphoid hyperplasia of gastric mucosa associated with Helicobacter pylori (HP) infection represents a preneoplastic condition of the mucosa associated lymphoid tissue (MALT), which may evolve to a B-cell lymphoma. While it is well established that the initial neoplastic proliferation of B cells is antigen-driven and dependent on the helper activity of HP-specific T cells, it needs to be elucidated which cytokine or soluble factor(s) promote B-cell activation and lymphomagenesis. Herein, we originally report that gastric MALT lymphoma express high levels of a proliferation inducing ligand (APRIL), a novel cytokine crucial in sustaining B-cell proliferation. By immunohistochemistry, we demonstrate that APRIL is produced almost exclusively by gastric lymphoma-infiltrating macrophages located in close proximity to neoplastic B cells. We also show that macrophages produce APRIL on direct stimulation with both HP and HP-specific T cells. Collectively, our results represent the first evidence for an involvement of APRIL in gastric MALT lymphoma development in HP-infected patients.

TpF1 from Treponema pallidum activates inflammasome and promotes the development of regulatory T cells.

Human syphilis is a multistage disease, with diverse and wide-ranging manifestations caused by Treponema pallidum. Despite the fact that a cell-mediated immune response takes part in the course of syphilis, T. pallidum often manages to evade host immunity and, in untreated individuals, may trigger chronic infection. With this study, we demonstrate for the first time, to our knowledge, that Treponema pallidum induces a regulatory T (Treg) response in patients with secondary syphilis and we found that the miniferritin TpF1, produced by the bacterium, is able to expand this response and promote the production of TGF-ß. Accordingly, TpF1 stimulates monocytes to release IL-10 and TGF-ß, the key cytokines in driving Treg cell differentiation. Interestingly, we also found that TpF1 stimulates monocytes to synthesize and release several proinflammatory cytokines, such as TNF-α, IL-6, and IL-1ß, the latter following the activation of the multiprotein complex inflammasome. Collectively, these data strongly support a central role for TpF1 both in the inflammation process, which occurs in particular during the early stage of syphilis, and in the long-term persistence of the spirochete within the host by promoting Treg response and TGF-ß production.

Increased IL-17A Serum Levels and Gastric Th17 Cells in Helicobacter pylori-Infected Patients with Gastric Premalignant Lesions.

Background:Helicobacter pylori infection is characterized by an inflammatory infiltrate that might be an important antecedent of gastric cancer. The purpose of this study was to evaluate whether interleukin (IL)-17 inflammation is elicited by gastric T cells in Helicobacter pylori patients with gastric intestinal metaplasia and dysplasia (IM/DYS). We also investigated the serum IL-17A levels in Helicobacter pylori patients with gastric intestinal metaplasia and dysplasia, and patients with Helicobacter pylori non-atrophic gastritis (NAG). Methods: the IL-17 cytokine profile of gastric T cells was investigated in six patients with IM/DYS and Helicobacter pylori infection. Serum IL-17A levels were measured in 45 Helicobacter pylori-infected IM/DYS patients, 45 Helicobacter pylori-infected patients without IM/DYS and in 45 healthy controls (HC). Results: gastric T cells from all IM/DYS patients with Helicobacter pylori were able to proliferate in response to Helicobacter pylori and to produce IL-17A. The Luminex analysis revealed that IL-17A levels were significantly increased in Helicobacter pylori IM/DYS patients compared to healthy controls and to Helicobacter pylori gastritis patients without IM/DYS (452.34 ± 369.13 pg/mL, 246.82 ± 156.06 pg/mL, 169.26 ± 73.82 pg/mL, respectively; p < 0.01, p < 0.05). Conclusions: the results obtained indicate that Helicobacter pylori is able to drive gastric IL-17 inflammation in IM/DYS Helicobacter pylori-infected patients, and that IL-17A serum levels are significantly increased in Helicobacter pylori-infected patients with IM/DYS.

HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response.

Intravesical Bacillus Calmette-Guérin (BCG) is the gold standard treatment for intermediate and high-risk non-muscle-invasive bladder cancer. BCG therapy is the most successful example of immunotherapy in cancer. Unfortunately, the treatment-related side effects are still relevant. Furthermore, non-responder patients are candidate to radical cystectomy in the absence of valuable alternative options. These aspects have prompted the search for newer biological response modifiers (BRM) with a better benefit/side effects ratio. The toll-like receptor (TLR) 2 ligand, Helicobacter pylori protein HP-NAP, has been shown to deserve a potential role as BRM. HP-NAP is capable of driving the differentiation of T helper (Th) 1 cells, both in vitro and in vivo, because of its ability to create an IL-12-enriched milieu. Herein, we report that local administration of HP-NAP decreases tumour growth by triggering tumour necrosis in a mouse model of bladder cancer implant. The effect is accompanied by a significant accumulation of both CD4+ and CD8+ IFN-γ-secreting cells, within tumour and regional lymph nodes. Noteworthy, HP-NAP-treated tumours show also a reduced vascularization due to the anti-angiogenic activity of IFN-γ induced by HP-NAP. Our findings strongly indicate that HP-NAP might become a novel therapeutic "bullet" for the cure of bladder tumours.

HP-NAP of Helicobacter pylori: The Power of the Immunomodulation.

The miniferritin HP-NAP of Helicobacter pylori was originally described as a neutrophil-activating protein because of the capacity to activate neutrophils to generate oxygen radicals and adhere to endothelia. Currently, the main feature for which HP-NAP is known is the ability to promote Th1 responses and revert the immune suppressive profile of macrophages. In this review, we discuss the immune modulating properties of the protein regarding the H. pylori infection and the evidence that support the potential clinical application of HP-NAP in allergy and cancer immunotherapy.

Promoting the preservation of strawberry by supercritical CO2 drying.

This work aimed to investigate the supercritical CO2 (ScCO2) drying of strawberries and its effect on enzymatic, chemical and microbial stability. Process conditions influenced the final weight loss (WL), water activity (aw) and the inactivation of polyphenol oxidase (PPO) and peroxidase (POD). At 40 °C, an efficient drying (WL > 92 %, aw < 0.34) and a complete enzymatic (POD and PPO activity) inactivation can be achieved using several combinations of pressure, time and flow rate. ScCO2 dried strawberry at 40 °C, 13.3 MPa, 7 h and 19 kg/h flow rate maintain the total content of Vitamin C (358.5 mg/100 g), 95 % of total anthocyanin (61.68 mg/100 g) and 76 % of total flavonoids (25.85 mg/100 g) in comparison with fresh samples. Foodborne pathogens (E.coli O157:H7, Salmonella enterica and Listeria monocytogenes) inoculated at high concentration (≥6 log CFU/g) were undetected after the process. Overall results are promising for the development of a novel low temperature drying process for the production of healthy and safe snack.

The antigen CD300e drives T cell inflammation in adipose tissue and elicits an antibody response predictive of the insulin sensitivity recovery in obese patients.

Obesity and insulin resistance (IR), the key features of metabolic syndrome, are closely associated with a state of chronic, low-grade inflammation. Bariatric surgery leads to a considerable reduction in the adipose tissue mass and systemic inflammation along with a reduction of IR, with a whole-body metabolic improvement. However, a sizable portion of people experience an IR relapse within few years of remission.Numerous studies have attempted to explore the best clinical predictors of the improvement of insulin sensitivity and the maintenance of glucose homeostasis after bariatric surgery, but no simple fasting blood test has been found to be effective in predicting the short and long-term beneficial effects on glycaemia.With the present study, we investigated T-cell and antibody responses against CD300e, an antigen highly expressed in the adipose tissue of patients with obesity before the bariatric surgery-induced weight loss. We found both in fat tissue and in peripheral blood anti-CD300e-specific T helper 1 responses. Moreover, we evidenced in the sera of individuals with obesity an antibody response towards CD300e and revealed the existence of a significant correlation between the level of antibodies before surgery and the maintenance of glucose control after the intervention.

Structure and immunomodulatory property relationship in NapA of Borrelia burgdorferi.

NapA from Borrelia burgdorferi is a member of the Dps-like protein family with specific immunomodulatory properties; in particular, NapA is able to induce the expression of IL-23 in neutrophils and monocytes, as well as the expression of IL-6, IL-1ß, and transforming growth factor beta (TGF-ß) in monocytes, via Toll-like receptor (TLR) 2. Such an activity on innate immune cells triggers a synovial fluid Th17 response. Here we report the crystal structure of NapA, determined at 2.6Å resolution, which shows that the quaternary structure of the protein is that of a dodecamer with 23 symmetry, typical of the proteins of the family. We also demonstrate that the N- and C-terminal tails, which are flexible and not visible in the crystal, are not relevant for its pro-Th17 activity. Based on the crystal structure and on the comparison with the structure of the orthologous protein from Helicobacter pylori, HP-NAP, we hypothesize that the charge distributions on the two proteins' surfaces are responsible for the interaction with TLR2 and for the different behaviors in modulating the immune response.

MicroRNA expression profiling in human Barrett's carcinogenesis.

Barrett's esophagus (BE) is characterized by the native stratified squamous epithelium (N) lining the esophagus being replaced by a columnar epithelium with intestinal differentiation (Barrett's mucosa; BM). BM is considered as the main risk factor for esophageal adenocarcinoma (Barrett's adenocarcinoma; BAc). MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting messenger RNAs and they are reportedly dysregulated in BM. To test the hypothesis that a specific miRNA expression signature characterizes BM development and progression, we performed miRNA microarray analysis comparing native esophageal mucosa with all the phenotypic lesions seen in the Barrett's carcinogenic process. Specimens were collected from 14 BE patients who had undergone esophagectomy, including: 14 with N, 14 with BM, 7 with low-grade intraepithelial neoplasia, 5 with high-grade intra-epithelial neoplasia and 11 with BAc. Microarray findings were further validated by quantitive real-time polymerase chain reaction and in situ hybridization analyses using a different series of consecutive cases (162 biopsy samples and 5 esophagectomies) of histologically proven, long-segment BE. We identified a miRNA signature of Barrett's carcinogenesis consisting of an increased expression of 6 miRNAs and a reduced expression of 7 miRNAs. To further support these results, we investigated target gene expression using the Oncomine database and/or immunohistochemical analysis. We found that target gene expression correlated significantly with miRNA dysregulation. Specific miRNAs are directly involved in BE progression to cancer. miRNA profiling significantly expands current knowledge on the molecular history of Barrett's carcinogenesis, also identifying molecular markers of cancer progression.

Helicobacter pylori-derived neutrophil-activating protein increases the lifespan of monocytes and neutrophils.

An invariable feature of Helicobacter pylori-infected gastric mucosa is the persistent infiltration of inflammatory cells. The neutrophil-activating protein (HP-NAP) has a pivotal role in triggering and orchestrating the phlogistic process associated with H. pylori infection. Aim of this study was to address whether HP-NAP might further contribute to the inflammation by increasing the lifespan of inflammatory cells. We report that HP-NAP is able to prolong the lifespan of monocytes, in parallel with the induction of the anti-apoptotic proteins A1, Mcl-1, Bcl-2 and Bcl-X(L). This effect does not result from a direct action on the apoptotic machinery, but rather it requires the release of endogenous pro-survival factors, such as interleukin-1beta, which probably acts in synergy with other unidentified mediators. We also report that HP-NAP promotes the survival of Ficoll-purified neutrophils in a monocyte-dependent fashion: indeed, mononuclear cell depletion of Ficoll-purified neutrophils completely abolished the pro-survival effect by HP-NAP. In conclusion, our data reinforce the notion that HP-NAP has a pivotal role in sustaining a prolonged activation of myeloid cells.