Disruption of the Suprachiasmatic Nucleus in Fibroblast Growth Factor Signaling-Deficient Mice.

Fibroblast growth factor (Fgf) 8 is essential for the development of multiple brain regions. Previous studies from our laboratory showed that reduced Fgf8 signaling led to the developmental alterations of neuroendocrine nuclei that originated within the diencephalon, including the paraventricular (PVN) and supraoptic (SON) nuclei. To further understand the role of Fgf8 in the development of other hypothalamic nuclei, we examined if Fgf8 and its cognate receptor, Fgfr1, also impact the integrity of the suprachiasmatic nuclei (SCN). The SCN control an organism's circadian rhythm and contain vasoactive intestinal peptide (VIP)-producing neurons as the main input neurons. Mice hypomorphic for Fgf8, Fgfr1, or both were examined for their SCN volume and the number of VIP neurons on postnatal day (PN) 0; adult hypomorphic mice were further examined for SCN function by quantifying SCN neuronal activation using cFos as a marker. On PN0, mice homozygous for Fgf8 hypomorphy displayed the most severe reduction of the SCN volume and VIP neurons. Those heterozygous for Fgf8 hypomorphy alone or Fgf8 combined with Fgfr1 hypomorphy, called double heterozygotes (DH), showed normal SCN volume but significantly reduced VIP neurons, albeit less severely than the homozygotes. Adult wild type, heterozygous Fgf8 hypomorphs (F8 Het), and DH mice were also examined for SCN cFos activation at three time points: 1 h (morning), 6 h (afternoon), and 11 h (evening) after light onset. In F8 Het mice, a significant change in the pattern of cFos immunostaining that may reflect delayed morning SCN activation was observed. Overall, our studies provide evidence supporting that deficiencies in Fgf8 not only impact the structural integrity of the SCN but also the pattern of SCN activation in response to light

Spondylarthropathies (including psoriatic arthritis): 244. Validity of Colour Doppler and Spectral Doppler Ultrasound of Sacroilicac Joints Againts Physical Examination as Gold Standard

Background: Sacroiliac joints (SJ) involvement is a distinctive and charasteristic feature of Spondyloarthritis (SpA) and x-ray is the test routinely used to make a diagnosis. However, x-ray reveals late structural damage but cannot detect active inflammation. The objective of this study was to assess the validity of Doppler ultrasound in SJ. Methods: Prospective blinded and controlled study of SJ, in which three populations were compared. We studied 106 consecutive cases, who were divided into three groups: a) 53 patients diagnosed with SpA who had inflammatory lumbar and gluteal pain assessed by a rheumatologist; b) 26 patients diagnosed with SpA who didn't have SJ tenderness and had normal physical examination; c) control group of 27 subjects (healthy subjetcs or with mechanical lumbar pain). All patients included that were diagnosed with SpA met almost the European Spondyloarthropathy Study Group (ESSG) classification criteria. Physical examination of the SJ included: sacral sulcus tenderness, iliac gapping, iliac compression, midline sacral thrust test, Gaenslen's test, and Patrick s test were used as gold standard. Both SJ were examined with Doppler ultrasound (General Electric Logiq 9, Wauwatosa WI, USA) fitted with a 9-14 Mhz lineal probe. The ultrasonographer was blinded to clinical data. Doppler in SJ was assessed as positive when both Doppler colour and resistance index (RI) < 0.75 within the SJ area were present. Statistical analysis was performed estimating sensitivity and specificity against gold standard. The Kappa correlation coefficient was used for reliability study. Results: 106 cases (53 female, 55 male; mean age 36 10 years) were studied. There were no statistical differences between groups related to age or sex. Physical examination of SJ was positive in 38 patients (59 sacroiliac joints). US detected Doppler signal within SJ in 37 patients (58 SJ): 33 of them were symptomatic SpA (52 SJ), one of them were asymptomatic SpA (1 SJ) and one was a healthy control (1 SJ). The accuracy of US when compared to clinical data as gold standard at subject level in the overall group was: sensitivity of 68.6% and specificity of 85.7%, positive predictive value of 70.5% and negative predictive value of 84.5%. A positive likelihood ratio of 4.8, a negative likelihood ratio of 0.36 and a kappa coefficient of 0.55 were achieved. Conclusions: Doppler US of SJ seems to be a valid method to detect active SJ inflammation. Disclosure statement: The authors have declared no conflicts of interes

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Functional Authentication of a Novel Gastropod Gonadotropin-Releasing Hormone Receptor Reveals Unusual Features and Evolutionary Insight

<div><p>A gonadotropin-releasing hormone (GnRH)-like molecule was previously identified in a gastropod, <i>Aplysia californica</i>, and named ap-GnRH. In this study, we cloned the full-length cDNA of a putative ap-GnRH receptor (ap-GnRHR) and functionally authenticated this receptor as a bona fide ap-GnRHR. This receptor contains two potential translation start sites, each accompanied by a Kozak sequence, suggesting the translation of a long and a short form of the receptor is possible. The putative ap-GnRHR maintains the conserved structural motifs of GnRHR-like receptors and shares 45% sequence identity with the octopus GnRHR. The expression of the putative ap-GnRHR short form is ubiquitous in all tissues examined, whereas the long form is only expressed in parts of the central nervous system, osphradium, small hermaphroditic duct, and ovotestis. The cDNA encoding the long or the short receptor was transfected into the <i>Drosophila</i> S2 cell line and subject to a radioreceptor assay using ¹²⁵I-labeled ap-GnRH as the radioligand. Further, the transfected cells were treated with various concentrations of ap-GnRH and measured for the accumulation of cAMP and inositol monophosphate (IP1). Radioreceptor assay revealed that only the long receptor bound specifically to the radioligand. Further, only the long receptor responded to ap-GnRH with an increased accumulation of IP1, but not cAMP. Our studies show that despite the more prevalent expression of the short receptor, only the long receptor is the functional ap-GnRHR. Importantly, this is only the second report on the authentication of a protostome GnRHR, and based on the function and the phylogenetic grouping of ap-GnRHR, we suggest that this receptor is more similar to protostome corazonin receptors than chordate GnRHRs.</p></div

Seasonal changes of gonadotropin-releasing hormone in the Atlantic hagfish Myxine glutinosa

To investigate seasonal reproduction in Myxine glutinosa, we measured total brain gonadotropin-releasing hormone (GnRH) and determined gonadal stages of hagfish collected from the Gulf of Maine once a month for 12 months. Thirty hagfish from each of three different size classes of small (20-35 cm), medium (35-45 cm), and large (50-60+ cm) were sampled for brains and gonads. In the medium and large class hagfish there was an increase in GnRH concentrations during April and May that correlated with male and female gonadal maturity. Also in these size classes of female hagfish, there was a similar rise in GnRH in November and then again in January that preceded the highest incidence of large eggs (stage 7). The elevated GnRH may be influencing the onset of ovarian recrudescence which has been shown in other vertebrates. These data suggest an association of the concentration of brain GnRH with gonadal maturity and provide supportive evidence of a possible seasonal reproductive cycle in M. glutinosa shown in recent studies of [J. Exp. Zool. 301 A (2004) 352], correlating steroid production with gonadal maturation. (C) 2004 Elsevier Inc. All rights reserved

Nucleotide and deduced amino acid sequences of the full-length putative ap-GnRHR (Genbank Accession # AHE78444.1).

<p>The nucleotides (lower case letters) and amino acids (upper case letters) are numbered accordingly. The seven predicted transmembrane domains are underlined. The two putative translation start sites are highlighted in green, and the asterisk (*) denotes the stop codon. The nucleotides corresponding to the truncated polyadenylation signal (aata) are double-underlined.</p

ap-GnRH did not stimulate cAMP accumulation in S2 cells transfected with either ap-GnRHR-L or ap-GnRHR-S.

<p>Data are expressed as fold stimulation over cAMP levels in the untreated cells. Forskolin was used as a positive control. N = 3 assays run in triplicates.</p

Amino acid sequence alignment of ap-GnRHR with representative GnRHR-related receptors.

<p>Shaded amino acids indicate the transmembrane (TM) domains. The predicted intracellular loops (IL) and extracellular loops (EL) are numbered accordingly. The two putative start sites (M) are each denoted by a “+”. Ms-CRZR—corazonin receptor, <i>Manduca sexta</i> (AAR14318); Oct-GnRHR—Gonadotropin-releasing hormone receptor, <i>Octopus vulgaris</i> (GNRHR_OCTVU); Ci-GnRHRI—gonadotropin-releasing hormone receptor 1, <i>Ciona intestinalis</i> (NP_001028997); Ci-GnRHRII—gonadotropin-releasing hormone receptor 2, <i>Ciona intestinalis</i> (NP_001028996); Pm-GnRHRI—gonadotropin releasing-hormone receptor, <i>Petromyzon marinus</i> (AF439802_1); Mm-GnRH—gonadotropin-releasing hormone receptor isoform <i>Mus musculus</i> (NP_001297580). Identical, highly conserved, and less conserved amino acid residues are denoted by an asterisk (*), a colon (:), and a period (.), respectively. Ampersand (&) denotes cysteine residues involved in the disulfide bridge formation.</p