Presence of tumor cells in intra-operative blood salvage autotransfusion samples from hepatocellular carcinoma liver transplantation: analysis using highly sensitive microfluidics technology.

BACKGROUND: The application of intra-operative blood salvage autotransfusion(IBSA) in liver transplantation(LT) for hepatocellular carcinoma(HCC) remains controversial due to the theoretical risk of tumour cell(TC) reintroduction. Current studies evaluating for presence of TC are limited by suboptimal detection techniques. This study aims to analyze the presence of TC in HCC LT autologous blood using microfluidics technology. METHODS: A prospective study of HCC patients who underwent LT from February 2018-April 2019 was conducted. Blood samples were collected peri-operatively. TCs were isolated using microfluidics technology and stained with antibody cocktails for confirmation. RESULTS: A total of 15 HCC LT patients were recruited. All recipients had tumour characteristics within the University of California, San Francisco(UCSF) criteria pre-operatively. TC was detected in all of the autologous blood samples collected from the surgical field. After IOCS wash, five patients had no detectable TC, while 10 patients had detectable TC; of these two remained positive for TC after Leukocyte Depletion Filter(LDF) filtration. CONCLUSION: The risk of tumour cell reintroduction using IBSA in HCC LT patients can be reduced with a single LDF. Future studies should evaluate the proliferation capacity and tumorigenicity of HCC TC in IBSA samples, and the effects of TC reintroduction in patients with pre-existing HCC TCs.

Characterization of on-target adverse events caused by TRK inhibitor therapy.

BACKGROUND: The tropomyosin receptor kinase (TRK) pathway controls appetite, balance, and pain sensitivity. While these functions are reflected in the on-target adverse events (AEs) observed with TRK inhibition, these AEs remain under-recognized, and pain upon drug withdrawal has not previously been reported. As TRK inhibitors are approved by multiple regulatory agencies for TRK or ROS1 fusion-positive cancers, characterizing these AEs and corresponding management strategies is crucial. PATIENTS AND METHODS: Patients with advanced or unresectable solid tumors treated with a TRK inhibitor were retrospectively identified in a search of clinical databases. Among these patients, the frequency, severity, duration, and management outcomes of AEs including weight gain, dizziness or ataxia, and withdrawal pain were characterized. RESULTS: Ninety-six patients with 15 unique cancer histologies treated with a TRK inhibitor were identified. Weight gain was observed in 53% [95% confidence interval (CI), 43%-62%] of patients and increased with time on TRK inhibition. Pharmacologic intervention, most commonly with glucagon-like peptide 1 analogs or metformin, appeared to result in stabilization or loss of weight. Dizziness, with or without ataxia, was observed in 41% (95% CI, 31%-51%) of patients with a median time to onset of 2 weeks (range, 3 days to 16 months). TRK inhibitor dose reduction was the most effective intervention for dizziness. Pain upon temporary or permanent TRK inhibitor discontinuation was observed in 35% (95% CI, 24%-46%) of patients; this was more common with longer TRK inhibitor use. TRK inhibitor reinitiation was the most effective intervention for withdrawal pain. CONCLUSIONS: TRK inhibition-related AEs including weight gain, dizziness, and withdrawal pain occur in a substantial proportion of patients receiving TRK inhibitors. This safety profile is unique relative to other anticancer therapies and warrants careful monitoring. These on-target toxicities are manageable with pharmacologic intervention and dose modification.

Novel therapies in urothelial carcinoma: a biomarker-driven approach.

Urothelial malignancies, including carcinomas of the bladder, ureters, and renal pelvis comprised ∼8% of new cancer cases in the USA in 2016. In the metastatic setting, 15% of patients exhibit long-term survival following cisplatin-based chemotherapy and in patients with recurrent disease, response rates to second-line chemotherapy are generally 15%-20% with a 3-month progression-free survival. However, recent advances in immunotherapy represent an opportunity to significantly improve patient outcomes. Moreover, the advent of next-generation sequencing has resulted in both an improved understanding of the fundamental genetic changes that characterize urothelial carcinoma (UC) and identification of several candidate biomarkers of response to various therapies. Incorporation of prospective genotyping into clinical trials will allow for the identification and enrichment of patients most likely to respond to specific targeted therapies and chemotherapy. Combining different therapeutic classes to enhance outcomes is also an area of active research in UC.

Same admission laparoscopic cholecystectomy for acute cholecystitis: is the "golden 72 hours" rule still relevant?

BACKGROUND: Studies have shown that same admission laparoscopic cholecystectomy (SALC) is superior to delayed laparoscopic cholecystectomy for acute cholecystitis (AC). While some proposed a"golden 72-hour" for SALC, the optimal timing remains controversial. The aim of the study was to compare the outcomes of SALC in AC patients with different time intervals from symptom onset. METHODS: A retrospective analysis of 311 patients who underwent SALC for AC from June 2010-June 2015 was performed. Patients were divided into three groups based on the time interval between symptom onset and surgery: <4 days (E-SALC), 4-7 days (M-SALC), >7 (L-SALC). RESULTS: The mean duration of symptoms was 2(1-3), 5(4-7) and 9 (8-13) days for E-SALC, M-SALC and L-SALC, respectively (p < 0.001). Conversion rates were higher in the L-SALC group [E-SALC, 8.2% vs M-SALC, 9.6% vs L-SALC, 21.4%] (p = 0.048). The total length of stay was longer in patients with longer symptom duration [E-SALC, 4 (2-33) vs M-SALC, 2 (2-23) vs L-SALC, 7 (2-49)] (p < 0.001). CONCLUSION: Patients with AC presenting beyond 7 days of symptoms have higher conversion rates and longer length of stay associated with SALC. However, patients with less than a week of symptoms should be offered SALC.

Outcomes of resection of giant hepatocellular carcinoma in a tertiary institution: does size matter?

BACKGROUND: The surgical management of giant hepatocellular carcinoma (G-HCC), or HCC of ≥10 cm in diameter, remains controversial. The aim of this study was to compare the outcomes of surgical resection of, respectively, G-HCC and small HCC (S-HCC), or HCC measuring <10 cm. METHODS: A retrospective review of all patients (n = 86) diagnosed with HCC and submitted to resection in a tertiary hospital during the period from January 2007 to June 2012 was conducted. Overall survival (OS), recurrence rates and perioperative mortality at 30 days were compared between patients with, respectively, G-HCC and S-HCC. Prognostic factors for OS were analysed. RESULTS: The sample included 23 patients with G-HCC (26.7%) and 63 with S-HCC (73.3%) based on histological tumour size. Patient demographics and comorbidities were comparable. Median OS was 39.0 months in patients with G-HCC and 65.0 months in patients with S-HCC (P = 0.213). Although size did not affect OS in this cohort, the presence of satellite lesions [hazard ratio (HR) 3.70, P = 0.012] and perioperative blood transfusion (HR 2.85, P = 0.015) were negative predictors for OS. CONCLUSIONS: Surgical resection of G-HCC provides OS comparable with that after resection of S-HCC.

Germline single nucleotide polymorphisms associated with response of urothelial carcinoma to platinum-based therapy: the role of the host.

BACKGROUND: Variations in urothelial carcinoma (UC) response to platinum chemotherapy are common and frequently attributed to genetic and epigenetic variations of somatic DNA. We hypothesized that variations in germline DNA may contribute to UC chemosensitivity. PATIENTS AND METHODS: DNA from 210 UC patients treated with platinum-based chemotherapy was genotyped for 80 single nucleotide polymorphisms (SNPs). Logistic regression was used to examine the association between SNPs and response, and a multivariable predictive model was created. Significant SNPs were combined to form a SNP score predicting response. Eleven UC cell lines were genotyped as validation. RESULTS: Six SNPs were significantly associated with 101 complete or partial responses (48%). Four SNPs retained independence association and were incorporated into a response prediction model. Each additional risk allele was associated with a nearly 50% decrease in odds of response [odds ratio (OR) = 0.51, 95% confidence interval 0.39-0.65, P = 1.05 × 10(-7)). The bootstrap-adjusted area under the curves of this model was greater than clinical prognostic factors alone (0.78 versus 0.64). The SNP score showed a positive trend with chemosensitivity in cell lines (P = 0.115). CONCLUSIONS: Genetic variants associated with response of UC to platinum-based therapy were identified in germline DNA. A model using these genetic variants may predict response to chemotherapy better than clinical factors alone.

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).

Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

Commentary on "DNA damage response and repair gene alterations are associated with improved survival in patients with platinum-treated advanced urothelial carcinoma."

PURPOSE: Platinum-based chemotherapy remains the standard treatment for advanced urothelial carcinoma by inducing DNA damage. We hypothesize that somatic alterations in DNA damage response and repair (DDR) genes are associated with improved sensitivity to platinum-based chemotherapy. EXPERIMENTAL DESIGN: Patients with diagnosis of locally advanced and metastatic urothelial carcinoma treated with platinum-based chemotherapy who had exon sequencing with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay were identified. Patients were dichotomized based on the presence/absence of alterations in a panel of 34 DDR genes. DDR alteration status was correlated with clinical outcomes and disease features. RESULTS: One hundred patients were identified, of which 47 harbored alterations in DDR genes. Patients with DDR alterations had improved progression-free survival (9.3 vs. 6.0 months, log-rank P = 0.007) and overall survival (23.7 vs. 13.0 months, log-rank P = 0.006). DDR alterations were also associated with higher number mutations and copy-number alterations. A trend toward positive correlation between DDR status and nodal metastases and inverse correlation with visceral metastases were observed. Different DDR pathways also suggested variable effect on clinical outcomes. CONCLUSIONS: Somatic DDR alteration is associated with improved clinical outcomes in platinum-treated patients with advanced urothelial carcinoma. Once validated, it can improve patient selection for clinical practice and future study enrollment.

ZBP-89, a Krüppel-like zinc finger protein, inhibits epidermal growth factor induction of the gastrin promoter.

We have shown previously that a GC-rich element (GGGGCGGGGTGGGGGG) conferring epidermal growth factor (EGF) responsiveness to the human gastrin promoter binds Sp1 and additional undefined complexes. A rat GH4 cell line expression library was screened by using a multimer of the gastrin EGF response element, and three overlapping cDNA clones were identified. The full-length rat cDNA encoded an 89-kDa zinc finger protein (ZBP-89) that was 89% identical to a 49-kDa human factor, ht(beta), that binds a GTGGG/CACCC element in T-cell receptor promoters. The conservation of amino acids between the zinc fingers indicates that ZBP-89 is a member of the C2H2 zinc finger family subclass typified by the Drosophila Krüppel protein. ZBP-89 is ubiquitously expressed in normal adult tissues. It binds specifically to the gastrin EGF response element and inhibits EGF induction of the gastrin promoter. Collectively, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of the gastrin gene.

Immunotherapy for fungal infections with special emphasis on central nervous system infections.

Opportunistic fungal infections are major causes of morbidity and mortality in the immunocompromized. Fungi have evolved complex and coordinated mechanisms to survive in the environment and the mammalian host. Fungi must adapt to "stressors" in the host, including nutrient scarcity, pH and reactive oxygen and nitrogen intermediates, in addition to evading host immunity. Knowledge of the immunopathogenesis of fungal infections has paved the way to promising strategies for immunotherapy. These include strategies that increase phagocyte number, activate innate host defense pathways in phagocytes and dendritic cells and stimulate antigen-specific immunity (e.g, vaccines). Immunotherapy must be tailored to specific immunocompromized states. Our review focuses on cryptococcosis and coccidioidomycosis because of the propensity of these diseases to involve the central nervous system (CNS). The CNS has long been considered "immunologically privileged" in the sense of being isolated from normal immune surveillance. This notion is only partially accurate. Immune-based therapies for fungal CNS disease are at an exploratory level and merit further evaluation in clinical trials.

Laparoscopic cholecystectomy for acute cholecystitis: an analysis of early versus delayed cholecystectomy and predictive factors for conversion.

BACKGROUND: There is an increasing preference for early laparoscopic cholecystectomy (ELC) as compared to delayed LC (DLC) in the management of acute cholecystitis (AC). Conversion to open cholecystectomy (LOC) remains an important outcome. We aim to compare ELC and DLC outcomes and identify LOC predictors. METHODS: Retrospective analysis of 466 patients who underwent LC for AC from June 2010 to June 2015 was performed. Patients were divided into ELC and DLC groups, defined as LC performed within 7 days and between 4 to 24 weeks of symptom onset, respectively. Peri-operative outcomes and predictors for LOC were analyzed. RESULTS: Conversion rates were comparable [ELC, 8.6% vs. DLC, 8.0%] (P=0.867). While median operative time was longer in ELC (101.5 min [83.0-130.1]) than DLC (88.0 min [62.3-118.8]) (P<0.001), intraoperative (ELC, 1.9% vs. DLC, 3.0%; P=0.541) and postoperative morbidity (ELC, 13.5% vs. DLC, 12.5%; P=0.688) was comparable. Median total length of stay (LOS) was shorter in ELC (4 days [3-6]) than DLC (5 days [4-9]) (P<0.001). Univariate analysis showed increased age (LC, 57 [45-66] vs. LOC, 60 [56-72]; P=0.016), presence of comorbidities (LC, 69.0% vs. LOC, 87.8%; P=0.009), previous abdominal surgery (LC, 6.1% vs. LOC, 17.1%; P=0.014), fever (P=0.001), Murphy's sign (P=0.005) and lower albumin (LC, 42.0 [39.0-45.0] vs. LOC, 40.0 [36.0-43.0]; P=0.003) to be predictors for LOC. CONCLUSIONS: ELC provides shorter LOS and eliminates the risk of gallstone-related morbidity while awaiting surgery. It should be advocated for patients with AC. The presence of comorbidities, increased age, previous abdominal surgery and low albumin are predictors for conversion.

Delayed Presentation of Acute Cholecystitis: Comparative Outcomes of Same-Admission Versus Delayed Laparoscopic Cholecystectomy.

INTRODUCTION: Studies have shown that same-admission laparoscopic cholecystectomy (SALC) is superior to delayed laparoscopic cholecystectomy (DLC) for acute cholecystitis (AC). However, no studies have compared both modalities in patients with delayed presentation. The aim of the study was to compare outcomes between SALC and DLC in AC patients with more than 7-day symptom duration. METHODS: A retrospective analysis of 83 AC patients who underwent LC after presenting with >7 days of symptoms from June 2010 to June 2015 was performed. Patients were divided into L-SALC and L-DLC, defined as LC performed within the same admission and between 4 and 24 weeks after discharge, respectively. Peri-operative outcomes were evaluated. RESULTS: In L-SALC patients, the intra-operative severity was higher (p < 0.001) and median operative time was longer (L-SALC, 107 min (46-220) vs L-DLC, 95 mins (25-186)) (p = 0.048). Conversion rates were also higher in L-SALC than that in L-DLC (L-SALC, 21.4% vs L-DLC, 4.9%) (p = 0.048). While post-operative morbidity was similar, L-SALC was associated with a longer post-operative length of stay as compared to L-DLC (L-SALC, 2 (1-17) vs L-DLC, 1 (1-6)) (p < 0.001). CONCLUSION: DLC provides lower conversion rates and shorter length of stay in AC patients presenting beyond 7 days of symptoms. This group of patients should be offered DLC.

Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts.

Recent studies indicate that hepatitis C virus (HCV) proteins can mediate innate immune response and inflammation in conjunctival fibroblasts which contributes to the pathology of dry eye condition associated with chronic HCV infection. The present study investigates the phagocytic potential of human conjunctival fibroblasts (HCFj) for HCV core protein. HCFj cells were incubated with HCV core antigen for different periods of time, and fluorescent micrographs were taken to observe protein internalisation. HCFj cells were capable of internalising HCV core antigen within 1 h; this gives an insight into another molecular mechanism which may contribute towards HCV-associated conjunctival inflammation.

Genetic vaccination-induced immune responses to the human immunodeficiency virus protein Rev: emergence of the interleukin 2-producing helper T lymphocyte.

Rev M10 is a trans-dominant negative inhibitor of HIV replication. Hence, stable transduction of CD4+ T cells with Rev M10 represents a novel gene therapy aimed at inhibiting HIV replication within these cells, thereby slowing the progression of AIDS. However, the immune system may recognize Rev M10 as foreign and target transduced cells for elimination. In the current study, mice were genetically immunized with a plasmid encoding Rev M10, to (1) identify immune parameters that may be induced by Rev M10 gene transfer, (2) determine the impact of repeated introduction of the Rev M10-encoding plasmid on the immune response to the transgene product, and (3) determine if cotransfection with a plasmid encoding TGFbeta1 would suppress the response. Kinetic studies revealed that Rev-specific IL-2-producing helper T lymphocytes (HTLs) appeared following the second genetic immunization, peaked after the third, and persisted at peak levels for at least 6 weeks. Rev-specific HTLs were CD4+, and the development of these cells was ablated by cotransfection with TGFbeta1. Other cytokines were not readily detectable when immune splenocytes were restimulated with Rev in vitro, and Rev-specific IgG antibodies were not present in the sera of these mice. To our knowledge, this represents the first report that genetic immunization with Rev M10 induces an immune response that is dominated by IL-2-producing HTLs. Further, this study demonstrates the potential utility of introducing immunosuppressive genes as a means to control the immune response to foreign transgene products.

Messenger RNA editing of the human serotonin 5-HT2C receptor.

RNA encoding the rat serotonin 5-HT2C receptor undergoes editing whereby one to four adenosines are converted to inosines. This conversion can change up to three codons out of a stretch of five in the second intracellular loop of the receptor. RNA editing of the rat 5-HT2C receptor that changes all three codons was shown previously to alter intracellular signaling by 5-HT without changing its receptor-binding affinity. We analyzed 5-HT2C receptor editing in human brain and hypothalamic RNA samples and confirmed that all four adenosine editing sites observed in rat were also present in human samples. Additionally, we identified a novel editing site in the middle edited codon that extends the repertoire of 5-HT2C receptors by six additional protein isoforms. We observed that editing reduces both the binding affinity and functional potency of agonists for recombinant human 5-HT2C receptor isoforms. This effect on binding affinity was proportional to the agonist's intrinsic activity, with full agonists most affected, and antagonists showing no effect. These data suggest that RNA editing may alter coupling energetics within the ternary complex, thereby altering agonist binding affinities, G protein coupling, and functional responses. RNA editing may thus provide a novel mechanism for regulating 5-HT synaptic signaling and plasticity.

Perceiving, remembering, and communicating structure in events.

How do people perceive routine events, such as making a bed, as these events unfold in time? Research on knowledge structures suggests that people conceive of events as goal-directed partonomic hierarchies. Here, participants segmented videos of events into coarse and fine units on separate viewings; some described the activity of each unit as well. Both segmentation and descriptions support the hierarchical bias hypothesis in event perception: Observers spontaneously encoded the events in terms of partonomic hierarchies. Hierarchical organization was strengthened by simultaneous description and, to a weaker extent, by familiarity. Describing from memory rather than perception yielded fewer units but did not alter the qualitative nature of the descriptions. Although the descriptions were telegraphic and without communicative intent, their hierarchical structure was evident to naive readers. The data suggest that cognitive schemata mediate between perceptual and functional information about events and indicate that these knowledge structures may be organized around object/action units.

Studies on free stream turbulence as related to gas turbine heat transfer. A review of authors' past work and future implications.

A review of the past work done on free stream turbulence (FST) as applied to gas turbine heat transfer and its implications for future studies are presented. It is a comprehensive approach to the results of many individual studies in order to derive the general conclusions that could be inferred from all rather than discussing the results of each individual study. Three experimental and four modeling studies are reviewed. The first study was on prediction of heat transfer for film cooled gas turbine blades. An injection model was devised and used along with a 2-D low Reynolds number k-epsilon model of turbulence for the calculations. Reasonable predictions of heat transfer coefficients were obtained for turbulence intensity levels up to 7%. Following this modeling study a series of experimental studies were undertaken. The objective of these studies was to gain a fundamental understanding of mechanisms through which FST augments the surface heat transfer. Experiments were carried out in the boundary layer and in the free stream downstream of a gas turbine combustor simulator, which produced initial FST levels of 25.7% and large length scales (About 5-10 cm for a boundary layer 4-5 cm thick). This result showed that one possible mechanism through which FST caused an increase in heat transfer is by increasing the number of ejection events. In a number of modeling studies several well-known k-epsilon models were compared for their predictive capability of heat transfer and skin friction coefficients under moderate and high FST. Two data sets, one with moderate levels of FST (about 7%) and one with high levels of FST (about 25%) were used for this purpose. Although the models did fine in their predictions of cases with no FST (baseline cases) they failed one by one as FST levels were increased. Under high FST (25.7% initial intensity) predictions of Stanton number were between 35-100% in error compared to the measured values. Later a new additional production term indicating the interaction between the turbulent kinetic energy (TKE) and mean velocity gradients was introduced into the TKE equation. The predicted results of skin friction coefficient and Stanton number were excellent both in moderate and high FST cases. In fact these model also gave good predictions of TKE profiles whereas earlier unmodified models did not predict the correct TKE profiles even under moderate turbulence intensities. Although this new production term seems to achieve the purpose, it is the authors' belief that it is diffusion term of the TKE equation, which needs to be modified in order to fit the physical events in high FST boundary layer flows. The results of these studies are currently being used to come up with new diffusion model for the TKE equation.

Are bilateral superior vena cavae a risk factor for single ventricle palliation?

BACKGROUND: Performing superior vena cava-to-pulmonary artery anastomosis, in the presence of bilateral superior vena cavae, can be technically challenging. Our clinical observation has been that bilateral superior vena cavae are a risk factor for poor outcome in children needing single ventricle palliation. METHODS: Detailed operative, angiographic, and follow-up data were analyzed in 39 children undergoing bilateral cavopulmonary anastomosis (b-CPA). Overall outcome was compared to 274 children having a unilateral cavopulmonary anastomoses (u-CPA). RESULTS: Nine patients (23%) with bilateral superior vena cavae were found to have thrombus in the cavopulmonary circulation after the b-CPA. Postoperative mean arterial oxygen saturation was significantly lower in those who had thrombus [69%+/-10% versus 82%+/-7%, (p < 0.01)]. Thrombus formation was associated with mortality. The indexed superior vena cavae size was not a risk factor for thrombosis. In follow-up studies the connecting pulmonary artery segment between the two cavopulmonary anastomosis was smaller than the pulmonary arteries adjacent to the hilum. Survivors of a b-CPA were less frequently converted to a Fontan circulation at 5 years of follow up (Kaplan-Meier 5-year estimates, 39% for b-CPA versus 74% for u-CPA [p = 0.02]). CONCLUSIONS: Bilateral superior vena cava-to-pulmonary artery anastomosis is associated with an increased risk of thrombus formation and unfavorable growth in the central pulmonary arteries. Modifications of surgical technique may alter flow patterns, thereby optimizing growth and diminishing the risk of thrombus formation. Anticoagulation therapy may be an important adjunct in children undergoing a b-CPA.