FAK promotes stromal PD-L2 expression associated with poor survival in pancreatic cancer

BACKGROUND: Pancreatic Cancer is one of the most lethal cancers, with less than 8% of patients surviving 5 years following diagnosis. The last 40 years have seen only small incremental improvements in treatment options, highlighting the continued need to better define the cellular and molecular pathways contributing to therapy response and patient prognosis. METHODS: We combined CRISPR, shRNA and flow cytometry with mechanistic experiments using a Kras(G12D)p53(R172H) mouse model of pancreatic cancer and analysis of publicly available human PDAC transcriptomic datasets. RESULTS: Here, we identify that expression of the immune checkpoint, Programmed Death Ligand 2 (PD-L2), is associated with poor prognosis, tumour grade, clinical stage and molecular subtype in patients with Pancreatic Ductal Adenocarcinoma (PDAC). We further show that PD-L2 is predominantly expressed in the stroma and, using an orthotopic murine model of PDAC, identify cancer cell-intrinsic Focal Adhesion Kinase (FAK) signalling as a regulator of PD-L2 stromal expression. Mechanistically, we find that FAK regulates interleukin-6, which can act in concert with interleukin-4 secreted by CD4 T-cells to drive elevated expression of PD-L2 on tumour-associated macrophages, dendritic cells and endothelial cells. CONCLUSIONS: These findings identify further complex heterocellular signalling networks contributing to FAK-mediated immune suppression in pancreatic cancer

Tetrahedral framework of inverse opal photonic crystals defines the optical response and photonic band gap

By forming anatase TiO2 inverse opals by infiltration of an opal photonic crystal, we demonstrate that the optical response and angle-resolved blue-shift of the band-gap of the inverse opal structure are defined by a particular three-dimensional structure of the infilled voids. The optical structure of TiO2 inverse opals usually displays significant deviation from its physical structure and from the theoretically predicted position of the photonic band-gap. Following rigorous structural characterization of the parent opal template and TiO2 inverse opals, alternative explanations for the signature of optical transmission through inverse opals are proposed. These approaches posit that, for light-matter interaction, an inverse opal is not precisely the inverse of an opal. Accurate parameters for the structure and material properties can be obtained by invoking a Bragg FCC selection rule-forbidden (-211) plane, which is not a realistic model for diffraction in the IO. Alternatively, by assuming optical interactions with just the periodic arrangement of tetrahedral filled interstitial sites in the structure of the inverse opal, a complete reconciliation with the spectral blue-shift with the angle, photonic band gap, and material parameters is obtained when a reduced unit cell is defined based on interstitial void filling. The analysis suggests a reduced interplanar spacing (d = 1/√3 D, for pore diameter D), based on the actual structure of an inverse opal in general, rather than a definition based on the inverse of an FCC packed opal. This approach provides an accurate and general description for predicting the spectral response and material parameters of ordered inverse opal photonic crystal materials

Effect of equine chorionic gonadotropin treatment during a progesterone-based timed artificial insemination program on reproductive performance in seasonal-calving lactating dairy cows

peer-reviewedThe aim of this study was to investigate the effect of progesterone (P4)-based timed artificial insemination (TAI) programs on fertility in seasonal-calving, pasture-based dairy herds. A total of 1,421 lactating dairy cows on 4 spring-calving farms were stratified based on days in milk (DIM) and parity and randomly allocated to 1 of 3 treatments: (1) control: no hormonal treatment; cows inseminated at detected estrus; (2) P4-Ovsynch: cows received a 7-d P4-releasing intravaginal device (PRID Delta; CEVA Santé Animale, Libourne, France) with 100 μg of a gonadotropin-releasing hormone (GnRH) analog (Ovarelin; CEVA Santé Animale) at PRID insertion, a 25-mg injection of PGF2α (Enzaprost; CEVA Santé Animale) at PRID removal, GnRH at 56 h after device removal and TAI 16 h later; (3) P4-Ovsynch+eCG: the same as P4-Ovsynch, but cows received 500 IU of equine chorionic gonadotropin (eCG; Syncrostim; CEVA Santé Animale) at PRID removal. At 10 d before mating start date (MSD), all cows that were ≥35 DIM were examined by transrectal ultrasound to assess presence or absence of a corpus luteum; body condition score (BCS) was also recorded. Pregnancy diagnosis was performed by transrectal ultrasonography 30 to 35 d after insemination. Overall pregnancy/AI (P/AI) was not different between groups (50.9, 49.8, and 46.3% for control, P4-Ovsynch, and P4-Ovsynch+eCG, respectively) but the 21-d pregnancy rate was increased by the use of synchronization (35.0, 51.7, and 47.2%, respectively). Compared with the control group, synchronization significantly reduced the interval from MSD to conception (34.6, 23.0, and 26.5 d, respectively) and consequently reduced the average days open (98.0, 86.0, and 89.0 d). Across all treatment groups, DIM at the start of synchronization affected P/AI (42.3, 49.5, and 53.9% for 80 DIM, respectively), but neither parity (46.5, 50.4, and 48.4% for parity 1, 2, and ≥3, respectively) nor BCS (44.0, 49.4, and 58.6% for ≤2.50, 2.75–3.25, and ≥3.50, respectively) affected the likelihood of P/AI. Two-way interactions between treatment and DIM, parity, or BCS were not detected. In conclusion, the use of TAI accelerated pregnancy establishment in cows in a pasture-based system by reducing days open, but eCG administration at PRID removal did not affect P/AI

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: II. Effects on metabolic and reproductive endocrinology and identification of physiological markers of puberty and sexual maturation

peer-reviewedThe aim of this study was (1) to examine the effect of plane of nutrition during the first and second 6 mo of life on systemic concentrations of reproductive hormones and metabolites in Holstein-Friesian dairy bulls, and (2) to establish relationships with age at puberty and postpubertal semen production potential. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high or low plane of nutrition for the first 6 mo of life. At 24 wk of age, bulls were reassigned, within treatment, either to remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: high-high, high-low, low-low, and low-high. Monthly blood samples were analyzed for metabolites (albumin, urea, total protein, β-hydroxybutyrate, glucose, nonesterified fatty acid, triglycerides and creatinine), insulin, insulin-like growth factor-1, leptin, adiponectin, FSH, and testosterone. A GnRH challenge was carried out at 16 and 32 wk of age (n = 9 bulls per treatment). Blood was collected at 15-min intervals for 165 min, with GnRH administered (0.05 mg/kg of body weight, i.v.) immediately after the third blood sample. Blood samples were subsequently analyzed for LH, FSH, and testosterone. Stepwise regression was used to detect growth and blood measurements to identify putative predictors of age at puberty and subsequent semen quality traits. Metabolic hormones and metabolites, in general, reflected metabolic status of bulls. Although FSH was unaffected by diet, it decreased with age both in monthly samples and following GnRH administration. Testosterone was greater in bulls on the high diet before and after 6 mo of age. Testosterone concentrations increased dramatically after 6 mo of age. Luteinizing hormone was unaffected by diet following GnRH administration but basal serum LH was greater in bulls on a high diet before 6 mo of age. In conclusion, the plane of nutrition offered before 6 mo of age influenced metabolic profiles, which are important for promoting GnRH pulsatility, in young bull

Effect of Exposure to Seminal Plasma Through Natural Mating in Cattle on Conceptus Length and Gene Expression

peer-reviewedA growing body of evidence suggests that paternal factors have an impact on offspring development. These studies have been mainly carried out in mice, where seminal plasma (SP) has been shown to regulate endometrial gene expression and impact embryo development and subsequent offspring health. In cattle, infusion of SP into the uterus also induces changes in endometrial gene expression, however, evidence for an effect of SP on early embryo development is lacking. In addition, during natural mating, the bull ejaculates in the vagina; hence, it is not clear whether any SP reaches the uterus in this species. Thus, the aim of the present study was to determine whether SP exposure leads to improved early embryo survival and developmental rates in cattle. To this end, Day 7 in vitro produced blastocysts were transferred to heifers (12–15 per heifer) previously mated to vasectomized bulls (n = 13 heifers) or left unmated (n = 12 heifers; control). At Day 14, heifers were slaughtered, and conceptuses were recovered to assess size, morphology and expression of candidate genes involved in different developmental pathways. Additionally, CL volume at Day 7, and weight and volume of CL at Day 14 were recorded. No effect of SP on CL volume and weight not on conceptus recovery rate was observed. However, filamentous conceptuses recovered from SP-exposed heifers were longer in comparison to the control group and differed in expression of CALM1, CITED1, DLD, HNRNPDL, PTGS2, and TGFB3. In conclusion, data indicate that female exposure to SP during natural mating can affect conceptus development in cattle. This is probably achieved through modulation of the female reproductive environment at the time of mating. Keywords: seminal plasma, embryo development, corpus luteu

Epinephrine and dDAVP administration in patients with congenital nephrogenic diabetes insipidus Evidence for a pre-cyclic AMP V2 receptor defective mechanism

Epinephrine and dDAVP administration in patients with congenital nephrogenic diabetes insipidus. Evidence for a pre-cyclic AMP V2 receptor defective mechanism. We recently showed that the administration of the antidiuretic V2 specific agonist, l-desamino[8-D-arginine]vasopressin (dDAVP), to seven male patients with congenital nephrogenic diabetes insipidus (CNDI) did not cause a decrease in blood pressure nor an increase in plasma renin activity or factor VIIIc or von Willebrand factor release. In normal subjects, plasma renin activity, coagulation factors and plasma cyclic AMP are stimulated not only by dDAVP but also by the administration of epinephrine. In the present study, we measured tissue plasminogen activator (activity and antigenicity), von Willebrand factor multimers, plasma and urinary cyclic AMP concentrations following dDAVP or epinephrine administration. We infused epinephrine into three male patients with CNDI. Factor VIIIc and tissue plasminogen activator augmented by 75 to 100% and von Willebrand Factor multimers were increased; plasma renin activity and plasma cyclic AMP concentration increased by 200%. None of these values changed when the same subjects as well as eleven other male patients with CNDI received dDAVP. Furthermore, dDAVP administration increased plasma cyclic AMP concentrations in normal subjects, but not in 14 male patients with CNDI. These results demonstrate the specificity of the extrarenal V2 receptor defect expressed in our patients. The lack of a plasma cyclic AMP response to the administration of dDAVP would suggest an altered pre-cyclic AMP stimulation mechanism

FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer

Objective: Immunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack of response. Here, we aimed to further investigate the immunoregulatory function of focal adhesion kinase (FAK) in PDAC, with specific emphasis on regulation of the type-II interferon response that is critical in promoting T-cell tumour recognition and effective immunosurveillance. Design: We combined CRISPR, proteogenomics and transcriptomics with mechanistic experiments using a KrasG12Dp53R172H mouse model of pancreatic cancer and validated findings using proteomic analysis of human patient-derived PDAC cell lines and analysis of publicly available human PDAC transcriptomics datasets. Results: Loss of PDAC cell-intrinsic FAK signalling promotes expression of the immunoproteasome and Major Histocompatibility Complex class-I (MHC-I), resulting in increased antigen diversity and antigen presentation by FAK-/- PDAC cells. Regulation of the immunoproteasome by FAK is a critical determinant of this response, optimising the physicochemical properties of the peptide repertoire for high affinity binding to MHC-I. Expression of these pathways can be further amplified in a STAT1-dependent manner via co-depletion of FAK and STAT3, resulting in extensive infiltration of tumour-reactive CD8 T-cells and further restraint of tumour growth. FAK-dependent regulation of antigen processing and presentation is conserved between mouse and human PDAC, but is lost in cells/tumours with an extreme squamous phenotype. Conclusion: Therapies aimed at FAK degradation may unlock additional therapeutic benefit for the treatment of PDAC through increasing antigen diversity and promoting antigen presentation