Markers for ovarian cancer: regan isoenzyme and other glycoproteins.

Since embryonic genes are not generally active in normal adult subjects and because certain of these genes are activated in cancer leading to ectopic synthesis, it is the difference between the ectopic level and the normal adult concentrations of embryonic gene products which we seek in developing "markers" for ovarian cancer. The carcinoplacental alkaline phosphatases corresponding to the term gestational phenotypes correlate positively with ovarian cancer as does hCG. Other fetal and placental glycoproteins whose presence is noted in ovarian cancer include CEA, alpha-FP, and Bj?rklund's antigen. Antigens of mucinous cystadenocarcinoma have not yet been examined for their possible fetal or placental origins. The degree of concordance of expression of Regan isoenzyme and hCG is variable. Profiles of glycoproteins would appear to offer an opportunity to inquire more deeply into the nature of ovarian cancer and from this inquiry, one can expect to develop a system of markers which can be of clinical use.     

Remote ischemic preconditioning to reduce contrast-induced acute kidney injury in chronic kidney disease: a randomized controlled trial

Background

The impact of contrast-induced acute kidney injury (CI-AKI) on patients with chronic renal disease is well-known. Remote ischemic preconditioning (RIPC) is a non-invasive method that can reduce the risk of CI-AKI, but studies on RIPC have had different results. The aim of the present study was to assess the potential impact of RIPC on CI-AKI.

Methods

In a randomized, double blinded, controlled trial, 132 patients with chronic renal dysfunction (glomerular filtration rate?<?60?mL/min/m²) who underwent coronary angiography or angioplasty received adequate hydration. RIPC was performed in 66 patients by applying an upper arm blood pressure cuff. The cuff was inflated four times for 5?min to 50?mmHg above the systolic blood pressure, followed by deflation for 5?min. In the control group, the blood pressure cuff was inflated only to 10?mmHg below the patient’s diastolic blood pressure. The primary endpoint was an increase in serum cystatin C?≥?10% from baseline to 48–72?h after exposure to the contrast.

Results

The primary endpoint was achieved in 48 (36.4%) patients (24 in each group). RIPC did not show any significant effect on the occurrence of the primary endpoint (P?=?1). In addition, when the results were analyzed based on the Mehran risk score for subgroups of patients, RIPC did not reduce the occurrence of the primary endpoint (P?=?0.97).

Conclusions

In patients at moderate-to-high risk of developing CI-AKI when an adequate hydration protocol is performed, RIPC does not have an additive effect to prevent the occurrence of CI-AKI.

Trial registration

The clinical trial was registered on (Identification number IRCT2016050222935N2, on December 19, 2016 as a retrospective IRCT).

     

Quantitation of oestrogen receptors: use of solid-phase antisteroid antibodies to quantify binding sites and determination of dissociation constant.

The dextran-coated charcoal assay method of McGuire and DeLaGarza is a well-known method for quantitation of oestrogen receptors in breast tumour tissues. Castañeda and Liao reported that the solid-phase antisteroid antibodies are superior to dextran-coated charcoal for quantitating the receptor bound steroids. In this report we compare the results obtained using both methods to quantitate oestrogen receptors and determine the affinity for steroid binding in seven human breast tumours and the uteri of rat and calf.     

SERPINB2 and miR-146a/b are coordinately regulated and act in the suppression of psoriasis-associated inflammatory responses in keratinocytes

Psoriasis is a chronic inflammatory skin disease with numerous involved factors. miR-146a and miR-146b (miR-146a/b) are anti-inflammatory miRNAs that are increased in psoriatic skin. SERPINB2 has been shown to be upregulated in the inflammation and infections. Here we aimed to study the relationship between miR-146a/b and SERPINB2 and to delineate the role of SERPINB2 in association of plaque psoriasis. We report increased SERPINB2 expression in the skin of psoriasis patients, which was in a positive relationship with psoriasis severity and in a negative relationship with miR-146a/b in psoriatic lesions. In cultured keratinocytes, both cellular and secreted SERPINB2 levels were strongly induced in response to IFN-γ and TNF-α. Interestingly, SERPINB2 mRNA was downregulated by IL-17A and the combination of TNF-α and IL-17A at time points when miR-146a was increased. The predicted binding site for miR-146a/b in 3’ untranslated region of SERPINB2 revealed no activity in luciferase assay, while siRNA silencing of miR-146a/b direct targets IRAK1 and CARD10 resulted in reduced expression of SERPINB2, suggesting that miR-146a/b indirectly control SERPINB2 expression in the skin. The siRNA silencing of SERPINB2 increased the expression of IL-8, CXCL5 and CCL5 and migration of neutrophils revealing its anti-inflammatory role in keratinocytes. Our data together suggest that SERPINB2 and miR-146a/b are part of disease-related network of molecules that are coordinately regulated and act in controlling the inflammatory responses in psoriatic skin.     

Screening for hybridomas producing antibodies to steroid hormone receptors: interference from phenol red in the hybridoma culture supernates.

The culture supernates from a variety of mouse hybridomas which were known to secrete antibodies to antigens unrelated to steroid hormone receptors were examined for routine use as experimental controls for monoclonal anti-oestrogen receptor (ER) antibodies. During this process, we made an observation which is important for all investigations on monoclonal anti-receptor antibodies. Our results indicated that the phenol red dye present in these hybridoma culture fluids by binding to the immunoglobulins (Ig) secreted by the hybridoma cells, may impart to the Ig-dye complex, a capacity to interact with ER/progesterone receptors(PR) which are present in the cytosols or in the sections of ER+ tumors. The Ig-dye may thus mimic anti-receptor antibody activity. Growth media containing phenol red and Ig-free bovine calf serum supplement or culture fluids from a non-Ig secreting hybridoma failed to show similar binding to ER+ tissues. This observation documents the need for Ig-dye complex formation to obtain such ER-Ig interaction. Complete dialysis of phenol red from the Ig positive supernates removed the observed interference of the dye. In this report we describe the nature of this interference and the results obtained before and after the removal of the dye from Ig+ culture fluids. In addition, we suggest some remedial measures that should be considered, while screening and testing the specificity of any hybridoma culture product for anti-receptor antibody activity (particularly, anti-ER or anti-PR antibodies).     

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2^E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57^KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.Recent large-scale multidimensional genomic analyses of many cancers have established a framework in which biological functions and genetic interactions of established and novel cancer genes can be explored (1, 2). We initially identified PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) as being significantly mutated in human melanomas (3), an observation that was corroborated by the recently completed TCGA melanoma study (4). PREX2 is a guanine nucleotide exchanger (GEF) for Rac1 (5, 6) and is known to bind to the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) (7). PREX2 has recently been shown to regulate insulin signaling and glucose homeostasis through modulation of the PI3K (phosphatidyl inositol 3 kinase) pathway, and also to regulate Rac1 mediated cellular invasion in a manner that cross-talks with PTEN signaling (8). Further expanding the significance of genetic perturbations of PREX2 in cancer, a recent report by the International Cancer Genome Consortium (ICGC) described the identification PREX2 as a significantly mutated gene in pancreatic ductal adenocarcinoma (PDAC) (9). Interestingly, PREX2 harbors a wide spectrum of mutations including missense and truncating mutations in PDAC, similar to observations in melanoma (3, 9).To date, the most obvious connection between PREX2 and cancer relevant pathways is through its physical interaction with PTEN (7). PTEN catalyzes the conversion of phosphatidylinositol-3,4,5-trisphosphate to phosphatidylinositol-4,5 bisphosphate. PTEN acts as a tumor suppressor and plays important roles in multiple cellular processes primarily by antagonizing PI3-kinase-AKT signaling (10–13). Pathologically, PTEN is inactivated via multiple mechanisms in about a third of melanoma tumors resulting in activation of the downstream PI3K/Akt signaling pathway (14, 15). Despite these connections to cancer signaling pathways, the exact mechanism of tumorigenesis by PREX2 mutations remains unknown. Here, we elucidated a previously unidentified mechanism of action of PREX2 mutations in melanoma pathogenesis.To study PREX2 mutations in vivo, we generated an inducible transgenic mouse model that expresses one of the truncating PREX2 mutants observed in melanoma patients, and showed that melanoma development was accelerated in this genetic context. Using integrated gene expression analysis, we identified several cellular pathways including cell cycle regulation and cytoskeletal organization to be deregulated in PREX2 mutant tumors. Biochemically, we showed that truncating PREX2 mutations increase its Rac1 guanine nucleotide exchange (GEF) activity, abolish binding to the tumor suppressor PTEN and activate the PI3K/Akt signaling pathway. Finally, we showed that PREX2 mutation or PTEN deletion induces DNA hypomethylation and down-regulation of expression of CDKN1C (p57^KIP2), a critical cell cycle regulator. In conclusion, this study demonstrates the oncogenic capability of PREX2 truncations in vivo and identifies a mechanistic link to an established oncogenic signaling pathway and downstream regulation of a tumor suppressor to impact melanoma pathogenesis.     

Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity

Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNF-induced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3(-/-) mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the "two-faced" nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others.