Molecular Targets of Triple-Negative Breast Cancer: Where Do We Stand?

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Due to its heterogeneity and lack of hormone receptor expression, this subtype is more likely to metastasize and resist treatment attempts than are other forms of breast cancer. Due to the absence of targetable receptors, chemotherapy and breast conserving surgery have been the predominant treatment options for patients. However, resistance to chemotherapy and local recurrence of the tumors is frequent. Emerging immunotherapies have begun to change treatment plans for patients diagnosed with TNBC. In this review, we discuss the various immune pathways identified in TNBC and the role they play as targets for new potential treatment choices. Various therapeutic options that inhibit key pathways in cellular growth cycles, DNA repair mechanisms, epithelial mesenchymal transition, and immunosuppression have been shown to improve survival in patients with this disease. With promising results thus far, continued studies of immunotherapy and neoadjuvant therapy options for TNBC are likely to alter the treatment course for these diagnoses in the future.

The Emerging Global Tobacco Treatment Workforce: Characteristics of Tobacco Treatment Specialists Trained in Council-Accredited Training Programs from 2017 to 2019.

Tobacco use is projected to kill 1 billion people in the 21st century. Tobacco Use Disorder (TUD) is one of the most common substance use disorders in the world. Evidence-based treatment of TUD is effective, but treatment accessibility remains very low. A dearth of specially trained clinicians is a significant barrier to treatment accessibility, even within systems of care that implement brief intervention models. The treatment of TUD is becoming more complex and tailoring treatment to address new and traditional tobacco products is needed. The Council for Tobacco Treatment Training Programs (Council) is the accrediting body for Tobacco Treatment Specialist (TTS) training programs. Between 2016 and 2019, n = 7761 trainees completed Council-accredited TTS training programs. Trainees were primarily from North America (92.6%) and the Eastern Mediterranean (6.1%) and were trained via in-person group workshops in medical and academic settings. From 2016 to 2019, the number of Council-accredited training programs increased from 14 to 22 and annual number of trainees increased by 28.5%. Trainees have diverse professional backgrounds and work in diverse settings but were primarily White (69.1%) and female (78.7%) located in North America. Nearly two-thirds intended to implement tobacco treatment services in their setting; two-thirds had been providing tobacco treatment for 1 year or less; and 20% were sent to training by their employers. These findings suggest that the training programs are contributing to the development of a new workforce of TTSs as well as the development of new programmatic tobacco treatment services in diverse settings. Developing strategies to support attendance from demographically and geographically diverse professionals might increase the proportion of trainees from marginalized groups and regions of the world with significant tobacco-related inequities.

Visualizing the invisible: class excursions to ignite children's enthusiasm for microbes.

We have recently argued that, because microbes have pervasive - often vital - influences on our lives, and that therefore their roles must be taken into account in many of the decisions we face, society must become microbiology-literate, through the introduction of relevant microbiology topics in school curricula (Timmis et al. 2019. Environ Microbiol 21: 1513-1528). The current coronavirus pandemic is a stark example of why microbiology literacy is such a crucial enabler of informed policy decisions, particularly those involving preparedness of public-health systems for disease outbreaks and pandemics. However, a significant barrier to attaining widespread appreciation of microbial contributions to our well-being and that of the planet is the fact that microbes are seldom visible: most people are only peripherally aware of them, except when they fall ill with an infection. And it is disease, rather than all of the positive activities mediated by microbes, that colours public perception of 'germs' and endows them with their poor image. It is imperative to render microbes visible, to give them life and form for children (and adults), and to counter prevalent misconceptions, through exposure to imagination-capturing images of microbes and examples of their beneficial outputs, accompanied by a balanced narrative. This will engender automatic mental associations between everyday information inputs, as well as visual, olfactory and tactile experiences, on the one hand, and the responsible microbes/microbial communities, on the other hand. Such associations, in turn, will promote awareness of microbes and of the many positive and vital consequences of their actions, and facilitate and encourage incorporation of such consequences into relevant decision-making processes. While teaching microbiology topics in primary and secondary school is key to this objective, a strategic programme to expose children directly and personally to natural and managed microbial processes, and the results of their actions, through carefully planned class excursions to local venues, can be instrumental in bringing microbes to life for children and, collaterally, their families. In order to encourage the embedding of microbiology-centric class excursions in current curricula, we suggest and illustrate here some possibilities relating to the topics of food (a favourite pre-occupation of most children), agriculture (together with horticulture and aquaculture), health and medicine, the environment and biotechnology. And, although not all of the microbially relevant infrastructure will be within reach of schools, there is usually access to a market, local food store, wastewater treatment plant, farm, surface water body, etc., all of which can provide opportunities to explore microbiology in action. If children sometimes consider the present to be mundane, even boring, they are usually excited with both the past and the future so, where possible, visits to local museums (the past) and research institutions advancing knowledge frontiers (the future) are strongly recommended, as is a tapping into the natural enthusiasm of local researchers to leverage the educational value of excursions and virtual excursions. Children are also fascinated by the unknown, so, paradoxically, the invisibility of microbes makes them especially fascinating objects for visualization and exploration. In outlining some of the options for microbiology excursions, providing suggestions for discussion topics and considering their educational value, we strive to extend the vistas of current class excursions and to: (i) inspire teachers and school managers to incorporate more microbiology excursions into curricula; (ii) encourage microbiologists to support school excursions and generally get involved in bringing microbes to life for children; (iii) urge leaders of organizations (biopharma, food industries, universities, etc.) to give school outreach activities a more prominent place in their mission portfolios, and (iv) convey to policymakers the benefits of providing schools with funds, materials and flexibility for educational endeavours beyond the classroom.

Striking Similarities Between Botrytis cinerea From Non-agricultural and From Agricultural Habitats.

Investigations into life history of microorganisms that cause plant diseases have been limited mostly to contexts where they are in interaction with plants, and with cropped or otherwise managed vegetation. Therefore, knowledge about the diversity of plant pathogens, about potential reservoirs of inoculum and about the processes that contribute to their survival and adaptation is limited to these contexts. The agro-centric perspective of plant pathogen life histories is incoherent with respect to the capacity of many of them to persist as saprophytes on various substrates. In this context we have investigated the ubiquity of the broad host range necrotrophic fungus Botrytis cinerea, outside of agricultural settings and have determined if the populations in these natural habitats can be distinguished phenotypically and phylogenetically from populations isolated from diseased crops. Over a period of 5 years, we isolated B. cinerea from 235 samples of various substrates collected in France including rainfall, snowpack, river, and lake water, epilithic biofilms in mountain streams, leaf litter and plant debris, rock surfaces, bird feathers and healthy wild plants from outside of agricultural fields. All substrates except rock surfaces harbored B. cinerea leading us to establish a collection of purified strains that were compared to B. cinerea from diseased tomato, grapes and various other crops in France. Phylogenetic comparisons of 321 strains from crop plants and 100 strains from environmental substrates based on sequences of 9 microsatellite markers revealed that strains from crops and the environment could not be distinguished. Furthermore, the genetic diversity of strains outside of agriculture was just as broad as within agriculture. In tests to determine the aggressiveness of strains on tomato stems, the mean disease severity caused by strains from environmental substrates was statistically identical to the severity of disease caused by strains from tomato, but was significantly greater than the severity caused by strains from grape or other crops. Our results suggest that highly diverse populations of this plant pathogen persist outside of agriculture in association with substrates other than plants and that this part of their life history is compatible with its capacity to maintain its potential as plant pathogen.

Assessing the Aerial Interconnectivity of Distant Reservoirs of Sclerotinia sclerotiorum.

Many phytopathogenic fungi are disseminated as spores via the atmosphere from short to long distances. The distance of dissemination determines the extent to which plant diseases can spread and novel genotypes of pathogens can invade new territories. Predictive tools including models that forecast the arrival of spores in areas where susceptible crops are grown can help to more efficiently manage crop health. However, such models are difficult to establish for fungi with broad host ranges because sources of inoculum cannot be readily identified. Sclerotinia sclerotiorum, the pandemic agent of white mold disease, can attack >400 plant species including economically important crops. Monitoring airborne inoculum of S. sclerotiorum in several French cropping areas has shown that viable ascospores are present in the air almost all the time, even when no susceptible crops are nearby. This raises the hypothesis of a distant origin of airborne inoculum. The objective of the present study was to determine the interconnectivity of reservoirs of S. sclerotiorum from distant regions based on networks of air mass movement. Viable airborne inoculum of S. sclerotiorum was collected in four distinct regions of France and 498 strains were genotyped with 16 specific microsatellite markers and compared among the regions. Air mass movements were inferred using the HYSPLIT model and archived meteorological data from the global data assimilation system (GDAS). The results show that up to 700 km could separate collection sites that shared the same haplotypes. There was low or no genetic differentiation between strains collected from the four sites. The rate of aerial connectivity between two sites varied according to the direction considered. The results also show that the aerial connectivity between sites is a better indicator of the probability of the incoming component (PIC) of inoculum at a given site from another one than is geographic distance. We identified the links between specific sites in the trajectories of air masses and we quantified the frequencies at which the directional links occurred as a proof-of-concept for an operational method to assess the arrival of airborne inoculum in a given area from distant origins.

Tobacco-Free Policy Outcomes for an Inpatient Substance Abuse Treatment Center.

We assessed changes in patient census, smoking prevalence, and intention to remain abstinent associated with the voluntary enactment of a campus-wide tobacco-free policy in a for-profit residential addictions treatment center. Employing nationally recognized tobacco-free policy strategies, steps toward implementation were tailored to addictions treatment settings. Census data and survey of client tobacco use data were collected for 1 year before the policy began and for 1 year following the policy. Average daily census increased for both the inpatient and extended residential treatment programs after enactment of the tobacco-free policy. Number of tobacco users admitted to treatment increased significantly, while the number of tobacco users who left treatment early did not significantly change. In addition to reducing smoking, the policy was associated with a significant increase of patients reporting the intention to remain abstinent after discharge. Contrary to common concern, a tobacco-free policy implemented in an inpatient residential addictions treatment setting did not negatively affect census rates. This study suggests potential clinical and organizational benefits for campus-wide tobacco-free policies.

Regulation of Wnt/ß-catenin signaling by herpesviruses.

The Wnt/ß-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/ß-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/ß-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/ß-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/ß-catenin pathway to promote a virus-centric agenda.

A comprehensive next generation sequencing-based virome assessment in brain tissue suggests no major virus - tumor association.

Next generation sequencing (NGS) can globally interrogate the genetic composition of biological samples in an unbiased yet sensitive manner. The objective of this study was to utilize the capabilities of NGS to investigate the reported association between glioblastoma multiforme (GBM) and human cytomegalovirus (HCMV). A large-scale comprehensive virome assessment was performed on publicly available sequencing datasets from the Cancer Genome Atlas (TCGA), including RNA-seq datasets from primary GBM (n = 157), recurrent GBM (n = 13), low-grade gliomas (n = 514), recurrent low-grade gliomas (n = 17), and normal brain (n = 5), and whole genome sequencing (WGS) datasets from primary GBM (n = 51), recurrent GBM (n = 10), and normal matched blood samples (n = 20). In addition, RNA-seq datasets from MRI-guided biopsies (n = 92) and glioma stem-like cell cultures (n = 9) were analyzed. Sixty-four DNA-seq datasets from 11 meningiomas and their corresponding blood control samples were also analyzed. Finally, three primary GBM tissue samples were obtained, sequenced using RNA-seq, and analyzed. After in-depth analysis, the most robust virus findings were the detection of papillomavirus (HPV) and hepatitis B reads in the occasional LGG sample (4 samples and 1 sample, respectively). In addition, low numbers of virus reads were detected in several datasets but detailed investigation of these reads suggest that these findings likely represent artifacts or non-pathological infections. For example, all of the sporadic low level HCMV reads were found to map to the immediate early promoter intimating that they likely originated from laboratory expression vector contamination. Despite the detection of low numbers of Epstein-Barr virus reads in some samples, these likely originated from infiltrating B-cells. Finally, human herpesvirus 6 and 7 aligned viral reads were identified in all DNA-seq and a few RNA-seq datasets but detailed analysis demonstrated that these were likely derived from the homologous human telomeric-like repeats. Other low abundance viral reads were detected in some samples but for most viruses, the reads likely represent artifacts or incidental infections. This analysis argues against associations between most known viruses and GBM or mengiomas. Nevertheless, there may be a low percentage association between HPV and/or hepatitis B and LGGs.

Kaposi's sarcoma-associated herpesvirus G-protein coupled receptor activates the canonical Wnt/ß-catenin signaling pathway.

BACKGROUND: KSHV is a tumorigenic γ-herpesvirus that has been identified as the etiologic agent of Kaposi's sarcoma (KS), a multifocal highly vascularized neoplasm that is the most common malignancy associated with acquired immunodeficiency syndrome (AIDS). The virus encodes a constitutively active chemokine receptor homologue, vGPCR that possesses potent angiogenic and tumorigenic properties, and is critical for KSHV pathobiology. To date, a number of signaling pathways have been identified as key in mediating vGPCR oncogenic potential. FINDINGS: In this study, we identify a novel pathway, the Wnt/ß-catenin pathway, which is dysregulated by vGPCR expression in endothelial cells. Expression of vGPCR in endothelial cells enhances the nuclear accumulation of ß-catenin, that correlates with an increase in ß-catenin transcriptional activity. Activation of ß-catenin signaling by vGPCR is dependent on the PI3K/Akt pathway, as treatment of vGPCR-expressing cells with a pharmacological inhibitor of PI3K, leads to a decreased activation of a ß-catenin-driven reporter, a significant decrease in expression of ß-catenin target genes, and reduced endothelial tube formation. CONCLUSIONS: Given the critical role of Wnt/ß-catenin signaling in angiogenesis and tumorigenesis, the findings from this study suggest a novel mechanism in KSHV-induced malignancies.

Kaposi sarcoma-associated herpesvirus g protein-coupled receptor enhances endothelial cell survival in part by upregulation of bcl-2.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) encoded G protein-coupled receptor (vGPCR) is a constitutively active lytic phase protein with significant homology to the human interleukin-8 receptor. vGPCR is necessary and sufficient to induce angiogenesis as well as the spindle cell proliferation characteristic of Kaposi sarcoma (KS) lesions. We previously demonstrated that Bcl-2, an antiapoptotic protein, is upregulated in KS lesions. The aim of this study was to determine if vGPCR enhances endothelial cell survival through upregulation of Bcl-2 expression and to elucidate the signaling pathways involved. METHODS: Primary human umbilical vein endothelial cells were transduced with a recombinant retrovirus expressing vGPCR and then subjected to serum starvation. Cell viability and apoptosis were analyzed by fluorescence-activated cell sorting. Bcl-2 expression was determined by real-time quantitative reverse transcription polymerase chain reaction and immunoblotting. Specific pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) were employed to elucidate the signaling pathways involved. Bcl-2 expression was knocked down using small interfering RNA (siRNA). RESULTS: Endothelial cells expressing vGPCR showed increased survival after serum starvation and upregulation of Bcl-2 messenger RNA (mRNA) and protein. The vGPCR-induced increases in both Bcl-2 mRNA and protein levels were dependent on PI3K signaling but not on mTOR. Moreover, siRNA inhibition of Bcl-2 resulted in significant abrogation of the observed vGPCR-mediated cell survival advantage. CONCLUSIONS: Taken together, the results demonstrate that Bcl-2 is a mediator of vGPCR-induced endothelial cell survival and is a downstream effector of Akt in this process.

Mesenchymal stem cells as a novel vaccine platform.

Vaccines are the most efficient and cost-effective means of preventing infectious disease. However, traditional vaccine approaches have thus far failed to provide protection against human immunodeficiency virus (HIV), tuberculosis, malaria, and many other diseases. New approaches to vaccine development are needed to address some of these intractable problems. In this report, we review the literature identifying stimulatory effects of mesenchymal stem cells (MSC) on immune responses and explore the potential for MSC as a novel, universal vaccination platform. MSC are unique bone marrow-derived multipotent progenitor cells that are presently being exploited as gene therapy vectors for a variety of conditions, including cancer and autoimmune diseases. Although MSC are predominantly known for anti-inflammatory properties during allogeneic MSC transplant, there is evidence that MSC can actually promote adaptive immunity under certain settings. MSC have also demonstrated some success in anti-cancer therapeutic vaccines and anti-microbial prophylactic vaccines, as we report, for the first time, the ability of modified MSC to express and secrete a viral antigen that stimulates antigen-specific antibody production in vivo. We hypothesize that the unique properties of modified MSC may enable MSC to serve as an unconventional but innovative, vaccine platform. Such a platform would be capable of expressing hundreds of proteins, thereby generating a broad array of epitopes with correct post-translational processing, mimicking natural infection. By stimulating immunity to a combination of epitopes, it may be possible to develop prophylactic and even therapeutic vaccines to tackle major health problems including those of non-microbial and microbial origin, including cancer, or an infectious disease like HIV, where traditional vaccination approaches have failed.

Human cytomegalovirus infection dysregulates the canonical Wnt/ß-catenin signaling pathway.

Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected in utero. During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/ß-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/ß-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of ß-catenin and expression of ß-catenin target genes in these cells. HCMV infection leads to ß-catenin protein accumulation in a discrete juxtanuclear region. Levels of ß-catenin in membrane-associated and cytosolic pools, as well as nuclear ß-catenin, are reduced after infection; while transcription of the ß-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/ß-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.

Pseudomonas syringae naturally lacking the canonical type III secretion system are ubiquitous in nonagricultural habitats, are phylogenetically diverse and can be pathogenic.

The type III secretion system (T3SS) is an important virulence factor of pathogenic bacteria, but the natural occurrence of variants of bacterial plant pathogens with deficiencies in their T3SS raises questions about the significance of the T3SS for fitness. Previous work on T3SS-deficient plant pathogenic bacteria has focused on strains from plants or plant debris. Here we have characterized T3SS-deficient strains of Pseudomonas syringae from plant and nonplant substrates in pristine nonagricultural contexts, many of which represent recently described clades not yet found associated with crop plants. Strains incapable of inducing a hypersensitive reaction (HR(-)) in tobacco were detected in 65% of 126 samples from headwaters of rivers (mountain creeks and lakes), snowpack, epilithic biofilms, wild plants and leaf litter and constituted 2 to 100% of the P. syringae population associated with each sample. All HR(-) strains lacked at least one gene in the canonical hrp/hrc locus or the associated conserved effector locus, but most lacked all six of the genes tested (hrcC, hrpL, hrpK1, avrE1 and hrpW1) and represented several disparate phylogenetic clades. Although most HR(-) strains were incapable of causing symptoms on cantaloupe seedlings as expected, strains in the recently described TA-002 clade caused severe symptoms in spite of the absence of any of the six conserved genes of the canonical T3SS according to PCR and Southern blot assays. The phylogenetic context of the T3SS variants we observed provides insight into the evolutionary history of P. syringae as a pathogen and as an environmental saprophyte.

Peptide inhibition of human cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV)- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB), a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. RESULTS: Using the Wimley-White Interfacial Hydrophobicity Scale (WWIHS), several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF) were infected with the Towne-GFP strain of HCMV (0.5 MOI), preincubated with peptides at a range of concentrations (78 nm to 100 µM), and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 µM, and 51% and 62% inhibition at concentrations of 5 µM and 2.5 µM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100 µM and 50 µM, respectively, and 60% at a concentration of 2.5 µM. While peptides 264-291 and 297-315, individually failed to inhibit viral infection, when combined, they showed 67% inhibition of HCMV infection at a concentration of 0.125 µM each. CONCLUSIONS: Peptides designed to target putative fusogenic domains of gB provide a basis for the development of novel therapeutics that prevent HCMV infection.

Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases.

BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3beta as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3beta. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3beta phosphorylation and the subsequent expression of beta-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of beta-catenin alone was insufficient to stimulate cell motility.

Naturally occurring nonpathogenic isolates of the plant pathogen Pseudomonas syringae lack a type III secretion system and effector gene orthologues.

Pseudomonas syringae causes plant diseases, and the main virulence mechanism is a type III secretion system (T3SS) that translocates dozens of effector proteins into plant cells. Here we report the existence of a subgroup of P. syringae isolates that do not cause disease on any plant species tested. This group is monophyletic and most likely evolved from a pathogenic P. syringae ancestor through loss of the T3SS. In the nonpathogenic isolate P. syringae 508 the genomic region that in pathogenic P. syringae strains contains the hrp-hrc cluster coding for the T3SS and flanking effector genes is absent. P. syringae 508 was also surveyed for the presence of effector orthologues from the closely related pathogenic strain P. syringae pv. syringae B728a, but none were detected. The absence of the hrp-hrc cluster and effector orthologues was confirmed for other nonpathogenic isolates. Using the AvrRpt2 effector as reporter revealed the inability of P. syringae 508 to translocate effectors into plant cells. Adding a plasmid-encoded T3SS and the P. syringae pv. syringae 61 effector gene hopA1 increased in planta growth almost 10-fold. This suggests that P. syringae 508 supplemented with a T3SS could be used to determine functions of individual effectors in the context of a plant infection, avoiding the confounding effect of other effectors with similar functions present in effector mutants of pathogenic isolates.

The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle.

Pseudomonas syringae is a plant pathogen well known for its capacity to grow epiphytically on diverse plants and for its ice-nucleation activity. The ensemble of its known biology and ecology led us to postulate that this bacterium is also present in non-agricultural habitats, particularly those associated with water. Here, we report the abundance of P. syringae in rain, snow, alpine streams and lakes and in wild plants, in addition to the previously reported abundance in epilithic biofilms. Each of these substrates harbored strains that corresponded to P. syringae in terms of biochemical traits, pathogenicity and pathogenicity-related factors and that were ice-nucleation active. Phylogenetic comparisons of sequences of four housekeeping genes of the non-agricultural strains with strains of P. syringae from disease epidemics confirmed their identity as P. syringae. Moreover, strains belonging to the same clonal lineage were isolated from snow, irrigation water and a diseased crop plant. Our data suggest that the different substrates harboring P. syringae modify the structure of the associated populations. Here, we propose a comprehensive life cycle for P. syringae--in agricultural and non-agricultural habitats--driven by the environmental cycle of water. This cycle opens the opportunity to evaluate the importance of non-agricultural habitats in the evolution of a plant pathogen and the emergence of virulence. The ice-nucleation activity of all strains from snow, unlike from other substrates, strongly suggests that P. syringae plays an active role in the water cycle as an ice nucleus in clouds.

Ovarian cancers overexpress the antimicrobial protein hCAP-18 and its derivative LL-37 increases ovarian cancer cell proliferation and invasion.

The role of the pro-inflammatory peptide, LL-37, and its pro-form, human cationic antimicrobial protein 18 (hCAP-18), in cancer development and progression is poorly understood. In damaged and inflamed tissue, LL-37 functions as a chemoattractant, mitogen and pro-angiogenic factor suggesting that the peptide may potentiate tumor progression. The aim of this study was to characterize the distribution of hCAP-18/LL-37 in normal and cancerous ovarian tissue and to examine the effects of LL-37 on ovarian cancer cells. Expression of hCAP-18/LL-37 was localized to immune and granulosa cells of normal ovarian tissue. By contrast, ovarian tumors displayed significantly higher levels of hCAP-18/LL-37 where expression was observed in tumor and stromal cells. Protein expression was statistically compared to the degree of immune cell infiltration and microvessel density in epithelial-derived ovarian tumors and a significant correlation was observed for both. It was demonstrated that ovarian tumor tissue lysates and ovarian cancer cell lines express hCAP-18/LL-37. Treatment of ovarian cancer cell lines with recombinant LL-37 stimulated proliferation, chemotaxis, invasion and matrix metalloproteinase expression. These data demonstrate for the first time that hCAP-18/LL-37 is significantly overexpressed in ovarian tumors and suggest LL-37 may contribute to ovarian tumorigenesis through direct stimulation of tumor cells, initiation of angiogenesis and recruitment of immune cells. These data provide further evidence of the existing relationship between pro-inflammatory molecules and ovarian cancer progression.

Kaposi's sarcoma associated herpesvirus G-protein coupled receptor activation of cyclooxygenase-2 in vascular endothelial cells.

BACKGROUND: Kaposi's sarcoma associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a highly vascularized neoplasm characterized by endothelial-derived spindle-shaped tumor cells. KSHV-infected microvascular endothelial cells demonstrate increased cyclooxygenase-2 (COX-2) expression and KS lesions have high levels of prostaglandin E2 (PGE2), a short-lived eicosanoid dependent on cyclooxygenase activity that has been linked to pathogenesis of other neoplasias. To determine whether increased COX-2 expression and PGE2 production is mediated by the angiogenic and tumorigenic KSHV-encoded G-protein coupled receptor (vGPCR), we developed a recombinant retrovirus to express vGPCR in Human Umbilical Vascular Endothelial Cells (HUVEC). RESULTS: In the present study, we show that vGPCR-expressing HUVEC exhibit a spindle-like morphology that is characteristic of KS endothelial cells and demonstrate selective induction of PGE2 and COX-2. By treating vGPCR-expressing HUVEC with selective and non-selective COX inhibitors, we show that vGPCR-induced PGE2 production is dependent on the expression of COX-2 but not COX-1. CONCLUSION: Taken together, these results demonstrate that vGPCR induces expression of COX-2 and PGE2 that may mediate the paracrine effects of this key viral protein in KS pathogenesis.

Erythropoietin, a hypoxia-regulated factor, elicits a pro-angiogenic program in human mesenchymal stem cells.

OBJECTIVE: The ability of erythropoietin (EPO) to elicit a pro-angiogenic effect on human mesenchymal stem cells (hMSC) was tested. hMSC are currently under study as therapeutic delivery agents that target tumor vessels. Hypoxia favors the differentiation of hMSC towards a pro-angiogenic program. However, the classical angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, are not fully capable of restoring this effect. The hypoxia-regulated factor, EPO, induces angiogenesis in endothelial cells. Here, EPO's pro-angiogenic effect on hMSC was analyzed. METHODS: hMSC were tested for EPO receptor expression by western blot, immunofluorescence, and flow cytometry assays. Downstream receptor signaling components JAK and STAT were measured by standard assays. Pro-angiogenesis effects mediated by EPO treatment of hMSC were measured by proliferation, cytokine, or pro-angiogenesis factor secretion, metalloprotease activation, migration, invasion, wound healing, and tubule formation assays. RESULTS: hMSC express the cognate EPO receptor and are capable of promoting angiogenesis following EPO treatment in all the angiogenesis assays tested. EPO-treated hMSC proliferate and secrete pro-angiogenesis factors more readily than untreated hMSC. EPO leads to increased hMSC chemotaxis, migration, and activation of matrix metalloprotease-2. This treatment causes greater recruitment of vessels as measured in an in vivo angiogenesis assay. CONCLUSION: EPO is capable of eliciting a pro-angiogenesis program in hMSC that instigates secretion of angiogenic factors and the subsequent recruitment of endothelium. This study defines a novel mechanism for tumor cell recruitment of blood vessels that is important to consider in the design of stem cell-based therapies.