No description available.

No description available.

No description available.

BAP1 (BRCA-Associated Protein1) is a tumor suppressor gene encoding a deubiquitinating enzyme (DUB) that regulates many facets of cellular biology. Genetic studies have demonstrated that somatic BAP1 mutations occur in numerous cancer types and that germline BAP1 mutations lead to a cancer susceptibility disorder that predisposes individuals to various tumors, in particular malignant mesothelioma (MM) and both uveal melanoma (UM) and cutaneous melanoma (CM). The Testa laboratory has identified several families (including one in Louisiana, designated Lou) with germline BAP1 mutations, in which there were recurrent cases of MM, UM, and CM. We generated a Bap1 mutant mouse model with a knockin mutation identical to that observed in the Lou family (Bap1+/Lou ) to test whether this mutation alone confers susceptibility to ultraviolet (UV) light-induced melanomagenesis either alone or in combination with a mutation found in a well-established Hepatocyte Growth Factor (HGF)/Scatter Factor transgenic mouse model. Neither Bap1+/Lou, HGF, nor Bap1+/Lou;HGF mice showed a significantly higher incidence or shorter latency of UV light-induced melanoma than wild type (WT) mice. The study also suggests that germline mutation of Bap1 alone does not cause an increased incidence of UV-induced melanomas under the conditions used in this investigation. Recent evidence indicates that BAP1 participates in the DNA damage repair response, suggesting that BAP1’s role in tumorigenesis could be particularly important in cancers associated with environmental carcinogens such as ultraviolet irradiation (UVR). To further investigate the role of BAP1 (Bap1 in the mouse) in DNA damage, we first knocked down BAP1 in human melanocytes as well as Melan-A and Melan-C mouse melanocytes and then exposed the cells to UVR, followed by analysis of DNA damage repair. UVR-induced and steady state levels of DNA damage were higher in BAP1-knockdown cells compared to shGFP-control cells. Levels of UVR-related DNA damage markers such as p53, γH2AX and CPD (cyclobutane pyrimidine dimers) were increased following BAP1 loss and UVR treatment. Cell cycle analysis by flow cytometry demonstrated that cells with knockdown of BAP1 and post UVR treatment showed a higher proportion of cells in S/G2 phase. Such an effect could be due to BAP1 loss and consequent inability to repair DNA damage and/or cell cycle progression. These data are consistent with a role for BAP1 in UVR-induced DNA damage repair. In MM, it is unclear to what extent BAP1 mutations cooperate tumorigenically with mutations of other tumor suppressor genes (TSGs) implicated in MM, such as CDKN2A and NF2. While germline mutations of BAP1 clearly predispose to MM, whether somatic mutations of BAP1 drive a more aggressive, metastatic tumor phenotype may depend on the disease type. For such studies, we used conditional knockout (CKO) mice along with intrathoracic (IT) or intraperitoneal (IP) injection of adenovirus expressing Cre recombinase (Adeno-Cre) to excise critical homozygously floxed TSGs in the mesothelial lining. These labor-intensive experiments demonstrated that while homozygous deletion of Bap1, Cdkn2a, or Nf2 alone in the pleural cavity (IT) of genetically engineered mouse (GEM) models gave rise to few or no MMs, inactivation of Bap1 cooperated with loss of either Nf2 or Cdkn2a to drive development of MM in ~20% of double CKO mice, and a high incidence (22/26, 85%) of MMs with short latency (12 weeks) was observed in Bap1;Nf2;Cdkn2a (triple)-CKO mice. The same trend was confirmed when the same gene combinations were homozygously deleted IP in these same GEM models, except that a much higher incidence of MM was observed in homozygously floxed Bap1 (Bap1f/f) mice injected IP versus IT, which may be due to a larger cell surface area of the peritoneum. Adeno-Cre treatment of normal mesothelial cells from Bap1f/f;Nf2 f/f;Cdkn2 f/f mice, but not from mice with knockout of one or any two of these tumor suppressor genes, resulted in robust spheroid formation in vitro, suggesting that homozygous deletion of all three of these TSGs is sufficient to drive a cancer stem cell-like potential. RNA-seq analysis of pleural MMs from triple-CKO mice revealed enrichment of many genes transcriptionally regulated by the polycomb repressive complex 2 (PRC2). Other genes upregulated in MMs from triple-CKO mice included Vegfd and Pak3, which encode proteins involved in angiogenic and cell motility pathways. In conclusion, we hypothesize that inherited mutations of BAP1 may increase susceptibility to certain environmental factors that may induce DNA damage and contribute to cancer development. Our data also indicate that cooperative somatic inactivation of Bap1, Nf2, and Cdkn2a results in rapid, highly aggressive MMs, and that deletion of Bap1 contributes to tumorigenesis, in part, by loss of PRC2-mediated gene repression of tumorigenic target genes and by acquisition of stem-cell potential. Thus, our studies suggest a potential avenue for therapeutic intervention. / Biomedical Sciences

Yes / Backgorund Prior data suggest pre-diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple-negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes. Method NOD/SCID mice were implanted with HER2+ MDA-MB-231/LN/2-4/H2N, trastuzumab-resistant HER2+ HCC1954 or a TNBC patient-derived xenograft (PDX). A daily low-dose aspirin regimen commenced until primary tumours reached ~250 mm3 and subsequently resected. MDA-MB-231/LN/2-4/H2N mice were monitored for metastasis utilising imaging. To interrogate the survival benefit of pre-treatment aspirin, 3 weeks post-resection, HCC1954/TNBC animals received standard-of-care (SOC) chemotherapy for 6 weeks. Primary tumour response to aspirin was interrogated using immunohistochemistry. Results Aspirin delayed time to metastasis in MDA-MB-231/LN/2-4/H2N xenografts and decreased growth of HER2+/TNBC primary tumours. Lymphangiogenic factors and lymph vessels number were decreased in HER2+ tumours. However, no survival benefit was seen in aspirin pre-treated animals (HCC1954/TNBC) that further received adjuvant SOC, compared with animals treated with SOC alone. In an effort to study mechanisms responsible for the observed reduction in lymphangiogenesis in HER2+ BC we utilised an in vitro co-culture system of HCC1954 tumour cells and mesenchymal stromal cells (MSC). Aspirin abrogated the secretion of VEGF-C in MSCs and also decreased the lymph/angiogenic potential of the MSCs and HCC1954 by tubule formation assay. Furthermore, aspirin decreased the secretion of uPA in HCC1954 cells potentially diminishing its metastatic capability. Conclusion Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo-preventative agent in the HER2+ or TNBC setting. / Funding is acknowledged from the Irish Cancer Society Collaborative Cancer Research Centre under BREAST- PREDICT grant CCRC13GAL (www.breas tpred ict.com). I.S.M, E.M and A.T.B, are members of the EurOPDX Consortium, and receive funding from the European Union's Horizon 2020 research and innovation programme, grant agreement no. #731105 (EurOPDX Research Infrastructure, www.europ dx.eu).

Feral swine are a nuisance species across the United States that costs around $1.5 billion each year in agricultural, environmental, and personal property damages. In the last ten years the population of feral swine is estimated to have quadrupled and novel population control methods are needed. Furthermore, feral swine are known carriers of zoonotic diseases such as brucellosis, which threatens both livestock biosecurity and public health. Recombinant multimeric gonadotropin-releasing hormone (mGnRH) has been previously used as a subunit vaccine to induce immunocontraception in feral pigs. However, potent adjuvants and large amounts of purified antigen are needed to elicit a robust anti-GnRH immune response and current delivery methods are limited. Brucella suis strain VTRS2 can be used as a novel platform to deliver mGnRH without the use of antibiotic resistant markers. Strain VTRS2 was created by deletion of the LPS biosynthesis gene wboA as well as the leuB gene required for leucine biosynthesis inside the nutrient-depleted intracellular environment occupied by Brucella. Mutations in wboA are known to attenuate Brucella strains such as the vaccine strain B. abortus RB51, however strain RB51 is rifampin resistant and has poor efficacy in swine. Strain VTRS2 confers significant protection against B. suis challenge in mice and additionally shows evidence of protection in feral swine. Furthermore, the mGnRH antigen can be delivered using the pNS4 plasmid (which expresses leuB under its native promoter) thus maintaining the plasmid in strain VTRS2 under leucine-deficient conditions while expressing recombinant antigen in the host. The murine model was used to determine the clearance kinetics of strain VTRS2-mGnRH and to measure vaccine efficacy against challenge by virulent B. suis 1330. Subsequently the effects of the VTRS2-mGnRH vaccine on fertility were assessed in breeding trials in mice. Strains VTRS2 and VTRS2-mGnRH were found to be protective against virulent Brucella suis challenge. Strain VTRS2-mGnRH elicited an anti-mGnRH antibody response in vaccinated mice, though an effect on fertility was not observed. An improved vaccine against brucellosis in swine, which also confers immunocontraception without the introduction of antibiotic resistance, could become an important tool in the management of this nuisance invasive species. / Ph. D. / Feral swine (<i>Sus scrofa</i>) are a major invasive species in the United States. Their population is estimated to have quadrupled in the past ten years and their geographic range has expanded to include at least 39 states. In addition to causing over $1.5 billion annually in agricultural, environmental, and property damages, feral swine also carry several diseases of public health and agricultural significance including influenza, leptospirosis, and brucellosis. Among these diseases, brucellosis, caused by the bacterial organism <i>Brucella suis</i>, is of particular concern because of its ability to cause reproductive losses in domestic swine and cattle as well as a debilitating febrile illness in humans. The disease has been eradicated from domestic livestock in the United States, and reintroduction could have severe consequences for both animal agriculture and public health. With the continued expansion of the feral swine population, the potential for spillover of disease into domestic livestock and humans increases. Additional tools are therefore needed to aid in both population and disease control. Immunocontraceptive vaccines have previously been used to reduce population growth in wildlife, and have been proposed for use in feral swine. Immunocontraceptives work by introducing an immunogenic form of a reproductive hormone which then causes an autoimmune response against the natural form of the hormone produced by the animal. This work describes the development of the <i>B. suis</i> vaccine strain VTRS2-mGnRHb, which was created from a virulent strain of <i>B. suis</i> to make an attenuated live vaccine capable of delivering the immunocontraceptive antigen mGnRH. The goals of strain VTRS2 were to act as a vaccine which protect against virulent <i>B. suis</i> challenge and which confers an infertility effect in the mouse model. An improved vaccine against brucellosis in feral swine, which also confers an infertility effect, could become an important tool in the management of this nuisance invasive species.

40% of severe acute respiratory syndrome coronavirus two (SARS-CoV-2) severe cases develop acute kidney injury (AKI). Current treatment for renal complications limits financial and material resources available. To explore alternative treatments and accelerate research in case of future coronavirus outbreaks, a mouse model of coronavirus disease 2019-associated AKI (C19-AKI) would represent a critical biomedical research tool. The surrogate model of C19-AKI (SMC) developed consisted of angiotensin-converting enzyme two (ACE2) knockout (KO) mice receiving 400 ng/kg/min of angiotensin (Ang) II by osmotic minipump for eight days with a single injection of lipopolysaccharide (LPS; 10 mg/kg) on the seventh day of Ang II and euthanasia 24 hours after LPS. Similarly, to C19-AKI, the SMC exhibited albuminuria, elevated blood urea nitrogen, electrolyte imbalance, neutrophil infiltration, and upregulation of the G-coupled protein receptor (GPR)84 and pro-inflammatory and injury markers. GPR84 was found in bronchoalveolar lavage fluid neutrophils of coronavirus disease 2019 (COVID-19) patients, suggesting a potential implication of GPR84 in the disease. We hypothesised that GPR84 deletion or antagonism with GLPG-1205 could attenuate SMC’s indices of renal injury and inflammation. GLPG-1205 and GPR84 KO had no effects in the SMC model, as suggested by unchanged albuminuria, electrolytes, and markers expression. Interestingly, neutrophil infiltration was attenuated by GLPG-1205 only. The SMC is an interesting tool for therapeutic development for infections associated with renal injury, such as SARS-CoV-2. GPR84 role in the SMC needs to be further assessed.

No description available.

No description available.

Aortic dissection is a detrimental disease with a high mortality. However, the mechanisms regulating the susceptibility to aortic dissection remain unknown. We hypothesize that endothelial oxidative stress due to the activation of the reactive oxygen species (ROS)-generating Nox2 enzyme may play an important role in the development of aortic dissection. To investigate this, we generated transgenic mice (C57BL/6J background) with endothelial specific over-expression of Nox2 (Nox2 Tg) under the control of a tie-2 promoter. Expression of the human Nox2 transgene was confirmed by qRT-PCR to be found only in endothelial cells (EC) isolated from transgenic mice, and not in Wt EC or vascular smooth muscle cells (VSMC) and macrophages isolated from either genotype. Wild-type (Wt) littermates and Nox2 Tg male mice (22-24 weeks old, n=11) were treated with saline or Ang II (1mg/kg/day) via subcutaneous mini-pump for 28 days. There was no significant difference in the pressor responses to Ang II between Wt and Nox2 Tg mice (Wt 121±7mmHg vs. Nox2-Tg 122±6mmHg). However, 5/11 Nox2 Tg mice developed aortic dissections compared to 0/11 Wt mice (P<0.05). Immunohistochemistry revealed significant increases in endothelial VCAM-1 expression, MMP activity and CD45+ inflammatory cell recruitment in the aortas of Nox2 Tg mice after 5 days of Ang II infusion. Inflammatory cell recruitment was confirmed by FACS analysis of cells from digested aortas (P<0.05). Explanted aortas from Nox2-Tg mice had significantly greater secreted pro-inflammatory cytokine, Cyclophilin A (CypA) both at baseline and after 5 days of Ang II infusion compared to Wt littermates. Compared to primary Wt EC and VSMC, Nox2-Tg primary EC, but not primary VSMC, had increased ROS production which was accompanied by increased endothelial CypA secretion and ERK1/2 activation. Furthermore, conditioned media from Nox2-Tg EC induced greater ERK1/2 phosphorylation compared to conditioned media from Wt controls. Knockdown of CypA from sEND.1 endothelial conditioned media by siRNA knockdown abolished VSMC Erk1/2 phosphorylation. In conclusion, we demonstrate for the first time that a specific increase in endothelial ROS through the over-expression of Nox2 was sufficient to induce aortic dissection in response to Ang II stimulation. Endothelial secreted CypA could be the signalling mechanism by which increased endothelial ROS regulates the inflammatory response and the susceptibility to aortic dissection.