Applicability evaluation of draft test protocol for functional inspection of solid fumigant applicators.

The applicability of draft test protocol for functional inspection of solid fumigant applicators has been checked. Two widespread machine models, normally used in horticultural farm of Liguria Region (north-west Italy) for broadcast soil fumigation were tested. Pre-inspection phase does not show specific problems; also the functional inspection phase, both for visual check operation -presence or not of some devices on the machines- and for test of they function if present, does not show specific hurdles. In particular the test of distribution uniformity, performed using rubber granules as blank test, give some indications on how to modify the current test protocol draft. The modification consist in change the admissible threshold; the maximum deviation of each distribution pipe, relative to the mean value, have to be varied from 10% (current draft) to 15%. Furthermore, it is suggested an alternate method to assess the distribution uniformity of rubber granules; this method considers the use of horizontal patternator fitted by grooves similar to that used to check the distribution uniformity of boom sprayers. The proposed method will facilitate the coefficient of variation (CV) calculation.The applicability of draft test protocol for functional inspection of solid fumigant applicators has been checked. Two widespread machine models, normally used in horticultural farm of Liguria Region (north-west Italy) for broadcast soil fumigation were tested. Pre-inspection phase does not show specific problems; also the functional inspection phase, both for visual check operation -presence or not of some devices on the machines- and for test of they function if present, does not show specific hurdles. In particular the test of distribution uniformity, performed using rubber granules as blank test, give some indications on how to modify the current test protocol draft. The modification consist in change the admissible threshold; the maximum deviation of each distribution pipe, relative to the mean value, have to be varied from 10% (current draft) to 15%. Furthermore, it is suggested an alternate method to assess the distribution uniformity of rubber granules; this method considers the use of horizontal patternator fitted by grooves similar to that used to check the distribution uniformity of boom sprayers. The proposed method will facilitate the coefficient of variation (CV) calculation

Modulation of DNA repair genes induced by TLR9 agonists: A strategy to eliminate “altered” cells?

We provided evidence that the TLR9 engagement of innate immune cells present in the tumor microenvironment by CpG-oligodeoxynucleotide (CpG-ODN) induces down-modulation of DNA repair gene expression in tumor cells, sensitizing cancer cells to DNA-damaging chemotherapy. These findings expand the benefits of CpG-ODN therapy beyond induction of a strong immune response

Critical Role of TLR9 in Acute Graft-versus-Host Disease

Abstract Graft-vs-host disease (GVHD) is a major complication after allogeneic bone marrow transplantation. Different studies have demonstrated that intestinal bacterial breakdown products and loss of gastrointestinal tract integrity, both induced by conditioning regiments, are critical in the pathogenesis of acute GVHD. Using C57BL/6 knockout mice, we evaluated the role of TLR4 and TLR9, which recognize bacterial LPS and DNA, respectively, in the GVHD associated with allogeneic bone marrow transplantation. When myeloablative-irradiated TLR9 knockout (TLR9−/−) mice were used as graft recipients, survival and clinical score of acute GVHD were improved as compared with the wild-type recipient mice (18/30 vs 1/31 mice still alive at day 70 in a total of four experiments); while no differences were observed using recipient TLR4 knockout (TLR4−/−) mice. The reduced mortality and morbidity in TLR9−/− mice related with reduced stimulatory activity of TLR9−/− spleen APCs after conditioning and reduced proliferation of allogeneic donor T cells. Experiments using TLR9+/+ into TLR9−/− and TLR9−/− into TLR9+/+ chimeric mice as recipients indicated a critical role for nonhematopoietic TLR9+/+ cells interacting with bacterial breakdown products released in myeloablated mice. Altogether these data reveal a novel important role of TLR9 in GVHD, a finding that might provide tools to reduce this complication of allogeneic transplantation

Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition

Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activit

Activation of Enteroendocrine Cells via TLRs Induces Hormone, Chemokine, and Defensin Secretion

Abstract Enteroendocrine cells are known primarily for their production of hormones that affect digestion, but they might also be implicated in sensing and neutralizing or expelling pathogens. We evaluate the expression of TLRs and the response to specific agonists in terms of cytokines, defensins, and hormones in enteroendocrine cells. The mouse enteroendocrine cell line STC-1 and C57BL/6 mice are used for in vitro and in vivo studies, respectively. The presence of TLR4, 5, and 9 is investigated by RT-PCR, Western blot, and immunofluorescence analyses. Activation of these receptors is studied evaluating keratinocyte-derived chemokine, defensins, and cholecystokinin production in response to their specific agonists. In this study, we show that the intestinal enteroendocrine cell line STC-1 expresses TLR4, 5, and 9 and releases cholecystokinin upon stimulation with the respective receptor agonists LPS, flagellin, and CpG-containing oligodeoxynucleotides. Release of keratinocyte-derived chemokine and β-defensin 2 was also observed after stimulation of STC-1 cells with the three TLR agonists, but not with fatty acids. Consistent with these in vitro data, mice showed increased serum cholecystokinin levels after oral challenge with LPS, flagellin, or CpG oligodeoxynucleotides. In addition to their response to food stimuli, enteroendocrine cells sense the presence of bacterial Ags through TLRs and are involved in neutralizing intestinal bacteria by releasing chemokines and defensins, and maybe in removing them by releasing hormones such as cholecystokinin, which induces contraction of the muscular tunica, favoring the emptying of the distal small intestine

Whole-transcriptome analysis links trastuzumab sensitivity of breast tumors to both HER2 dependence and immune cell infiltration.

While results thus far demonstrate the clinical benefit of trastuzumab, some patients do not respond to this therapy. To identify a molecular predictor of trastuzumab benefit, we conducted whole-transcriptome analysis of primary HER2+ breast carcinomas obtained from patients treated with trastuzumab-containing therapies and correlated the molecular portrait with treatment benefit. The estimated association between gene expression and relapse-free survival allowed development of a trastuzumab risk model (TRAR), with ERBB2 and ESR1 expression as core elements, able to identify patients with high and low risk of relapse. Application of the TRAR model to 24 HER2+ core biopsies from patients treated with neo-adjuvant trastuzumab indicated that it is predictive of trastuzumab response. Examination of TRAR in available whole-transcriptome datasets indicated that this model stratifies patients according to response to trastuzumab-based neo-adjuvant treatment but not to chemotherapy alone. Pathway analysis revealed that TRAR-low tumors expressed genes of the immune response, with higher numbers of CD8-positive cells detected immunohistochemically compared to TRAR-high tumors. The TRAR model identifies tumors that benefit from trastuzumab-based treatment as those most enriched in CD8-positive immune infiltrating cells and with high ERBB2 and low ESR1 mRNA levels, indicating the requirement for both features in achieving trastuzumab response