Targeting circulating monocytes with CCL2-loaded liposomes armed with an oncolytic adenovirus.

Oncolytic viruses (OVs) selectively replicate in and destroy cancer cells resulting in anti-tumor immunity. However, clinical use remains a challenge because of virus clearance upon intravenous delivery. OV packaging using a nanomedicine approach could overcome this. Here we encapsulate an oncolytic adenovirus (Ad[I/PPT-E1A]) into CCL2-coated liposomes in order to exploit recruitment of CCR2-expressing circulating monocytes into tumors. We demonstrate successful encapsulation of Ad[I/PPT-E1A] into CCL2-coated liposomes that were preferentially taken up by CCR2-expressing monocytes. No complex-related toxicities were observed following incubation with prostate tumor cells and the encapsulation did not affect virus oncolytic activity in vitro. Furthermore, intravenous administration of our nanomedicine resulted in a significant reduction in tumor size and pulmonary metastasis in prostate cancer-bearing mice whereby a 1000-fold less virus was needed compared to Ad[I/PPT-E1A] alone. Taken together our data provide an opportunity to target OVs via circulation to inaccessible tumors using liposome-assisted drug delivery.

Preoperative Nutrition Status and Postoperative Outcomes in Patients Undergoing Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) is a complex surgery to treat peritoneal surface malignancy (PSM). PSM and gastrointestinal (GI) resection from CRS can lead to significant GI symptoms and malnutrition. There is limited research into the nutrition status of this patient group and the impact of malnutrition on morbidity. OBJECTIVE: This study aims to determine if preoperative malnutrition, assessed using the Subjective Global Assessment (SGA), is associated with postoperative morbidity and increased length of stay (LOS) in patients undergoing CRS/HIPEC for PSM. METHODS: This study prospectively assessed the nutritional status of patients undergoing CRS/HIPEC using a validated nutrition assessment tool. Preoperative clinical symptoms, Peritoneal Cancer Index (PCI), intraoperative blood transfusions, operative time, GI resections, postoperative morbidity, and LOS, as well as pre- and postoperative nutritional interventions, were recorded. The impact of preoperative nutritional status was assessed in relation to postoperative complications and hospital LOS. RESULTS: The study included 102 participants; 34 patients (33%) were classified as malnourished (SGA = B or C). Preoperative weight loss (15% vs. 74%; p ≤ 0.001) and the presence of clinical symptoms (18% vs. 47%; p = 0.002) were significantly higher in malnourished patients. While PCI, intraoperative blood transfusions, and GI resections were independent predictors of morbidity, malnutrition was significantly associated with infectious complications and LOS. For each grade of worsening malnutrition, LOS increased by an average of 7.65 days. CONCLUSIONS: Preoperative malnutrition is prevalent in patients undergoing CRS/HIPEC and postoperative morbidity is common. Malnutrition is linked to LOS and plays a role in postoperative outcomes such as infection. Clear pre- and postoperative nutrition pathways are needed to optimize nutrition support and postoperative recovery.

Enhancing utility and understanding of evidence based practice through undergraduate nurse education.

BACKGROUND: The concept of evidence-based practice is globally relevant in current healthcare climates. However, students and teachers struggle with integrating evidence based practice effectively into a curriculum. This has implications for nurse education and in particular the way in which research is presented and delivered to students. A new undergraduate Evidence Based Practice module (Evidence Based Nursing 1) was developed in a large University within the United Kingdom. It commenced in October 2014 running in year one of a 3 year undergraduate nursing programme. This study sought to formally evaluate attitudes and beliefs, knowledge level and utilization of evidence based practice though using two validated questionnaires: Evidence Based Practice Beliefs Scale© and Evidence Based Practice Implementation Scale©. METHOD: This was a pilot study using quantitative pre and post-test design. Anonymised data was collected from Year 1 undergraduate student nurses in the September 2014 intake (n = 311) at two time points. Time 1: pre-module in September 2014; and Time 2: post -module in August 2015. All data was collected via Survey Monkey. RESULTS: Results demonstrate that the educational initiative positively impacted on both the beliefs and implementation of evidence based practice. Analysis highlighted statistically significant changes (p < 0.05) in both the Evidence Based Practice Beliefs Scale (7/16 categories) and the Evidence Based Practice Implementation Scale (13 / 18 categories). CONCLUSIONS: The significance of integrating evidence based practice into undergraduate nurse education curriculum cannot be underestimated if evidence based practice and its positive impact of patient care are to be appreciated in healthcare settings internationally.

The impact of a simulated intervention on attitudes of undergraduate nursing and medical students towards end of life care provision.

BACKGROUND: The concerns of undergraduate nursing and medical students' regarding end of life care are well documented. Many report feelings of emotional distress, anxiety and a lack of preparation to provide care to patients at end of life and their families. Evidence suggests that increased exposure to patients who are dying and their families can improve attitudes toward end of life care. In the absence of such clinical exposure, simulation provides experiential learning with outcomes comparable to that of clinical practice. The aim of this study was therefore to assess the impact of a simulated intervention on the attitudes of undergraduate nursing and medical students towards end of life care. METHODS: A pilot quasi-experimental, pretest-posttest design. Attitudes towards end of life care were measured using the Frommelt Attitudes Towards Care of the Dying Part B Scale which was administered pre and post a simulated clinical scenario. 19 undergraduate nursing and medical students were recruited from one large Higher Education Institution in the United Kingdom. RESULTS: The results of this pilot study confirm that a simulated end of life care intervention has a positive impact on the attitudes of undergraduate nursing and medical students towards end of life care (p < 0.001). CONCLUSIONS: Active, experiential learning in the form of simulation teaching helps improve attitudes of undergraduate nursing and medical students towards end of life. In the absence of clinical exposure, simulation is a viable alternative to help prepare students for their professional role regarding end of life care.

Determining the suitability of mass spectrometry for understanding the dissolution processes involved with pharmaceutical tablets.

RATIONALE: A current challenge for analytical chemists is the development of the measurement systems and approaches required to understand dynamic processes such as tablet dissolution. The design and development of oral tablets could be improved by the availability of detailed information about the rates of release of the individual tablet components. Small footprint mass spectrometry (MS) systems are gaining use for on-line reaction monitoring because of their ability to rapidly determine multiple reactant, intermediate, and product species. We have therefore assessed the utility of such MS systems to the study of dissolution processes. METHODS: Aqueous dissolution media containing phosphate and other non-volatile buffer salts were pumped from a standard USPII dissolution vessel through an active splitter and back. The splitter sampled the dissolution stream and diluted it into a make-up flow which was pumped to a small single quadrupole mass spectrometer. Single ion monitoring was used to quantify the ions of interest. Three different bio-relevant dissolution media were studied to gauge the effect of the sample matrix. RESULTS: Individual dissolution profiles were obtained from a tablet containing three drugs, and lactose as the soluble filler. This was successfully demonstrated with three different bio-relevant media designed to reflect the pH of the different sections of the human gastro-intestinal tract. Component concentrations as low as 0.06 µg/mL (representing 1% dissolution) were detected. The MS dissolution profiles correlated with the visual observation of tablet dissolution. MS gave linear responses with concentration for the individual components, although analysis of the tablet solution indicated that ion suppression is an area for further investigation. CONCLUSIONS: An on-line MS system was used to determine the individual dissolution profiles of three drugs and lactose as they were released from the same tablet. The level of each of these components in solution was determined every 10 seconds, and each had a similar release profile. The dissolution profiles were determined using inorganic buffer solutions at three different bio-relevant pHs.

A peptide derived from TIMP-3 inhibits multiple angiogenic growth factor receptors and tumour growth and inflammatory arthritis in mice.

The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the surface of vascular endothelial cells stimulates many steps in the angiogenic pathway. Inhibition of this interaction is proving of value in moderating the neovascularization accompanying age-related macular degeneration and in the treatment of cancer. Tissue inhibitor of metalloproteinases-3 (TIMP-3) has been shown to be a natural VEGFR-2 specific antagonist-an activity that is independent of its ability to inhibit metalloproteinases. In this investigation we localize this activity to the C-terminal domain of the TIMP-3 molecule and characterize a short peptide, corresponding to part of this domain, that not only inhibits all three VEGF-family receptors, but also fibroblast growth factor and platelet-derived growth factor receptors. This multiple-receptor inhibition may explain why the peptide was also seen to be a powerful inhibitor of tumour growth and also a partial inhibitor of arthritic joint inflammation in vivo.

Community and federal collaboration to assess pregnancy outcomes in Alaska Native women, 1997-2005.

The objectives are to report the estimated prevalence of pregnancy complications and adverse pregnancy outcomes in a defined population of Alaska Native women and also examine factors contributing to an intensive and successful collaboration between a tribal health center and the Centers for Disease Control and Prevention. Investigators abstracted medical record data from a random sample of singleton deliveries to residents of the study region occurring between 1997 and 2005. We used descriptive statistics to estimate the prevalence and 95% confidence intervals of selected pregnancy complications and adverse pregnancy outcomes. Records were examined for 505 pregnancies ending in a singleton delivery to 469 women. Pregnancy complication rates were 5.9% (95% CI 4.0, 8.4) for gestational diabetes mellitus, 6.1% (95% CI 4.2, 8.6%) for maternal chronic hypertension and 11.5% (95% CI 8.8, 14.6) for pregnancy associated hypertension, and 22.9% (95% CI 19.2-26.5 %) for anemia. The cesarean section rate was 5.5% (95% CI 3.5, 7.5) and 3.8% (95% CI 2.3, 5.8) of newborns weighed >4,500 g. Few previous studies reported pregnancy outcomes among Alaska Native women in a specific geographic region of Alaska and regarding the health needs in this population. We highlight components of our collaboration that contributed to the success of the study. Studies focusing on special populations such as Alaska Native women are feasible and can provide important information on health indicators at the local level.

Analysis of SARS-CoV-2 antibody seroprevalence in Northern Ireland during 2020-2021.

Background: With the spread of SARS-CoV-2 impacting upon public health directly and socioeconomically, further information was required to inform policy decisions designed to limit virus spread during the pandemic. This study sought to contribute to serosurveillance work within Northern Ireland to track SARS-CoV-2 progression and guide health strategy. Methods: Sera/plasma samples from clinical biochemistry laboratories were analysed for anti-SARS-CoV-2 antibodies. Samples were assessed using an Elecsys anti-SARS-CoV-2 or anti-SARS-CoV-2 S ECLIA (Roche) on an automated cobas e 801 analyser. Samples were also assessed via an anti-SARS-CoV-2 ELISA (Euroimmun). A subset of samples assessed via the Elecsys anti-SARS-CoV-2 ECLIA were subsequently analysed in an ACE2 pseudoneutralisation assay using a V-PLEX SARS-CoV-2 Panel 7 for IgG and ACE2 (Meso Scale Diagnostics). Results: Across three testing rounds (June-July 2020, November-December 2020 and June-July 2021 (rounds 1-3 respectively)), 4844 residual sera/plasma specimens were assayed for anti-SARS-CoV-2 antibodies. Seropositivity rates increased across the study, peaking at 11.6 % (95 % CI 10.4 %-13.0 %) during round 3. Varying trends in SARS-CoV-2 seropositivity were noted based on demographic factors. For instance, highest rates of seropositivity shifted from older to younger demographics across the study period. In round 3, Alpha (B.1.1.7) variant neutralising antibodies were most frequently detected across age groups, with median concentration of anti-spike protein antibodies elevated in 50-69 year olds and anti-S1 RBD antibodies elevated in 70+ year olds, relative to other age groups. Conclusions: With seropositivity rates of <15 % across the assessment period, it can be concluded that the significant proportion of the Northern Ireland population had not yet naturally contracted the virus by mid-2021.

Generation of a novel mouse model for the inducible depletion of macrophages in vivo.

Macrophages play an essential role in tissue homeostasis, innate immunity, inflammation, and wound repair. Macrophages are also essential during development, severely limiting the use of mouse models in which these cells have been constitutively deleted. Consequently, we have developed a transgenic model of inducible macrophage depletion in which macrophage-specific induction of the cytotoxic diphtheria toxin A chain (DTA) is achieved by administration of doxycycline. Induction of the DTA protein in transgenic animals resulted in a significant 50% reduction in CD68+ macrophages of the liver, spleen, and bone over a period of 6 weeks. Pertinently, the macrophages remaining after doxycycline treatment were substantially smaller and are functionally impaired as shown by reduced inflammatory cytokine production in response to lipopolysaccharide. This inducible model of macrophage depletion can now be utilized to determine the role of macrophages in both development and animal models of chronic inflammatory diseases.

Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius.

The facultatively anaerobic, thermophilic bacterium Geobacillus thermoglucosidasius is being developed as an industrial micro-organism for cellulosic bioethanol production. Process improvement would be gained by enhanced secretion of glycosyl hydrolases. Here we report the construction of a modular system for combining promoters, signal peptide encoding regions and glycosyl hydrolase genes to facilitate selection of the optimal combination in G. thermoglucosidasius. Initially, a minimal three-part E. coli-Geobacillus sp. shuttle vector pUCG3.8 was constructed using Gibson isothermal DNA assembly. The three PCR amplicons contained the pMB1 E. coli origin of replication and multiple cloning site (MCS) of pUC18, the Geobacillus sp. origin of replication pBST1 and the thermostable kanamycin nucleotidyltransferase gene (knt), respectively. G. thermoglucosidasius could be transformed with pUCG3.8 at an increased efficiency [2.8×10(5) c.f.u. (µg DNA)(-1)] compared to a previously reported shuttle vector, pUCG18. A modular cassette for the inducible expression and secretion of proteins in G. thermoglucosidasius, designed to allow the simple interchange of parts, was demonstrated using the endoglucanase Cel5A from Thermotoga maritima as a secretion target. Expression of cel5A was placed under the control of a cellobiose-inducible promoter (Pßglu) together with a signal peptide encoding sequence from a G. thermoglucosidasius C56-YS93 endo-ß-1,4-xylanase. The interchange of parts was demonstrated by exchanging the cel5A gene with the 3' region of a gene with homology to celA from Caldicellulosiruptor saccharolyticus and substituting Pßglu for the synthetic, constitutive promoter PUp2n38, which increased Cel5A activity five-fold. Cel5A and CelA activities were detected in culture supernatants indicating successful expression and secretion. N-terminal protein sequencing of Cel5A carrying a C-terminal FLAG epitope confirmed processing of the signal peptide sequence.

Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion.

Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors.

Effects of maternal smokeless tobacco use on selected pregnancy outcomes in Alaska Native women: a case-control study.

OBJECTIVE: To examine the potential effects of prenatal smokeless tobacco use on selected birth outcomes. DESIGN: A population-based, case-control study using a retrospective medical record review. POPULATION: Singleton deliveries 1997-2005 to Alaska Native women residing in western Alaska. METHODS: Hospital discharge codes were used to identify potential case deliveries and a random control sample. Data on tobacco use and confirmation of pregnancy outcomes were abstracted from medical records for 1123 deliveries. Logistic regression was used to examine associations between tobacco use and pregnancy outcomes. Adjusted odds ratios (OR), 95% confidence intervals (95% CI), and p-values were calculated. MAIN OUTCOMES MEASURES: Preterm delivery, pregnancy-associated hypertension, and placental abruption. RESULTS: In unadjusted analysis, smokeless tobacco use was not significantly associated with preterm delivery (OR 1.44, 95% CI 0.97-2.15). After adjustment for parity, pre-pregnancy body mass index, and maternal age, the point estimate was attenuated and remained non-significant. No significant associations were observed between smokeless tobacco use and pregnancy-associated hypertension (adjusted OR 0.92, 95% CI 0.56-1.51) or placental abruption (adjusted OR 1.11, 95% CI 0.53-2.33). CONCLUSIONS: Prenatal smokeless tobacco use does not appear to reduce risk of pregnancy-associated hypertension or to substantially increase risk of abruption. An association between smokeless tobacco and preterm delivery could not be ruled out. Components in tobacco other than nicotine likely play a major role in decreased pre-eclampsia risk in smokers. Nicotine adversely affects fetal neurodevelopment and our results should not be construed to mean that smokeless tobacco use is safe during pregnancy.

Changes in the immune landscape of TNBC after neoadjuvant chemotherapy: correlation with relapse.

Introduction: Patients with high-risk, triple negative breast cancer (TNBC) often receive neoadjuvant chemotherapy (NAC) alone or with immunotherapy. Various single-cell and spatially resolved techniques have demonstrated heterogeneity in the phenotype and distribution of macrophages and T cells in this form of breast cancer. Furthermore, recent studies in mice have implicated immune cells in perivascular (PV) areas of tumors in the regulation of metastasis and anti-tumor immunity. However, little is known of how the latter change during NAC in human TNBC or their impact on subsequent relapse, or the likely efficacy of immunotherapy given with or after NAC. Methods: We have used multiplex immunofluorescence and AI-based image analysis to compare the immune landscape in untreated and NAC-treated human TNBCs. We quantified changes in the phenotype, distribution and intercellular contacts of subsets of tumor-associated macrophages (TAMs), CD4+ and CD8+ T cells, and regulatory T cells (Tregs) in PV and non-PV various areas of the stroma and tumor cell islands. These were compared in tumors from patients who had either developed metastases or were disease-free (DF) after a three-year follow up period. Results: In tumors from patients who remained DF after NAC, there was a marked increase in stromal CD163+ TAMs, especially those expressing the negative checkpoint regulator, T-cell immunoglobulin and mucin domain 3 (TIM-3). Whereas CD4+ T cells preferentially located to PV areas in the stroma of both untreated and NAC-treated tumors, specific subsets of TAMs and Tregs only did so only after NAC. Distinct subsets of CD4+ and CD8+ T cells formed PV clusters with CD163+ TAMs and Tregs. These were retained after NAC. Discussion: Quantification of stromal TIM-3+CD163+ TAMs in tumor residues after NAC may represent a new way of identifying patients at high risk of relapse. PV clustering of immune cells is highly likely to regulate the activation and function of T cells, and thus the efficacy of T cell-based immunotherapies administered with or after NAC.

Evaluating The Precocial-altricial Axis of Motor Skill at Birth in A Preterm Pig Model.

The pace of locomotor development is a critical component of lifetime evolutionary fitness. Developmental researchers often divide species into two broad categories based on functional competence at birth: precocial infants who can independently stand and locomote soon after birth versus altricial infants who are either incapable of independent movement or can only do so in a rudimentary manner. However, investigating the lower level neuromotor and biomechanical traits that account for perinatal variation in motor development is complicated by the lack of experimental control inherent to all comparative analyses. Precocial and altricial animals often differ along a host of dimensions that can obfuscate the specific factors controlling motor development per se. Here, we propose an alternative approach of examining locomotor development in a nominally precocial species-the domestic pig (Sus scrofa)-in which gestation length has been experimentally manipulated, thereby creating "functionally altricial" cohorts for comparison. We have used standard biomechanical testing to evaluate balance and locomotor performance in preterm pigs born at 94% full-term gestation (N = 29 individuals) and compared these data to a similar dataset on age-matched full-term piglets (N = 15 individuals). Static balance tests showed that preterm pigs were characterized by increased postural sway, particularly in the fore-aft (anteroposterior) direction. Locomotor analyses showed that preterm piglets tended to take shorter, more frequent strides, use higher duty factors, and preferentially choose gait patterns that ensured they were supported by at least three limbs during most of the stride cycle, though differences between preterm and full-term animals were often modulated by variation in locomotor speed. Morphometric analysis showed no differences in relative extensor muscle mass between preterm and full-term animals, suggesting that neurological immaturity might be more determinant of preterm piglet motor dysfunctions than musculoskeletal immaturity per se (though much work remains to be done to fully document the neuromotor phenotype of the preterm infant pig model). In many ways, the postural and locomotor deficits shown by the preterm piglets paralleled the locomotor phenotype of altricial mammals. Overall, our study demonstrates the utility of a "within-species" design for studying the biomechanical correlates and neuromotor basis of evolutionary variation in motor skill at birth.

Technical note on the exploration of COVID-19 in autopsy material.

Interrogation of immune response in autopsy material from patients with SARS-CoV-2 is potentially significant. We aim to describe a validated protocol for the exploration of the molecular physiopathology of SARS-CoV-2 pulmonary disease using multiplex immunofluorescence (mIF).The application of validated assays for the detection of SARS-CoV-2 in tissues, originally developed in our laboratory in the context of oncology, was used to map the topography and complexity of the adaptive immune response at protein and mRNA levels.SARS-CoV-2 is detectable in situ by protein or mRNA, with a sensitivity that could be in part related to disease stage. In formalin-fixed, paraffin-embedded pneumonia material, multiplex immunofluorescent panels are robust, reliable and quantifiable and can detect topographic variations in inflammation related to pathological processes.Clinical autopsies have relevance in understanding diseases of unknown/complex pathophysiology. In particular, autopsy materials are suitable for the detection of SARS-CoV-2 and for the topographic description of the complex tissue-based immune response using mIF.

Maternal smokeless tobacco use in Alaska Native women and singleton infant birth size.

OBJECTIVE: To examine the effects of maternal prenatal smokeless tobacco use on infant birth size. DESIGN: A retrospective medical record review of 502 randomly selected deliveries. POPULATION AND SETTING: Singleton deliveries to Alaska Native women residing in a defined geographical region in western Alaska, 1997-2005. METHODS: A regional medical center's electronic records were used to identify singleton deliveries. Data on maternal tobacco exposure and pregnancy outcomes were abstracted from medical records. Logistic models were used to estimate adjusted mean birthweight, length and head circumference for deliveries to women who used no tobacco (n=121), used smokeless tobacco (n=237) or smoked cigarettes (n=59). Differences in mean birthweight, length and head circumference, 95% confidence intervals and p-values were calculated using non-users as the reference group. MAIN OUTCOME MEASURES: Infant birthweight, crown-heel length and head circumference. RESULTS: After adjustment for gestational age and other potential confounders, the mean birthweight of infants of smokeless tobacco users was reduced by 78 g compared with that of infants of non-users (p=0.18) and by 331 g in infants of smokers (p<0.01). No association was found between maternal smokeless tobacco use and infant length or infant head circumference. CONCLUSIONS: We found a modest but non-significant reduction in the birthweight of infants of smokeless tobacco users compared with infants of tobacco non-users. Because smokeless tobacco contains many toxic compounds that could affect other pregnancy outcomes, results of this study should not be construed to mean that smokeless tobacco use is safe during pregnancy.

Inhibition of neutrophil infiltration into A549 lung tumors in vitro and in vivo using a CXCR2-specific antagonist is associated with reduced tumor growth.

Neutrophils are important innate immune cells that are involved in microbial clearance at sites of infection and in wound healing. The microenvironment of tumors often resembles that of chronic inflammation and increased numbers of neutrophils have been observed in several tumors and, in some cases, these positively correlate with poor prognosis. Neutrophil recruitment into tumors appears to be dependent on chemokines that bind to CXCR1 and CXCR2 expressed by neutrophils. In our study, we used lung adenocarcinoma A549 multicellular tumor spheroids and A549 tumor xenografts along with a CXCR2-specific small molecule inhibitor (AZ10397767) to investigate the recruitment and function of human neutrophils in tumors. We found that A549 spheroids constitutively secrete high levels of CXCL chemokines and that neutrophil recruitment into A549 tumors in vitro and in vivo is largely dependent on CXCR2 activation. AZ10397767 significantly reduced the numbers of infiltrating neutrophils into both in vitro and in vivo tumor models, which was associated with slower growing tumors. Neutrophil infiltration into A549 tumor spheroids increased their size compared to noninfiltrated spheroids and neutrophil-derived factors increased the proliferation of A549 tumor cells and induced endothelial cell tubule formation in vitro. In contrast, we saw no reduction in microvascular density in AZ10397767-treated A549 tumors or in tumors grown in CXCR2(-/-) mice, suggesting that angiogenesis in these tumors is CXCR2-independent. Our data show that neutrophils can contribute to lung tumor growth and that CXCR2 antagonists may be a useful therapeutic agent in the treatment of lung carcinomas.

The periplasmic chaperone Skp is required for successful Salmonella Typhimurium infection in a murine typhoid model.

The alternative sigma factor σ(E) (rpoE) is essential for survival in vivo of Salmonella Typhimurium but is dispensable during growth in the laboratory. We have been identifying σ(E)-regulated genes and studying their regulation and function to elucidate their potential role in the severe attenuation of S. Typhimurium rpoE mutants. In this study we identify five promoters that control the rseP, yaeT (bamA), skp region. A confirmed σ(E)-dependent promoter, yaeTp1, and a second downstream promoter, yaeTp2, are located within the upstream gene rseP and direct expression of the downstream genes. The only known function of RseP is σ(E) activation, and it is therefore not expected to be essential for S. Typhimurium in vitro. However, it proved impossible to delete the entire rseP gene due to the presence of internal promoters that regulate the essential gene yaeT. We could inactivate rseP by deleting the first third of the gene, leaving the yaeT promoters intact. Like the rpoE mutant, the rseP mutant exhibited severe attenuation in vivo. We were able to delete the entire coding sequence of skp, which encodes a periplasmic chaperone involved in targeting misfolded outer-membrane proteins to the ß-barrel assembly machinery. The skp mutant was attenuated in mice after oral and parenteral infection. Virulence could be complemented by providing skp in trans but only by linking it to a heterologous σ(E)-regulated promoter. The reason the skp mutant is attenuated is currently enigmatic, but we know it is not through increased sensitivity to a variety of RpoE-activating host stresses, such as H(2)O(2), polymyxin B and high temperature, or through altered secretion of effector proteins by either the Salmonella pathogenicity island (SPI)-1 or the SPI-2 type III secretion system.

Elusive identities and overlapping phenotypes of proangiogenic myeloid cells in tumors.

It is now established that bone marrow-derived myeloid cells regulate tumor angiogenesis. This was originally inferred from studies of human tumor biopsies in which a positive correlation was seen between the number of tumor-infiltrating myeloid cells, such as macrophages and neutrophils, and tumor microvessel density. However, unequivocal evidence was only provided once mouse models were used to examine the effects on tumor angiogenesis by genetically or pharmacologically targeting myeloid cells. Since then, identifying the exact myeloid cell types involved in this process has proved challenging because of myeloid cell heterogeneity and the expression of overlapping phenotypic markers in tumors. As a result, investigators often simply refer to them now as "bone marrow-derived myeloid cells." Here we review the findings of various attempts to phenotype the myeloid cells involved and discuss the therapeutic implications of correctly identifying-and thus being able to target-this proangiogenic force in tumors.