Clinical Factors Associated With Long-Term Benefit in Patients With Metastatic Renal Cell Carcinoma Treated With Axitinib: Real-World AXILONG Study.

BACKGROUND: Axitinib monotherapy obtained approval in pre-treated mRCC patients and recently in combination with pembrolizumab or avelumab in the first-line setting. However, patient profiles that may obtain increased benefit from this drug and its combinations still need to be identified. PATIENTS AND METHODS: Retrospective multicentre analysis describing clinical characteristics associated with axitinib long-responder (LR) population by comparing two extreme-response sub-groups (progression-free survival [PFS] ≥9 months vs. disease progression/refractory patients [RP]). A multivariate logistic-regression model was used to analyse clinical factors. Efficacy and safety were also analysed. RESULTS: In total, 157 patients who received axitinib in second or subsequent line were evaluated (91 LR and 66 RP). Older age at start of axitinib and haemoglobin levels > LLN were independent predictive factors for LR in multivariate analyses. In LR patients, median (m) PFS was 18.1 months, median overall survival was 36.0 months and objective response rate (ORR) was 45.5%. In 59 LR patients receiving axitinib in second-line, mPFS was 18.7 months, mOS was 44.8 months and ORR was 43.9%. mOS was significantly longer in second line compared to subsequent lines (44.8 vs. 26.5 months; P = .009). In LR vs. RP, mPFS with sunitinib in first-line was correlated with mPFS with axitinib in second-line (27.2 vs. 10.9 months P < .001). The safety profile was manageable and consistent with known data. CONCLUSIONS: This study confirms the long-term benefits of axitinib in a selected population, helping clinicians to select the best sequential approach and patients who could obtain a greater benefit from axitinib.

GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles.

Genetically encoded calcium indicators allow monitoring subcellular Ca(2+) signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca(2+) sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg(2+), tunable Ca(2+) affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca(2+) indicators for organelles and becomes a valuable tool for in vivo Ca(2+) imaging applications.

The proto-oncogene c-myc regulates antibody secretion and Ig class switch recombination.

The immune response involves the generation of Ab-secreting cells and memory B cells through a process called terminal B lymphocyte differentiation. This program requires the transcriptional repressor Blimp-1, which inhibits c-myc expression and terminates proliferation. Although the role of c-Myc in cell proliferation is well characterized, it is not known whether it has other functions in terminal differentiation. In this study, we show that c-Myc not only regulates cell proliferation, but it is also essential for Ab-secreting cell function and differentiation in vivo. c-Myc-deficient B lymphocytes hypersecrete IgM and do not undergo Ig class switch recombination (CSR). CSR has been previously linked to proliferation, and in this study we mechanistically link class switching and proliferation via c-Myc. We observed that c-Myc regulates CSR by transcriptionally activating the B cell-specific factor activation-induced cytidine deaminase. By linking cell proliferation and CSR, c-Myc is thus a critical component for a potent immune response.

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells.

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.

[Single incision transumbilical laparoscopic appendectomy: initial experience]. / Apendicectomía laparoscópica mediante incisión única transumbilical: experiencia inicial.

INTRODUCTION: Appendicitis is the most common abdominal emergency. The treatment is surgical and single incision laparoscopic surgery (SILS) involves performing laparoscopic surgery through a single transumbilical point, in an attempt to improve the results of laparoscopic surgery. MATERIAL AND METHOD: A total of 73 patients with suspected acute appendicitis were operated on using the SILS technique between June 2009 and August 2010. All patients were operated on by the same surgical team, and the navel was the only point of entrance. Post-surgical pain was assessed using a numerical scale at the time of discharge. RESULTS: None of the patients required conversion to conventional laparoscopy. The mean surgical time was 40±14 (16-80) minutes. There were no complications during or after the surgery. The mean post-surgical pain score was 3±1 (1-7) and the mean hospital stay was 18±7 (9-42) hours. CONCLUSION: SILS is a safe and effective technique for appendicitis. In the future, the most common surgical procedures could be performed through the navel. This would be by surgeons, highly experienced in advance laparoscopic surgery in order to introduce this new technique safely without increasing morbidity and mortality.

The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is the third element in capacitative calcium entry.

STIM1 and Orai1 are the main players in capacitative calcium entry (CCE). STIM1 senses [Ca(2+)] inside the endoplasmic reticulum (ER) and, when it decreases, opens Orai1, a store-operated calcium channel (SOC) in the plasma membrane that promotes Ca(2+) entry and increases cytosolic Ca(2+). The final destination of the entering Ca(2+) is the ER, which refills very efficiently (capacitatively) with it. We propose here that SERCA is the third element of CCE, to which is tightly coupled to favour rapid Ca(2+) pumping from the high Ca(2+) microdomains, generated at the SOC's mouth, to the ER. We find that, on depletion of the intracellular Ca(2+) stores, SERCA co-localizes with STIM1 at puncta. Adequate coupling of CCE and ER Ca(2+) pumping requires correct proportions of STIM1, Orai1 and SERCA. Overexpression of Orai1 decreased modestly Ca(2+) entry, but produced a dramatic fall of Ca(2+) uptake into ER, which was rescued by STIM1 co-expression or by increasing external Ca(2+). In permeabilized cells, Ca(2+) uptake into the ER was indistinguishable in the Orai1-expressing and in the control cells. We propose that excess Orai1 uncouples SERCA from Ca(2+) entry in the intact cell by disturbing the fine topology of Ca(2+) pumping complexes within the ER-plasma membrane junctions.

The endoplasmic reticulum of dorsal root ganglion neurons contains functional TRPV1 channels.

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca(2+) channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1(ER)). We have studied the properties of TRPV1(ER) in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1(ER) with capsaicin or other vanilloids produced an increase of cytosolic Ca(2+) due to Ca(2+) release from the ER. The decrease of [Ca(2+)](ER) was directly revealed by an ER-targeted aequorin Ca(2+) probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1(ER) to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1(ER) was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca(2+) because the sensitivity to capsaicin was increased by loading the cells with the Ca(2+) chelator BAPTA. Decreasing [Ca(2+)](ER) did not affect the sensitivity of TRPV1(ER) to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Delta35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1(ER) for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatidylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1(ER) to agonists may be critical for neuron health, because otherwise Ca(2+) depletion of ER could lead to ER stress, unfolding protein response, and cell death.

Gastrojejunal anastomotic stenosis in laparoscopic gastric bypass with a circular stapler (21 mm): incidence, treatment and long-term follow-up.

BACKGROUND: One of the keys to the long-term success of laparoscopic gastric bypass (LGBP) is performing a small-diameter gastrojejunal anastomosis, which occasionally involves an increased incidence of stenosis. METHODS: Between May 2000 and October 2008, 676 patients underwent LGBP with a no. 21 circular stapler to create the gastrojejunoanastomosis (GJA). We define stenosis when clinical symptoms suggest an obstruction and it is impossible to pass a 10-mm endoscope through the GJA. The treatment of patients with stenosis was endoscopic dilation with 10-15-mm balloons. RESULTS: A total of 23 patients (3.4%) developed stenosis of whom 20 were females (3%) and three males (0.4%) with a mean age of 40.7+/-11.6 years (range, 16-71 years) and a body mass index of 48.1+/-6.9 kg/m2 (range, 34-78 kg/m2). The time between surgery and the onset of symptoms was 46.8+/-24.5 days (range, 15-93 days). The stricture was resolved in all patients with endoscopic dilation: 18 patients with one dilation, three patients with two dilations and two patients with three dilations. There were no complications. CONCLUSIONS: The incidence of gastrojejunal anastomotic stenosis in LGBP performed with a 21-mm circular stapler is low, and endoscopic dilation is an effective and complication-free treatment in 100% of cases.

Deletion of the SLUG (SNAI2) gene results in human piebaldism.

Slug is a zinc-finger neural crest transcription factor, encoded by the SLUG gene, which is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. In mouse, heterozygous and homozygous slug mutations result in anemia, infertility, white forehead blaze, and depigmentation of the ventral body, tail, and feet. This phenotype is very similar to the heterozygous W (KIT)-mutant mouse phenotype and to human piebaldism, which is characterized by a congenital depigmented patches and poliosis (white forelock). To investigate the possibility that some cases of human piebaldism might result from abnormalities of the human SLUG (SNAI2) gene, we carried out Southern blot analysis of the SLUG gene in 17 unrelated patients with piebaldism, who lack apparent KIT mutations. Three of these patients had evident heterozygous deletions of the SLUG gene encompassing the entire coding region. Real-time PCR confirmed the deletion in all cases. Fluoresence in situ hybridization (FISH) of genomic SLUG probes to metaphase chromosomes independently confirmed the deletion in one of the cases. These findings indicate that some cases of human piebaldism result from mutation of the SLUG gene on chromosome 8, and provide further strong evidence for the role of SLUG in the development of human melanocytes.

SLUG (SNAI2) deletions in patients with Waardenburg disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) is a congenital disorder caused by defective function of the embryonic neural crest. Depending on additional symptoms, WS is classified into four types: WS1, WS2, WS3 and WS4. WS1 and WS3 are caused by mutations in PAX3, whereas WS2 is heterogenous, being caused by mutations in the microphthalmia (MITF) gene in some but not all affected families. The identification of Slugh, a zinc-finger transcription factor expressed in migratory neural crest cells, as the gene responsible for pigmentary disturbances in mice prompted us to analyse the role of its human homologue SLUG in neural crest defects. Here we show that two unrelated patients with WS2 have homozygous deletions in SLUG which result in absence of the SLUG product. We further show that Mitf is present in Slug-deficient cells and transactivates the SLUG promoter, and that Slugh and Kit genetically interact in vivo. Our findings further define the locus heterogeneity of WS2 and point to an essential role of SLUG in the development of neural crest-derived human cell lineages: its absence causes the auditory-pigmentary symptoms in at least some individuals with WS2.

Zinc-finger transcription factor Slug contributes to the function of the stem cell factor c-kit signaling pathway.

The stem cell factor c-kit signaling pathway (SCF/c-kit) has been previously implicated in normal hematopoiesis, melanogenesis, and gametogenesis through the formation and migration of c-kit(+) cells. These biologic functions are also determinants in epithelial-mesenchymal transitions during embryonic development governed by the Snail family of transcription factors. Here we show that the activation of c-kit by SCF specifically induces the expression of Slug, a Snail family member. Slug mutant mice have a cell-intrinsic defect with pigment deficiency, gonadal defect, and impairment of hematopoiesis. Kit(+) cells derived from Slug mutant mice exhibit migratory defects similar to those of c-kit(+) cells derived from SCF and c-kit mutant mice. Endogenous Slug is expressed in migratory c-kit(+) cells purified from control mice but is not present in c-kit(+) cells derived from SCF mutant mice or in bone marrow cells from W/W(v) mice, though Slug is present in spleen c-kit(+) cells of W/W(v) (mutants expressing c-kit with reduced surface expression and activity). SCF-induced migration was affected in primary c-kit(+) cells purified from Slug-/- mice, providing evidence for a role of Slug in the acquisition of c-kit(+) cells with ability to migrate. Slug may thus be considered a molecular target that contributes to the biologic specificity to the SCF/c-kit signaling pathway, opening up new avenues for stem cell mobilization.