Immunomodulatory effects of G-CSF in cancer: Therapeutic implications.

Numerous preclinical studies have reported a pro-tumour role for granulocyte colony-stimulating factor (G-CSF) that is predominantly mediated by neutrophils and MDSCs, the major G-CSF receptor expressing populations. In the presence of G-CSF (either tumour-derived or exogenous) these myeloid populations commonly exhibit a T cell suppressive phenotype. However, the direct effects of this cytokine on other immune lineages, such as T and NK cells, are not as well established. Herein we discuss the most recent data relating to the effect of G-CSF on the major immune populations, exclusively in the context of cancer. Recent publications have drawn attention to the other tumour-promoting effects of G-CSF on myeloid cells, including NETosis, promotion of cancer stemness and skewed differentiation of bone marrow progenitors towards myelopoiesis. Although G-CSF is safely and commonly used as a supportive therapy to prevent or treat chemotherapy-associated neutropenia in cancer patients, we also discuss the potential impacts of G-CSF on other anti-cancer treatments. Importantly, considerations for immune checkpoint blockade are highlighted, as many publications report a T cell suppressive effect of G-CSF that may diminish the effectiveness of this immunotherapy.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. METHODS: To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. RESULTS: In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. CONCLUSIONS: BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Estrogen receptor beta expression in triple negative breast cancers is not associated with recurrence or survival.

BACKGROUND: Triple negative BCa (TNBC) is defined by a lack of expression of estrogen (ERα), progesterone (PgR) receptors and human epidermal growth factor receptor 2 (HER2) as assessed by protein expression and/or gene amplification. It makes up ~ 15% of all BCa and often has a poor prognosis. TNBC is not treated with endocrine therapies as ERα and PR negative tumors in general do not show benefit. However, a small fraction of the true TNBC tumors do show tamoxifen sensitivity, with those expressing the most common isoform of ERß1 having the most benefit. Recently, the antibodies commonly used to assess ERß1 in TNBC have been found to lack specificity, which calls into question available data regarding the proportion of TNBC that express ERß1 and any relationship to clinical outcome. METHODS: To confirm the true frequency of ERß1 in TNBC we performed robust ERß1 immunohistochemistry using the specific antibody CWK-F12 ERß1 on 156 primary TNBC cancers from patients with a median of 78 months (range 0.2-155 months) follow up. RESULTS: We found that high expression of ERß1 was not associated with increased recurrence or survival when assessed as percentage of ERß1 positive tumor cells or as Allred > 5. In contrast, the non-specific PPG5-10 antibody did show an association with recurrence and survival. CONCLUSIONS: Our data indicate that ERß1 expression in TNBC tumours does not associate with prognosis.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Effects in air-exposed corn silage of medium chain fatty acids on select spoilage microbes, zoonotic pathogens, and in vitro rumen fermentation.

Medium chain fatty acid (MCFA) treatment (0.75% C6, hexanoic; C8, octanoic; C10, decanoic; or equal proportion mixtures of C6:C8:C10:C12 or C8:C10/g; C12 = dodecanoic acid) of aerobically-exposed corn silage on spoilage and pathogenic microbes and rumen fermentation were evaluated in vitro. After 24 h aerobic incubation (37 °C), microbial enumeration revealed 3 log10 colony-forming units (CFU)/g fewer (P = 0.03) wild-type yeast and molds in C8:C10-treated silage than controls. Compared with controls, wild-type enterococci decreased (P < 0.01) in all treatments except the C6:C8:C10:C12 mixture; lactic acid bacteria were decreased (P < 0.01) in all treatments except C6 and the C6:C8:C10:C12 mixture. Total aerobes and inoculated Staphylococcus aureus or Listeria monocytogenes were unaffected by treatment (P > 0.05). Anaerobic incubation (24 h at 39 °C) of ruminal fluid (10 mL) with 0.02 g overnight air-exposed MCFA-treated corn silage revealed higher hydrogen accumulations (P = 0.03) with the C8:C10 mixture than controls. Methane, acetate, propionate, butyrate, or estimates of fermented hexose were unaffected. Acetate:propionate ratios were higher (P < 0.01) and fermentation efficiencies were marginally lower (P < 0.01) with C8- or C8:C10-treated silage than controls. Further research is warranted to optimize treatments to target unwanted microbes without adversely affecting beneficial microbes.

High-Resolution Genomic Comparisons within Salmonella enterica Serotypes Derived from Beef Feedlot Cattle: Parsing the Roles of Cattle Source, Pen, Animal, Sample Type, and Production Period.

Salmonella enterica is a major foodborne pathogen, and contaminated beef products have been identified as one of the primary sources of Salmonella-related outbreaks. Pathogenicity and antibiotic resistance of Salmonella are highly serotype and subpopulation specific, which makes it essential to understand high-resolution Salmonella population dynamics in cattle. Time of year, source of cattle, pen, and sample type (i.e., feces, hide, or lymph nodes) have previously been identified as important factors influencing the serotype distribution of Salmonella (e.g., Anatum, Lubbock, Cerro, Montevideo, Kentucky, Newport, and Norwich) that were isolated from a longitudinal sampling design in a research feedlot. In this study, we performed high-resolution genomic comparisons of Salmonella isolates within each serotype using both single-nucleotide polymorphism-based maximum-likelihood phylogeny and hierarchical clustering of core-genome multilocus sequence typing. The importance of the aforementioned features in clonal Salmonella expansion was further explored using a supervised machine learning algorithm. In addition, we identified and compared the resistance genes, plasmids, and pathogenicity island profiles of the isolates within each subpopulation. Our findings indicate that clonal expansion of Salmonella strains in cattle was mainly influenced by the randomization of block and pen, as well as the origin/source of the cattle, i.e., regardless of sampling time and sample type (i.e., feces, lymph node, or hide). Further research is needed concerning the role of the feedlot pen environment prior to cattle placement to better understand carryover contributions of existing strains of Salmonella and their bacteriophages. IMPORTANCESalmonella serotypes isolated from outbreaks in humans can also be found in beef cattle and feedlots. Virulence factors and antibiotic resistance are among the primary defense mechanisms of Salmonella, and are often associated with clonal expansion. This makes understanding the subpopulation dynamics of Salmonella in cattle critical for effective mitigation. There remains a gap in the literature concerning subpopulation dynamics within Salmonella serotypes in feedlot cattle from the beginning of feeding up until slaughter. Here, we explore Salmonella population dynamics within each serotype using core-genome phylogeny and hierarchical classifications. We used machine learning to quantitatively parse the relative importance of both hierarchical and longitudinal clustering among cattle host samples. Our results reveal that Salmonella populations in cattle are highly clonal over a 6-month study period and that clonal dissemination of Salmonella in cattle is mainly influenced spatially by experimental block and pen, as well by the geographical origin of the cattle.

Mouse Breast Carcinoma Monocytic/Macrophagic Myeloid-Derived Suppressor Cell Infiltration as a Consequence of Endothelial Dysfunction in Shb-Deficient Endothelial Cells Increases Tumor Lung Metastasis.

Metastasis reflects both the inherent properties of tumor cells and the response of the stroma to the presence of the tumor. Vascular barrier properties, either due to endothelial cell (EC) or pericyte function, play an important role in metastasis in addition to the contribution of the immune system. The Shb gene encodes the Src homology-2 domain protein B that operates downstream of tyrosine kinases in both vascular and immune cells. We have investigated E0771.lmb breast carcinoma metastasis in mice with conditional deletion of the Shb gene using the Cdh5-CreERt2 transgene, resulting in inactivation of the Shb-gene in EC and some hematopoietic cell populations. Lung metastasis from orthotopic tumors, tumor vascular and immune cell characteristics, and immune cell gene expression profiles were determined. We found no increase in vascular leakage that could explain the observed increase in metastasis upon the loss of Shb expression. Instead, Shb deficiency in EC promoted the recruitment of monocytic/macrophagic myeloid-derived suppressor cells (mMDSC), an immune cell type that confers a suppressive immune response, thus enhancing lung metastasis. An MDSC-promoting cytokine/chemokine profile was simultaneously observed in tumors grown in mice with EC-specific Shb deficiency, providing an explanation for the expanded mMDSC population. The results demonstrate an intricate interplay between tumor EC and immune cells that pivots between pro-tumoral and anti-tumoral properties, depending on relevant genetic and/or environmental factors operating in the microenvironment.

Astragallus mollissimus plant extract: a strategy to reduce ruminal methanogenesis.

Ruminal methanogenesis is considered an inefficient process as it can result in the loss of 4 to 12% of the total energy consumed by the ruminant. Recent studies have shown that compounds such as nitroethane, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-1-propionic acid are capable of inhibiting methane production during in vitro studies. However, all of these nitrocompounds came from a synthetic origin, which could limit their use. In contrast, some plants of the Astragallus genus produce a natural nitrocompound, although its anti-methanogenic effect has not been evaluated. To determine the anti-methanogenic effect, in vitro cultures of freshly collected mixed populations of ruminal microbes were supplemented with A. mollissimus extracts (MISER). Cultures supplemented with 2-nitroethanol, ethyl 2-nitroacetate, or nitroethane were used as positive controls whereas distilled water was added to the untreated control tubes. After a 24 h incubation period, the methane production was reduced by more than 98% for the samples treated with A. mollissimus extract (P < 0.05) compared to the untreated controls (10.2 ± 0.1 mmol mL-1 incubated liquid). Cultures supplemented with MISER produced a greater (P < 0.05) amount of total VFA, compared to the rest of treated and untreated cultures. Considering that there are significant differences between MISER treatment, positive controls and untreated cultures (P < 0.05) regarding the amounts of total gas, gas composition (CH4 and H2), and the amount of VFA produced, it is concluded that Astragallus mollissimus poses an alternative strategy to reduce ruminal methanogenesis. To further explore such alternative, it is necessary to determine if the metabolization byproducts are safe and/or useful for the animal.

FGF13 promotes metastasis of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes, due to the high propensity to develop distant metastases. Hence, new molecular targets for therapeutic intervention are needed for TNBC. We recently conducted a rigorous phenotypic and genomic characterization of four isogenic populations of MDA-MB-231 human triple-negative breast cancer cells that possess a range of intrinsic spontaneous metastatic capacities in vivo, ranging from nonmetastatic (MDA-MB-231_ATCC) to highly metastatic to lung, liver, spleen and spine (MDA-MB-231_HM). Gene expression profiling of primary tumours by RNA-Seq identified the fibroblast growth factor homologous factor, FGF13, as highly upregulated in aggressively metastatic MDA-MB-231_HM tumours. Clinically, higher FGF13 mRNA expression was associated with significantly worse relapse free survival in both luminal A and basal-like human breast cancers but was not associated with other clinical variables and was not upregulated in primary tumours relative to normal mammary gland. Stable FGF13 depletion restricted in vitro colony forming ability in MDA-MB-231_HM TNBC cells but not in oestrogen receptor (ER)-positive MCF-7 or MDA-MB-361 cells. However, despite augmenting MDA-MB-231_HM cell migration and invasion in vitro, FGF13 suppression almost completely blocked the spontaneous metastasis of MDA-MB-231_HM orthotopic xenografts to both lung and liver while having negligible impact on primary tumour growth. Together, these data indicate that FGF13 may represent a therapeutic target for blocking metastatic outgrowth of certain TNBCs. Further evaluation of the roles of individual FGF13 protein isoforms in progression of the different subtypes of breast cancer is warranted.

Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice.

BACKGROUND: Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data. METHODS: We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24midCD49fhi)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity. RESULTS: Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24+CD49fhi) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity. CONCLUSIONS: Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.

Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2+ve breast cancer metastasis.

BACKGROUND: Human epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of neratinib. METHODS: TBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting. RESULTS: TBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy. CONCLUSIONS: The TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of neratinib neoadjuvant therapy is warranted.

Bone morphogenetic protein signaling in breast cancer progression.

Breast cancer is the most prevalent type of cancer amongst women worldwide. The mortality rate for patients with early-stage breast cancer has been decreasing, however, the 5-year survival rate for patients with metastatic disease remains poor, currently at 27%. Here, we have reviewed the current understanding of the role of bone morphogenetic protein (BMP) signaling in breast cancer progression, and have highlighted the discordant results that are reported in different studies. We propose that some of these contradictory outcomes may result from signaling through either the canonical or non-canonical pathways in different cell lines and tumors, or from different tumor-stromal interactions that occur in vivo.

Breast tumour organoids: promising models for the genomic and functional characterisation of breast cancer.

Until recently, established cancer cell lines have been used extensively in breast cancer research, due largely to the difficulties associated with the manipulation and long-term maintenance in culture of primary tumour cells from patients. The recent development of organoid cultures has provided new opportunities to model and analyse patient samples, allowing the propagation of malignant cells under conditions that resemble the three-dimensional growth of breast tumours. They have proved efficacious in preserving the heterogeneity of primary samples and are emerging as a new model to further characterise the molecular features of breast cancer. Organoids formed from patient-derived cells are now in use for the evaluation of drug sensitivity and to validate disease-causing genomic variations. Here, the advantages and limitations of organoid cultures will be discussed and compared with the parallel development of other two- and three-dimensional culture strategies and with patient-derived xenografts. In particular, we will focus on the molecular characterisation of breast cancer organoids and provide some examples of how they have been used in functional studies.

Population Dynamics of Salmonella enterica within Beef Cattle Cohorts Followed from Single-Dose Metaphylactic Antibiotic Treatment until Slaughter.

Antibiotic use in cattle can select for multidrug-resistant Salmonella enterica, which is considered a serious threat by the U.S. Centers for Disease Control and Prevention. A randomized controlled longitudinal field trial was designed to determine the long-term effects of a single dose of ceftiofur or tulathromycin on Salmonella population characteristics in cattle feces and peripheral lymph nodes and on hides. A total of 134 beef cattle from two sources were divided among 12 pens, with cattle in each of the 3-pen blocks receiving a single dose of either ceftiofur or tulathromycin or neither (control) on day 0. Fecal samples were collected before treatment (day 0) and repeatedly following treatment until slaughter (day 99+). Hide and lymph node samples were collected at slaughter age. Salmonella prevalence, phenotypic antimicrobial resistance, serotype, and phylogenetic relationships were examined. Multilevel mixed logistic regression models indicated no significant effects (P ≥ 0.218) of metaphylactic antibiotics on the prevalence of Salmonella across sample types. However, there was a significant time effect observed, with prevalence increasing from spring through the midsummer months (P < 0.0001) in feces. The majority of Salmonella isolates were pansusceptible to a panel of 14 antibiotics both before and after treatment. Highly prevalent Salmonella serotypes were Salmonella enterica serovar Montevideo, Salmonella enterica serovar Anatum, Salmonella enterica serovar Cerro, and Salmonella enterica serovar Lubbock across all sample types. Strong pen and cattle source serotype clustering effects were observed among Salmonella isolates originating from fecal, lymph node, and hide samples; however, the potential role of Salmonella isolates from the pen environment prior to animal placement was not assessed in this study.IMPORTANCESalmonella is a leading bacterial foodborne pathogen, causing a significant number of human infections and deaths every year in the United States. Macrolides and 3rd-generation cephalosporins play critical roles in the treatment of human salmonellosis. Use of these antibiotics in beef cattle can select for resistant bacteria that may enter the food chain or spread from the farm via manure. There is a lack of longitudinal research concerning the long-term effects of metaphylactic antibiotic administration. Here, we assessed Salmonella population dynamics during the feeding period until slaughter following single-dose antibiotic treatment. We found no long-term effects of antibiotic use early in the cattle-feeding period on Salmonella prevalence and antimicrobial resistance at slaughter. We identified the pens in which cattle were housed as the factor that contributed most to Salmonella serotypes being shared; importantly, the dominant strain in each pen changed repeatedly over the entire feeding period.

G-CSF Receptor Blockade Ameliorates Arthritic Pain and Disease.

G-CSF or CSF-3, originally defined as a regulator of granulocyte lineage development via its cell surface receptor (G-CSFR), can play a role in inflammation, and hence in many pathologies, due to its effects on mature lineage populations. Given this, and because pain is an extremely important arthritis symptom, the efficacy of an anti-G-CSFR mAb for arthritic pain and disease was compared with that of a neutrophil-depleting mAb, anti-Ly6G, in both adaptive and innate immune-mediated murine models. Pain and disease were ameliorated in Ag-induced arthritis, zymosan-induced arthritis, and methylated BSA/IL-1 arthritis by both prophylactic and therapeutic anti-G-CSFR mAb treatment, whereas only prophylactic anti-Ly6G mAb treatment was effective. Efficacy for pain and disease correlated with reduced joint neutrophil numbers and, importantly, benefits were noted without necessarily the concomitant reduction in circulating neutrophils. Anti-G-CSFR mAb also suppressed zymosan-induced inflammatory pain. A new G-CSF-driven (methylated BSA/G-CSF) arthritis model was established enabling us to demonstrate that pain was blocked by a cyclooxygenase-2 inhibitor, suggesting an indirect effect on neurons. Correspondingly, dorsal root ganglion neurons cultured in G-CSF failed to respond to G-CSF in vitro, and Csf3r gene expression could not be detected in dorsal root ganglion neurons by single-cell RT-PCR. These data suggest that G-CSFR/G-CSF targeting may be a safe therapeutic strategy for arthritis and other inflammatory conditions, particularly those in which pain is important, as well as for inflammatory pain per se.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes.

Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Effects of thymol and carvacrol, alone or in combination, on fermentation and microbial diversity during in vitro culture of bovine rumen microbes.

Two essential oils (EO), thymol and carvacrol, were used in six ratio (100:00, 80:20, 60:40, 40:60, 20:80 and 00:100) combinations of both EO and in a dose of 0.2 g L-1 in bovine ruminal culture medium, 24-h cultures, to evaluate effects on total gas production (TGP), methane production, in vitro dry matter digestibility (IVDMD) and in vitro culture population dynamics of methanogenic and total bacteria. Total DNA extracted from ruminal microorganisms was subjected to denaturing gradient gel electrophoresis (DGGE)-polymerase chain reaction (PCR) to examine effects on bacterial populations. The effect of EO on TGP and IVDMD were assessed by comparison to untreated control cultures. In general, methane production by the microbial populations appeared to be higher with treatments containing the highest concentration of thymol than with treatments containing more carvacrol resulting in a tendency for greater methane-inhibiting activity achieved as the thymol concentration in the thymol:carvacrol mixtures decreased linearly. The population of total bacteria with a 74.5% Dice similarity coefficient for comparison of DGGE band patterns indicating shifts in bacterial constituents as EO ratios changed. No effects on TGP, IVDMD while only slight shifts in the methanogenic populations were seen with an overall 91.5% Dice similarity coefficient.

Effect of waste milk pasteurization on fecal shedding of Salmonella in preweaned calves.

The objective of the current research was to determine if pasteurization of nonsaleable waste milk influences fecal Salmonella concentrations and prevalence, or antimicrobial susceptibility and serotype of the cultured isolates. Holstein dairy calves (n = 211) were housed on a single commercial dairy in the southwestern United States and randomly allotted to be fed either pasteurized (PWM; n = 128 calves) or nonpasteurized waste milk (NPWM; n = 83 calves). Fecal samples were collected via rectal palpation or from freshly voided, undisturbed fecal pats, weekly during the first 4 wk of the animal's life and then again at weaning. Eight total collections were made and 1,117 fecal samples cultured for Salmonella. One isolate from each culture-positive fecal sample was preserved for antimicrobial susceptibility screening and serotyping. Sixty-nine percent of the fecal samples were culture positive for Salmonella with no difference due to treatment (67.7 and 69% Salmonella positive for PWM and NPWM treatments, respectively). Few fecal samples (178/1,117; 15.9%) contained Salmonella concentrations above the limit of detection (∼1 cfu/g of feces) with concentrations ranging from 1.0 to 6.46 cfu (log10)/g of feces. Concentration was not affected by treatment. Seventeen different serotypes were identified, the majority of which were Montevideo and Anatum. A greater percentage of Typhimurium (87 vs. 13%), Muenchen (88 vs. 12%), and Derby (91 vs. 9%) were recovered from calves fed PWM compared with NPWM-fed calves. Conversely, Newport (12.5 vs. 86%), Bredeney (22.2 vs. 77.8%), and Muenster (12.5 vs. 87.5%) were lower in PWM compared with NPWM treatments. The majority (66.7%) of isolates were susceptible to all of the antibiotics examined. Results from this one commercial dairy suggest that milkborne Salmonella is not an important vector of transmission in dairy neonates, nor does pasteurization of waste milk influence fecal shedding of this pathogen. Caution should be used, however, when extrapolating results to other farms as Salmonella contamination of milk on farm is well documented. The potential benefits of pasteurization in disease prevention outweigh the potential risks of feeding a nonpasteurized product and warrants incorporation into any calf-rearing program using nonsaleable waste milk for feeding young dairy neonates.

Bacterial communities related to 3-nitro-1-propionic acid degradation in the rumen of grazing ruminants in the Qinghai-Tibetan Plateau.

The objectives of this current study were to characterize the overall rumen bacterial community in grazing yak and two sheep species (Tibetan and Small Tail Han sheep) reared in the unique environmental conditions of the Qinghai-Tibetan Plateau, as well as the bacterial community associated with the detoxification of a phytotoxin, 3-nitro-1-propionic acid (NPA), during in vitro culture with 4.2 mM NPA. Using 16S rRNA gene high-throughput sequencing, it was found that the yak rumen harbored populations showing a higher bacterial diversity compared to Tibetan sheep. The rumen bacterial community in the three ruminant species differed from each other. PICRUSt analysis identified that the pathway involved in nitrogen metabolism was enriched in Tibetan sheep while that related to fatty acid biosynthesis was over-represented in the yak. The methane metabolism pathway was dominant in bacterial populations from the Small Tail Han sheep. Comparisons between freshly collected rumen fluid and populations subjected to consecutive 72 h batch cultures revealed substantial decreases in alpha diversity in populations cultured with NPA. Moreover, the relative abundances of some bacterial taxa changed significantly, with increased abundance of Proteobacteria and Actinobacteria. In addition, the overall community structure of the bacterial population in the freshly collected ruminal fluid was clearly different than that within populations observed in the 72 h batch cultures likely due to the impact of NPA treatments and the more restrictive growth conditions of the culture medium. In regard to PICRUSt analysis, the methane metabolism pathway became scarce in Tibetan and Small Tail Han sheep, whereas the energy and carbohydrate metabolic pathways such as nitrogen metabolism, ABC transporters and glycolysis/gluconeogenesis were found to be maintained across all populations. Results from the present study provide new information on the bacterial and functional composition within ruminal populations adapted to three economically important grazing ruminant species prominent on the Qinghai-Tibetan Plateau. The results further reveal that effects of NPA treatment on community structure can have an impact not only the metabolism of NPA but on other digestive functions as well.