Resolvin D1 enhances the resolution of lung inflammation caused by long-term Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa lung infection is a main cause of disability and mortality worldwide. Acute inflammation and its timely resolution are crucial for ensuring bacterial clearance and limiting tissue damage. Here, we investigated protective actions of resolvin (Rv) D1 in lung infection induced by the RP73 clinical strain of P. aeruginosa. RvD1 significantly diminished bacterial growth and neutrophil infiltration during acute pneumonia caused by RP73. Inoculum of RP73, immobilized in agar beads, resulted in persistent lung infection up to 21 days, leading to a non resolving inflammation reminiscent of human pathology. RvD1 significantly reduced bacterial titer, leukocyte infiltration, and lung tissue damage. In murine lung macrophages sorted during P. aeruginosa chronic infection, RvD1 regulated the expression of Toll-like receptors, downstream genes, and microRNA (miR)-21 and 155, resulting in reduced inflammatory signaling. In vitro, RvD1 enhanced phagocytosis of P. aeruginosa by neutrophils and macrophages, recapitulating its in vivo actions. These results unveil protective functions and mechanisms of action of RvD1 in acute and chronic P. aeruginosa pneumonia, providing evidence for its potent pro-resolution and tissue protective properties on airway mucosal tissue during infection.

Pathobiological implications of the d16HER2 splice variant for stemness and aggressiveness of HER2-positive breast cancer.

We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial-mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Low cells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene.

Human responses against HER-2-positive cancer cells in human immune system-engrafted mice.

BACKGROUND: Human immune system (HIS)-engrafted mice are new tools to investigate human immune responses. Here, we used HIS mice to study human immune responses against human HER-2-positive cancer cells and their ability to control tumour growth and metastasis. METHODS: BALB/c Rag2(-/-), Il2rg(-/-) mice were engrafted with CD34(+) or CD133(+) human cord blood hematopoietic stem cells (HSC) and vaccinated with human HER-2-positive cancer cells SK-OV-3 combined to human IL-12. RESULTS: Both CD34(+) or CD133(+) human HSC gave long-term engraftment and differentiation, both in peripheral blood and in lymphoid organs, and production of human antibodies. Vaccinated mice produced specific anti-HER-2 human IgG. An s.c. SK-OV-3 challenge was significantly inhibited (but not abolished) in both vaccinated and non-vaccinated HIS mice. Tumours were heavily infiltrated with human and murine cells, mice showed NK cells and production of human interferon-γ, that could contribute to tumour growth inhibition. Vaccinated HIS mice showed significantly inhibited lung metastases when compared with non-vaccinated HIS mice and to non-HIS mice, along with higher levels of tumour-infiltrating human dendritic cells. CONCLUSION: Anti-HER-2 responses were elicited through an adjuvanted allogeneic cancer cell vaccine in HIS mice. Human immune responses elicited in HIS mice effectively inhibited lung metastases.

Monomeric allergoid intragastric administration induces local and systemic tolerogenic response involving IL-10-producing CD4(+)CD25(+) T regulatory cells in mice.

The efficacy of sublingual immunotherapy, at present one of the treatments of choice for respiratory allergy, relies on the tolerance induced by oral mucosa-associated immune system; however, the gut-associated lymphoid tissue (GALT: Peyers patches and isolated lymphoid follicles) and mesenteric lymph nodes could also be involved, being stimulated by the ingested part of the allergen extract. The aim of the present study is to assess whether the exposure of the allergen exclusively to the GALT induces a tolerogenic response. For this purpose, mice were sensitized with ovalbumin or Par j 1 allergens. The corresponding gastric-resistant monomeric allergoids were then administered via orogastric gavage. After treatment, all mice were tested for: serum IgE, in vitro Th1 and Th2 cytokine release by allergen-stimulated peripheral blood lymphocytes, CD4(+)CD25(+) and CD4(+)CD25(+)IL-10(+) T cells in Peyers patches, mesenteric lymph nodes and spleen. Compared to the control, sensitized groups showed higher levels of serum IgE, lower frequency of CD4+CD25+IL-10+ T cells, at all sites, and higher amounts of in vitroreleased IL-4, IL-6 and TNF-alpha. Compared to the sensitized groups, higher frequency of CD4(+)CD25(+)IL-10(+) T cells was observed in the spleen of both Par-j 1 and OVA sensitized/treated groups and, only for ovalbumin-treated mice, in the Peyers patches and mesenteric lymph nodes, IgE and in vitro cytokines were significantly lower and equivalent to the control group. The results give the first evidence that the intragastric-restricted administration of gastric-resistant allergens restores local and peripheral tolerance in allergen-sensitized mice.

A multi-DNA preventive vaccine for p53/Neu-driven cancer syndrome.

The highly aggressive cancer syndrome of female mice carrying a p53 knockout allele and a rat HER-2/neu (Neu) transgene (BALB-p53Neu) can be prevented by a cell vaccine presenting three components: Neu, interleukin (IL)-12 production, and allogeneic major histocompatibility complex (MHC) alleles (Triplex cell vaccine). Here we tested a second-generation Triplex DNA-based vaccine (Tri-DNA), consisting of the combination of three gene components (a transmembrane-extracellular domain fragment of the Neu gene, IL-12 genes, and the H-2D(q) allogeneic MHC gene), carried by separate plasmids. The Tri-DNA vaccine was at least as effective as the Triplex cell vaccine for cancer immunoprevention, giving a similar delay in the onset of mammary cancer and complete protection from salivary cancer. Both vaccines induced anti-Neu antibodies of the murine IgG2a isotype at similar levels. The Tri-DNA vaccine gave more restricted immunostimulation, consisting of a fully helper T cell type 1 (Th1)-polarized response, with effective production of interferon (IFN)-gamma in response to the vaccine but no spontaneous production, and no induction of anti-Neu IgG3 antibodies. On the other hand, the Triplex cell vaccine induced both Th1 and Th2 cytokines, a strong increase in spontaneous IFN-gamma production, and high levels of IgG3 antibodies recognizing Neu-positive syngeneic cells. In conclusion, the Tri-DNA vaccine is as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Effects of castration on the development of prostate adenocarcinoma from its precursor HGPIN and on the occurrence of androgen-independent, poorly differentiated carcinoma in TRAMP mice.

Androgen ablation is thought to exert selective pressure for the development of androgen-independent forms of prostate cancer. This study was set up to investigate the effects of surgical castration on the development of prostate adenocarcinoma (ADC) from its precursor (high-grade prostate intraepithelial neoplasia (HGPIN)) and on the occurrence of androgen-independent, poorly differentiated carcinoma (PDC) in (C57Bl/6 transgenic adenocarcinoma of mouse prostate) TRAMP mice. It was found that castration cures HGPIN and ADC and prevents their further occurrence and growth, but has no effect on PDC. This indicates that in this model, PDC is not the progression of ADC favoured by androgen ablation and that its initiating cells are different from those of HGPIN and ADC.

A limited autoimmunity to p185neu elicited by DNA and allogeneic cell vaccine hampers the progression of preneoplastic lesions in HER-2/NEU transgenic mice.

Prevention of the progression of precancerous lesions by vaccines is virtually uncharted territory. Their potential, however, is being assessed in transgenic mice which develop autochthonous tumors with defined stages of progression. In this paper we show that the DNA micro-array technology significantly helps assessment of the preventive efficacy of a combined DNA and cell vaccine. All female rat Her-2/neu transgenic BALB/c (BALB-neuT) mice develop an invasive carcinoma in each of their mammary glands within 25 weeks of age. This is elicited by the activated transforming rat Her-2/neu oncogene embedded in their genome. We have previously shown that vaccination of mice bearing multiple in situ carcinomas with DNA plasmids which code for the extracellular and transmembrane domain of rat p185neu, the product of the rat Her-2/neu oncogene, followed by a boost with rat p185neu+ allogeneic cells engineered to secrete interferon-gamma, keeps 48% of mice tumor free until week 32. We have now extended our follow-up until mice reach one year of age and show that protection vanishes as time progresses. This observation suggests that the accuracy of the results studying immunotherapy against life-threatening tumors is a function of the length of the follow-up. The application of microarrays, and the concordance of morphologic and gene expression data led us to identify antibody as the main mechanism induced by vaccination. Protection is associated with a break of tolerance and a limited autoimmunity against the endogenous mouse p185neu.

Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells.

AIMS/HYPOTHESIS: Mutations in genes encoding HNF-4alpha, HNF-1alpha and IPF-1/Pdx-1 are associated with, respectively, MODY subtypes-1, -3 and -4. Impaired glucose-stimulated insulin secretion is the common primary defect of these monogenic forms of diabetes. A regulatory circuit between these three transcription factors has also been suggested. We aimed to explore how Pdx-1 regulates beta cell function and gene expression patterns. METHODS: We studied two previously established INS-1 stable cell lines permitting inducible expression of, respectively, Pdx-1 and its dominant-negative mutant. We used HPLC for insulin processing, adenovirally encoded aequorin for cytosolic [Ca2+], and transient transfection of human growth hormone or patch-clamp capacitance recordings to monitor exocytosis. RESULTS: Induction of DN-Pdx-1 resulted in defective glucose-stimulated and K+-depolarisation-induced insulin secretion in INS-1 cells, while overexpression of Pdx-1 had no effect. We found that DN-Pdx-1 caused down-regulation of fibroblast growth factor receptor 1 (FGFR1), and consequently prohormone convertases (PC-1/3 and -2). As a result, DN-Pdx-1 severely impaired proinsulin processing. In addition, induction of Pdx-1 suppressed the expression of glucagon-like peptide 1 receptor (GLP-1R), which resulted in marked reduction of both basal and GLP-1 agonist exendin-4-stimulated cellular cAMP levels. Induction of DN-Pdx-1 did not affect glucokinase activity, glycolysis, mitochondrial metabolism or ATP generation. The K+-induced cytosolic [Ca2+] rise and Ca2+-evoked exocytosis (membrane capacitance) were not abrogated. CONCLUSIONS/INTERPRETATION: The severely impaired proinsulin processing combined with decreased GLP-1R expression and cellular cAMP content, rather than metabolic defects or altered exocytosis, may contribute to the beta cell dysfunction induced by Pdx-1 deficiency.

The expression of LEC/CCL16, a powerful inflammatory chemokine, is upregulated in ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory disease of unknown aetiology and pathogenesis. The presence in the colonic mucosa of reactive cells expressing proinflammatory cytokines and chemokines is associated with high levels of IL-10, an anti-inflammatory cytokine. Our aim was to investigate the role of IL-10 and the beta chemokine LEC/CCL16 selectively up-regulated by IL-10 in inflammatory cell recruitment and cytokine and chemokine production during UC. We studied histologically, immunohistochemically and ultrastructurally colonic biopsies from 20 active UC patients and 10 control specimens taken far from any macroscopically detectable lesion in age and sex-matched patients with noninflammatory bowel disease. In active UC, immature dendritic cells (DCs) in the LP are associated with IL-10 in the T cell rich area. Furthermore, most of the LP-infiltrating macrophages strongly expressed LEC/CCL16, a chemokine upregulated by IL-10. To evaluate if LEC/CCL16 plays a role in the inflammatory reaction present in UC, we performed morphological studies in mice injected s.c. with syngeneic tumor cells engineered to produce LEC/CCL16. We found that the LEC protein locally released by LEC-gene-transfected tumor cells is a potent proinflammatory chemokine that induces the recruitment of a reactive infiltrate, and an angiogenic process mirroring that in human UC.

Desmopressin (DDAVP) induces NO production in human endothelial cells via V2 receptor- and cAMP-mediated signaling.

The hemostatic agent desmopressin (DDAVP) also has strong vasodilatory effects. DDAVP is a selective agonist for the vasopressin V2 receptor (V2R), which is coupled to cAMP-dependent signaling. DDAVP-induced vasodilation may be due to endothelial NO synthase (eNOS) activation. This hypothesis implies cAMP-mediated eNOS activation. It also implies wide extrarenal, endothelial V2R expression. We show that in human umbilical vein endothelial cells (HUVECs) the cAMP-raising agents forskolin and epinephrine increase NO production, as measured by a l-NMMA-inhibitable rise in cellular cGMP content. They also increase eNOS enzymatic activity, in a partly calcium-independent manner. cAMP-mediated eNOS activation is associated with phosphorylation of residue Ser1177, in a phosphatidyl inositol 3-kinase (PI3K)-independent manner. HUVECs do not express V2R. However, after heterologous V2R expression, DDAVP induces cAMP-dependent eNOS activation via Ser1177 phosphorylation. We have previously found V2R expression in cultured lung endothelial cells. By real time quantitative RT-PCR, we now find a wide V2R distribution notably in heart, lung and skeletal muscle. These results indicate that DDAVP and other cAMP-raising agents can activate eNOS via PI3K-independent Ser1177 phosphorylation in human endothelial cells. This mechanism most likely accounts for DDAVP-induced vasodilation.

Neutrophils in anti-cancer immunological strategies: old players in new games.

This review highlights the new "immunological identity" of neutrophils within the cytokine network and their role in biology of diseases, particularly in tumor biology. The latest preclinical evidence of their involvement in anti-cancer immunotherapeutic and prophylactic strategies will be discussed with particular reference to the real possibilities of transferring experimental results to a clinical setting.

Salivary carcinoma in HER-2/neu transgenic male mice: an angiogenic switch is not required for tumor onset and progression.

Morphologic examinations of salivary gland neoplasias arising in male BALB/c (H-2d) mice carrying the activated HER-2/neu (BALB-NeuT) indicate that expression of the oncogene product in the ductal-acinar structures results in a very human-like acinic cell adenocarcinoma with a smoldering course and infrequent metastatization. Typical and then atypical hyperplasia of ducts and acini preceded the rise of salivary tumors that originated from the confluence of multiple ductal hyperplastic foci, while hyperplastic acini behaved as an abortive preneoplastic lesion. The vascular network in normal, hyperplastic and neoplastic salivary tissue was analysed to see whether activation of the angiogenic process is essential in salivary gland carcinogenesis. Immunostaining with anti-endothelial cells (anti-CD31), anti-beta3 integrin and anti-laminin antibodies revealed that microvessel density was significantly higher in normal and hyperplastic than in neoplastic tissue, in which no signs of new vessel sprouting were found. Assessment of angiogenic factor expression indicates a low presence of VEGF in normal, hyperplastic and neoplastic epithelium, while bFGF was preferentially produced but not exported by neoplastic cells and remained in a cell-associated form. Our data suggest that normal salivary gland vascularization is able to support tumor onset and development with no need for an angiogenic switch.

The Rab3-interacting molecule RIM is expressed in pancreatic beta-cells and is implicated in insulin exocytosis.

The putative Rab3 effector RIM (Rab3-interacting molecule) was detected by Northern blotting, RT-PCR and Western blotting in native pancreatic beta-cells as well as in the derived cell lines INS-1E and HIT-T15. RIM was localized on the plasma membrane of INS-1E cells and beta-cells. An involvement of RIM in insulin exocytosis was indicated by transfection experiments of INS-1E cells with the Rab3 binding domain of RIM. This domain enhanced glucose-stimulated secretion in intact cells and Ca(2+)-stimulated exocytosis in permeabilized cells. Co-expression of Rab3A reversed the effect of RIM on exocytosis. These results suggest an implication of RIM in the control of insulin secretion.

Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells.

Insulin-secreting cells express four GTPases of the Rab3 family. After separation of extracts of INS-1 cells on a sucrose density gradient, the bulk of the A, B, and C isoforms was recovered in the fractions enriched in insulin-containing secretory granules. Rab3D was also mainly associated with secretory granules, but a fraction of this isoform was localized on lighter organelles. Analyses by confocal microscopy of immunostained HIT-T15 cells transfected with epitope-tagged constructs confirmed the distribution of the Rab3 isoforms. Transfection of HIT-T15 cells with GTPase-deficient mutants of the Rab3 isoforms decreased nutrient-induced insulin release to different degrees (D>B>A>>C), while overexpression of Rab3 wild types had minor or no effects. Expression of the same Rab3 mutants in PC12 cells provoked an inhibition of K+-stimulated secretion of dense core vesicles, indicating that, in beta-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxyterminal domain of A was replaced with the corresponding region of C inhibited insulin secretion as Rab3A. In contrast, a Rab3C/A chimera containing the amino-terminal domain of C was less potent and reduced exocytosis as Rab3C. This suggests that the degree of inhibition obtained after transfection of the Rab3 isoforms is determined by differences in the variable amino-terminal region.

Expression, localization and functional role of small GTPases of the Rab3 family in insulin-secreting cells.

We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulin-secreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in beta-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunofluorescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decreased by the overexpression of wild-type Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human beta-cells, a different Rab3 isoform but with homologous function may replace Rab3A.

Intracranial pressure dynamics in patients with acute brain damage: a critical analysis with the aid of a mathematical model.

The time pattern of intracranial pressure (ICP) in response to typical clinical tests (i.e., bolus injection and bolus withdrawal of 1 to 4 mL of saline in the craniospinal space) was studied in 18 patients with acute brain damage by means of a mathematical model. The model includes the main biomechanical factors assumed to affect intracranial pressure, particularly cerebrospinal fluid (CSF) dynamics, intracranial compliance, and cerebral hemodynamics. Best fitting between model simulation curves and clinical tracings was achieved using the Powell minimization algorithm and a least-square criterion function. The simulation results demonstrate that, in most patients, the ICP time pattern cannot be explained merely on the basis of CSF dynamics but also requires consideration of the contribution of cerebral hemodynamics and blood volume alterations. In particular, only in a few patients (about 40% of total) the ICP monotonically returns toward baseline following the clinical maneuver. In most of the examined cases (about 60%), ICP exhibits an anomalous response to the same maneuver, characterized by a delayed increase after bolus injection and a delayed decrease after withdrawal. The model is able to explain these responses, imputing them to active intracranial blood volume changes induced by mechanisms controlling cerebral blood flow. Finally, the role of the main intracranial biomechanical parameters in the genesis of the ICP time pattern is discussed and a comparison with previous theoretical studies performed.

Effect of acute and chronic cobalt administration on carotid body chemoreceptors responses.

Chronic cobalt exposure leads to release and production of erythropoietin and consequently to polycythemia. Accordingly, cellular elements sensitive to oxygen in the carotid body, would manifest responses during acute and chronic cobalt administration. The carotid body, detects gas changes (PO2, PCO2/pH) in the arterial blood and regulates ventilation and circulation by the afferent nerve discharge. We hypothesized that cobalt interacts with an oxygen sensitive mechanism in the carotid chemoreception and in erythropoietin producing cells. Twelve cats were anesthetized, paralysed and artificially ventilated; few fiber preparation of carotid sinus nerve were recorded during close intraarterial injection of cobalt. In another protocol, 12 rats received an intraperitoneal dose of CoCl2 (10 mg/kg) daily for 6 weeks. At the end, the carotid body was fixed in situ by superfusion. Ultrastructural and morphometric studies were made. Acute administration (0.08-2.3 mumol) promptly stimulated the chemoreceptor afferents. Type I cells increased significantly along with erythropoiesis in the chronic cobalt treated rats. The stimulatory effects of cobalt on the carotid body chemoreceptor showed that sensitive mechanisms in the kidney and in the carotid body are similar, and cobalt interacts with the physiological responses of oxygen.