Antitumor active trans­platinum complexes through metabolic stability and enhanced cellular accumulation.

Utilizing isoquinoline as a carrier ligand, we have evaluated the reactivity of selected trans­platinum planar amine (TPA) carboxylate compounds by varying the leaving carboxylate group (acetate, hydroxyacetate, and lactate) in an effort to optimize the cytotoxic and metabolic efficiency. To measure the pharmacological properties of these compounds, a combination of systematic biophysical and biological studies were carried out mainly involving substitution reaction with NAM (N-acetyl-methionine), effects on DNA structural perturbation, cytotoxicity, cellular accumulation, metabolic stability, and cell cycle effects. TPA compounds showed minimal losses in cytotoxic efficacy and outperformed cisplatin after pre-incubation with serum, while displaying a distinct micromolar cytotoxic activity with minimal DNA binding and unaltered cell cycle. Monitoring the TPA compounds with NAM suggests the following trend for the reactivity: hydroxyacetate > lactate > acetate. The same trend was seen for the cytotoxicity in tumor cells and DNA binding, while the rate of drug inactivation/protein binding in cells was not significantly different among these leaving groups. Thus, our results show superior cellular efficacy of TPA compounds and distinct micromolar cytotoxic activities different than cisplatin. Moreover, we found the TPA compounds had prolonged survival and decreased tumor burden compared to the control mice in a relevant human ovarian cancer mouse model with A2780 cells expressing luciferase. Therefore, we propose that further optimization of the basic TPA structure can give further enhanced in vivo activity and may eventually be translated into the development of clinically relevant non-traditional platinum drugs.

Next-generation sequencing methodologies to detect low-frequency mutations: "Catch me if you can".

Mutations, the irreversible changes in an organism's DNA sequence, are present in tissues at a variant allele frequency (VAF) ranging from ∼10-8 per bp for a founder mutation to ∼10-3 for a histologically normal tissue sample containing several independent clones - compared to 1%- 50% for a heterozygous tumor mutation or a polymorphism. The rarity of these events poses a challenge for accurate clinical diagnosis and prognosis, toxicology, and discovering new disease etiologies. Standard Next-Generation Sequencing (NGS) technologies report VAFs as low as 0.5% per nt, but reliably observing rarer precursor events requires additional sophistication to measure ultralow-frequency mutations. We detail the challenge; define terms used to characterize the results, which vary between laboratories and sometimes conflict between biologists and bioinformaticists; and describe recent innovations to improve standard NGS methodologies including: single-strand consensus sequence methods such as Safe-SeqS and SiMSen-Seq; tandem-strand consensus sequence methods such as o2n-Seq and SMM-Seq; and ultrasensitive parent-strand consensus sequence methods such as DuplexSeq, PacBio HiFi, SinoDuplex, OPUSeq, EcoSeq, BotSeqS, Hawk-Seq, NanoSeq, SaferSeq, and CODEC. Practical applications are also noted. Several methods quantify VAF down to 10-5 at a nt and mutation frequency (MF) in a target region down to 10-7 per nt. By expanding to > 1 Mb of sites never observed twice, thus forgoing VAF, other methods quantify MF < 10-9 per nt or < 15 errors per haploid genome. Clonal expansion cannot be directly distinguished from independent mutations by sequencing, so it is essential for a paper to report whether its MF counted only different mutations - the minimum independent-mutation frequency MFminI - or all mutations observed including recurrences - the larger maximum independent-mutation frequency MFmaxI which may reflect clonal expansion. Ultrasensitive methods reveal that, without their use, even mutations with VAF 0.5-1% are usually spurious.

Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging.

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here, we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs, which are the most potent subsets, it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol, please refer to Liang et al. (2020) and Qiu et al. (2021).

Erythroid enucleation: a gateway into a "bloody" world.

Erythropoiesis is an intricate process starting in hematopoietic stem cells and leading to the daily production of 200 billion red blood cells (RBCs). Enucleation is a greatly complex and rate-limiting step during terminal maturation of mammalian RBC production involving expulsion of the nucleus from the orthochromatic erythroblasts, resulting in the formation of reticulocytes. The dynamic enucleation process involves many factors ranging from cytoskeletal proteins to transcription factors to microRNAs. Lack of optimum terminal erythroid maturation and enucleation has been an impediment to optimum RBC production ex vivo. Major efforts in the past two decades have exposed some of the mechanisms that govern the enucleation process. This review focuses in detail on mechanisms implicated in enucleation and discusses the future perspectives of this fascinating process.

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

Nitric oxide-donor/PARP-inhibitor combination: A new approach for sensitization to ionizing radiation.

Recently, clinical development of PARP inhibitors (PARPi) expanded from using them as a single agent to combining them with DNA-damaging therapy to derive additional therapeutic benefit from stimulated DNA damage. Furthermore, inhibiting PARP in cancers with BRCA1/2 mutations has been shown to be an effective synthetic lethality approach either as a single agent or in combination with the different DNA damaging agents: chemotherapy or ionizing radiation (IR). However, inherited BRCA1/2 mutations account only for 5-10% of breast cancers, 10-15% of ovarian cancers, and lesser for the other cancers. Hence, for most of the cancer patients with BRCA1/2-proficient tumors, sensitization to DNA-damaging agents with PARPi is significantly less effective. We recently demonstrated that moderate, non-toxic concentrations of NO-donors inhibited BRCA1 expression, with subsequent inhibition of error-free HRR and increase of error-prone non-homologous end joining (NHEJ). We also demonstrated that the effect of NO-dependent block of BRCA1 expression can only be achieved in the presence of oxidative stress, a condition that characterizes the tumor microenvironment and is also a potential effect of IR. Hence, NO-donors in combination with PARPi, with effects limited by tumor microenvironment and irradiated area, suggest a precise tumor-targeted approach for radio-sensitization of BRCA1/2-proficient tumors. The combination with NO-donors allows PARPi to be successfully applied to a wider variety of tumors. The present work demonstrates a new drug combination (NO-donors and PARP-inhibitors) which demonstrated a high potency in sensitization of wide variety of tumors to ionizing radiation treatment.

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169.

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.

Fyn kinase is a novel modulator of erythropoietin signaling and stress erythropoiesis.

The signaling cascade induced by the interaction of erythropoietin (EPO) with its receptor (EPO-R) is a key event of erythropoiesis. We present here data indicating that Fyn, a Src-family-kinase, participates in the EPO signaling-pathway, since Fyn-/- mice exhibit reduced Tyr-phosphorylation of EPO-R and decreased STAT5-activity. The importance of Fyn in erythropoiesis is also supported by the blunted responsiveness of Fyn-/- mice to stress erythropoiesis. Fyn-/- mouse erythroblasts adapt to reactive oxygen species (ROS) by activating the redox-related-transcription-factor Nrf2. However, since Fyn is a physiologic repressor of Nrf2, absence of Fyn resulted in persistent-activation of Nrf2 and accumulation of nonfunctional proteins. ROS-induced over-activation of Jak2-Akt-mTOR-pathway and repression of autophagy with perturbation of lysosomal-clearance were also noted. Treatment with Rapamycin, a mTOR-inhibitor and autophagy activator, ameliorates Fyn-/- mouse baseline erythropoiesis and erythropoietic response to oxidative-stress. These findings identify a novel multimodal action of Fyn in the regulation of normal and stress erythropoiesis.

Factors predicting reclassification of variants of unknown significance.

Genetic variants of unknown significance (VUS) are an increasingly common result of genetic testing. VUS present dilemmas for treatment and surveillance. Family history may play a role in VUS reclassification over time. METHODS: All genetic tests performed at a tertiary referral center 2006-2015 were evaluated for the presence of VUS. Patients with VUS were evaluated for demographics, clinical characteristics, family history, and gene characteristics. RESULTS: In total, 2291 individuals were tested from 1639 families; 150 VUS were identified. Twenty-eight VUS reclassified, 21 to benign and 7 to pathogenic. Logistic regression demonstrated the number of family members with associated phenotypic disease was a significant predictor of reclassification. CONCLUSION: The likelihood of VUS reclassification can be predicted by increased positive family history of disease. Most VUS reclassify to benign, but one-fourth reclassify to pathogenic. The actual risk of a VUS should be assessed based on family history and routinely checked for reclassification.

Robotic Colorectal Surgery Learning Curve and Case Complexity.

PURPOSE: To understand the role of case complexity in the learning curve for robotic colorectal surgery. MATERIALS AND METHODS: Sixty-two patients who underwent robot-assisted colorectal surgery were retrospectively reviewed. Each case was assigned a category of complexity ranging from I to IV. Overall, groups and categories of segmental colectomy, rectopexy, and proctectomy for cancer were analyzed according to case volume. Forty-eight patients who underwent similar laparoscopic cases during the same period were also reviewed for comparison. RESULTS: Level I complexity cases were identified in 30% of the first 15 cases compared to 3% after the first 15 cases (P < .01). Level IV complexity cases were identified in 10% of the first 15 cases and 34% after 15 cases (P = .03). Mean operative time for the overall group was 426 minutes (range 178-766, standard deviation [SD] = 152) in the first 15 cases and 373 minutes (range 190-593, SD = 109) after more than 15 cases (P = NS). Mean operative time for rectal cancer procedures decreased from 518 minutes (range 425-752, SD = 88) to 410 minutes (range 220-593, SD = 98) after 15 cases (P = .02). Mean operative time for rectopexy decreased from 361 minutes (range 276-520, SD = 85) to 258 minutes (range 215-318, SD = 34) after 15 cases (P = .03). Overall complications were reduced after 15 cases (6.3%) compared with the first 15 cases (27%) (P = .04). When comparing laparoscopic and open cases, laparoscopic cases were associated with a significant shorter operative time (P = < .00001) as well as overall cost (P = < .00001). CONCLUSION: Complex robotic colorectal surgery can be performed early in the experience, with reduced operative time. Overall complications are reduced after 15 robotic cases. This study shows that improvement in robotic surgery operating time and surgical outcomes occur along with application of the technology to more difficult cases, not as a function of choosing less complex cases.

Transcription factors FOXO in the regulation of homeostatic hematopoiesis.

PURPOSE OF REVIEW: Work in the past decade has revealed key functions of the evolutionary conserved transcription factors Forkhead box O (FOXO) in the maintenance of homeostatic hematopoiesis. Here the diverse array of FOXO functions in normal and diseased hematopoietic stem and progenitor cells is reviewed and the main findings in the past decade are highlighted. Future work should reveal FOXO-regulated networks whose alterations contribute to hematological disorders. RECENT FINDINGS: Recent studies have identified unanticipated FOXO functions in hematopoiesis including in hematopoietic stem and progenitor cells (HSPC), erythroid cells, and immune cells. These findings suggest FOXO3 is critical for the regulation of mitochondrial and metabolic processes in hematopoietic stem cells, the balanced lineage determination, the T and B homeostasis, and terminal erythroblast maturation and red blood cell production. In aggregate these findings highlight the context-dependent function of FOXO in hematopoietic cells. Recent findings also question the nature of FOXO's contribution to heme malignancies as well as the mechanisms underlying FOXO's regulation in HSPC. SUMMARY: FOXO are safeguards of homeostatic hematopoiesis. FOXO networks and their regulators and coactivators in HSPC are greatly complex and less well described. Identifications and characterizations of these FOXO networks in disease are likely to uncover disease-promoting mechanisms.

Polynuclear Platinum Complexes. Structural Diversity and DNA Binding.

Polynuclear platinum complexes (PPCs) represent a discrete structural class of DNA-binding agents with excellent antitumor properties. The use of at least two platinum coordinating units automatically means that multifunctional DNA binding modes are possible. The structural variability inherent in a polynuclear platinum structure can be harnessed to produce discrete modes of DNA binding, with conformational changes distinct from and indeed inaccessible to, the mononuclear agents such as cisplatin. Since our original contributions in this field a wide variety of dinuclear complexes especially have been prepared, their DNA binding studied, and potential relevance to cytotoxicity examined. This chapter focuses on how DNA structure and reactivity is modulated through interactions with PPCs with emphasis on novel aspects of such structure and reactivity. How these major changes are further reflected in damaged DNA-protein binding and cellular effects are reviewed. We further review, for the first time, the great structural diversity achieved in PPC complex design and summarize their major DNA binding effects.

XLF/Cernunnos: An important but puzzling participant in the nonhomologous end joining DNA repair pathway.

DNA double strand breaks (DSBs) are one of the most deleterious DNA lesions that promote cell death, genomic instability and carcinogenesis. The two major cellular mechanisms that repair DSBs are Nonhomologous End-Joining (NHEJ) and Homologous Recombination Repair (HRR). NHEJ is the predominant pathway, in which XLF (also called Cernunnos) is a key player. Patients with XLF mutation exhibit microcephaly, lymphopenia, and growth retardation, and are immunodeficient and radiosensitive. During NHEJ, XLF interacts with XRCC4-Ligase IV, stimulates its ligase activity, and forms DNA-binding filaments of alternating XLF and XRCC4 dimers that may serve to align broken DNA and promote ligation of noncomplementary ends. Despite its central role in NHEJ, the effects of XLF deficiency are surprisingly variable in different biological contexts, and different individual cell lines. This review summarizes the role of XLF in NHEJ, and the unexpected complexity of its interplay with other repair factors in supporting radiosurvival and V(D)J recombination.

Can Transfusions Be Eliminated in Major Abdominal Surgery? Analysis of a Five-Year Experience of Blood Conservation in Patients Undergoing Pancreaticoduodenectomy.

Pancreaticoduodenectomy (PD) has historically required perioperative blood transfusion in 40 to 60 per cent of cases. Growing data suggest that transfusions may be deleterious in the surgical patient. We recently initiated a minimal transfusion approach to PD consisting of limited postoperative blood draws, early iron supplementation, changes in surgical technique, and elimination of hemoglobin transfusion triggers. Predictors of perioperative transfusion were analyzed in 130 consecutive patients undergoing PD by a single surgeon between 2008 and 2013, divided into two eras with 65 patients each. Patients in each era were similar with respect to age, comorbidities, American Society of Anesthesiologists class, body mass index, and diagnosis. The transfusion rate for the entire group was 22 per cent. Nonsignificant predictors of perioperative transfusion include American Society of Anesthesiologists class ≥3 (P = 0.41), vascular resections (P = 0.56), body mass index ≥30 (P = 0.72), and intraoperative blood loss (P = 0.89). Significant predictors of transfusion include PD performed in Era 1 as well as preoperative hemoglobin levels <10 g/dL. In Era 1, 38 per cent of patients required transfusion compared with 6 per cent in Era 2 (P < 0.01). Shorter length of stay and a trend toward decreased pancreatic fistulae were seen in Era 2. Transfusions can be almost completely eliminated in PD and this may contribute to improved outcomes.

Nucleolar targeting by platinum: p53-independent apoptosis follows rRNA inhibition, cell-cycle arrest, and DNA compaction.

TriplatinNC is a highly positively charged, substitution-inert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G1 arrest immediately, but cells treated in S or G2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G1, but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure-activity paradigms for antitumor complexes.

Involvement of p53 in the repair of DNA double strand breaks: multifaceted Roles of p53 in homologous recombination repair (HRR) and non-homologous end joining (NHEJ).

p53 is a tumor suppressor protein that prevents oncogenic transformation and maintains genomic stability by blocking proliferation of cells harboring unrepaired or misrepaired DNA. A wide range of genotoxic stresses such as DNA damaging anti-cancer drugs and ionizing radiation promote nuclear accumulation of p53 and trigger its ability to activate or repress a number of downstream target genes involved in various signaling pathways. This cascade leads to the activation of multiple cell cycle checkpoints and subsequent cell cycle arrest, allowing the cells to either repair the DNA or undergo apoptosis, depending on the intensity of DNA damage. In addition, p53 has many transcription-independent functions, including modulatory roles in DNA repair and recombination. This chapter will focus on the role of p53 in regulating or influencing the repair of DNA double-strand breaks that mainly includes homologous recombination repair (HRR) and non-homologous end joining (NHEJ). Through this discussion, we will try to establish that p53 acts as an important linchpin between upstream DNA damage signaling cues and downstream cellular events that include repair, recombination, and apoptosis.

Outcomes of vascular resection in pancreaticoduodenectomy: single-surgeon experience.

Extension of pancreatic adenocarcinoma into adjacent vasculature often necessitates resection of the portal vein (PV) and/or superior mesenteric vein (SMV) during pancreaticoduodenectomy (PD). The vein is reconstructed primarily by end-to-end anastomosis of vein remnants or venoplasty or by use of autologous or synthetic vein grafts. The objective of this study was to review outcomes in patients undergoing PD for pancreatic adenocarcinoma, specifically comparing the short- and long-term outcomes between the patients undergoing vascular resection and those undergoing standard PD. All patients undergoing PD for pancreatic adenocarcinoma by a single surgeon between 2007 and 2012 were reviewed. Of the 61 patients identified, 18 patients underwent vascular resection of the PV (four patients), SMV (10 patients), or both (four patients). The remaining 43 patients had standard PD. Demographic, perioperative, pathological, and long-term outcomes data were collected and both vascular and standard groups were compared. Both groups had similar demographics. The vascular group had significantly longer operative times (529 vs 406 minutes; P < 0.01) with a trend to greater estimated blood loss (0.64 vs 0.53 L; P = 0.06). Pathological analysis showed no difference between the two groups with regard to lymph node status/ratio and rate of R0 resection (94 vs 91%; P = 0.57); however, the size of the tumor was significantly greater in the vascular group (4.2 vs 3 cm; P < 0.01). Short-term outcomes were similar in the vascular group and standard group, respectively, with no difference in pancreatic fistula rate (6 vs 7%; P = 1.0), transfusion rate (44 vs 35%; P = 0.57), and median length of stay (8 vs 7 days; P = 0.10), and there was no 30-day mortality in either group. Based on Kaplan-Meier methods, the median recurrence-free survival was 18 versus 23 months (P = 0.37) in the vascular and standard groups, respectively, and the overall survival was almost identical in both groups, each with a median of 31 months (P = 0.91). In our experience, mesenteric and PV resection during PD was performed safely and without compromise of short- or longer-term outcomes. It can be performed safely and patients have no significant difference in perioperative outcomes or overall survival.

Surgical management of advanced adrenocortical carcinoma: a 21-year population-based analysis.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. When diagnosed in advanced stages of the disease, the outcomes of surgical resection are not well understood. The objective of this study is to determine the impact of surgery in patients with advanced ACC. Using the Surveillance, Epidemiology and End Results database, we identified patients diagnosed with Stage III and IV ACC between 1988 and 2009. A total of 320 patients with Stage III and IV disease were included in our analysis. In patients treated with surgical resection, the Stage III 1- and 5-year survival rates were 77 and 40 per cent, respectively, whereas the Stage IV 1- and 5-year survival rates were 54 and 27.6 per cent, respectively. Patients treated without surgery had poor survival at 1 year for both Stage III (13%) and Stage IV (16%) (P < 0.01 compared with the surgical groups). Lymph node dissection was performed in 26 per cent of the patients with advanced ACC and was associated with improved survival in univariate analysis of Stage IV patients. Overall, our results indicate that favorable survival outcomes can be achieved even in patients with Stage III and IV disease and surgery should be considered in patients with advanced ACC.

Hepaticojejunostomy using short-limb Roux-en-Y reconstruction.

IMPORTANCE: When performing biliary reconstruction, one of the long-standing tenets of surgery is that Roux-en-Y (RY) reconstruction should use a long hepatic limb to decrease the risk for postoperative cholangitis. However, this practice is not well supported and may also make postoperative biliary endoscopy difficult. While some authors recommend Roux limbs of up to 75 cm, we have routinely used a Roux length of 20 cm to facilitate possible postoperative endoscopic access. OBJECTIVE: To review our experience with short-limb RY hepaticojejunostomy (HJ) and examine the short-term and long-term outcomes following this procedure, as well as the success of future biliary interventions. DESIGN: Retrospective medical record review of all patients who underwent short-limb RYHJ by 2 surgeons (N.N.N. and S.D.C.). SETTING: Tertiary care, university-affiliated teaching hospital. PARTICIPANTS: One hundred patients who underwent RYHJ were identified, with 30 of those patients being excluded owing to creation of an RYHJ to intrahepatic bile ducts with concomitant liver resection. MAIN OUTCOMES AND MEASURES: Patient records were reviewed to determine the incidence of postoperative cholangitis and biliary stricture. Secondary outcomes were the need for postoperative biliary endoscopy and success rates for endoscopic biliary interventions. RESULTS Seventy patients underwent short-limb RYHJ over an 11-year period (2001-2012). Indications included benign stricture (n = 18), malignant stricture (n = 12), choledochal cyst (n = 5), choledocholithiasis (n = 3), idiopathic cholangitis (n = 2), and deceased donor or live donor liver transplant (n = 30). Seven patients, including 4 liver transplant patients, developed clinical or radiographic evidence of postoperative biliary stricture, and all patients underwent successful endoscopic cholangiography. Four of these patients required dilation and/or stone extraction, which were accomplished endoscopically in all cases. CONCLUSIONS AND RELEVANCE: Short-limb RYHJ is safe and associated with a low incidence of postoperative complications. In addition, biliary intervention, when indicated, can be performed endoscopically with a high degree of success. In the absence of any evidence demonstrating longer limbs to be superior, we recommend using short-limb RY reconstruction for HJ.

Attenuation of increased secretory leukocyte protease inhibitor, matricellular proteins and angiotensin II and left ventricular remodeling by candesartan and omapatrilat during healing after reperfused myocardial infarction.

While secretory-leukocyte-protease-inhibitor (SLPI) may promote skin wound healing, its role in infarct healing after reperfused myocardial infarction (RMI) remains unclear. Short-term intravenous angiotensin II (AngII) receptor blocker therapy with candesartan (CN) attenuates increased SLPI and markers of early matrix/left ventricular (LV) in acute RMI. To determine whether reducing effects of AngII with CN or the vasopeptidase inhibitor omapatrilat (OMA) during the healing phase after RMI attenuates SLPI and other mediators of healing and matrix/LV remodeling, we measured these in Sprague-Dawley rats randomized to oral placebo, CN (30 mg/kg/day) or OMA (10 mg/kg/day) therapy during healing between days 2 and 23 after RMI and sham. On day-25, RMI-placebo showed significant LV remodeling, systolic/diastolic dysfunction and impaired passive compliance, and ischemic zone increases in SLPI, secreted-protein-acidic-and-rich-in-cysteine (SPARC) and osteopontin (OPN) mRNA and protein. In addition, metalloproteinase (MMP)-9 and -2, a-disintegrin-and-metalloproteinase (ADAM)-10 and -17, inducible-nitric-oxide-synthase (iNOS), pro-inflammatory cytokines interleukin (IL)-6, and tumor necrosis factor-α, transforming growth factor (TGF)-ß(1) and its signaling molecule p-Smad-2, myeloperoxidase (MPO), AngII, MPO-positive granulocytes, MAC387-positive macrophages and monocytes, scar collagens, cardiomyocyte and fibroblast apoptosis, and microvascular no-reflow also increased whereas anti-inflammatory cytokine IL-10 decreased. Both CN and OMA attenuated all the changes except IL-10, which normalized. Thus, CN or OMA treatment during healing after RMI results in attenuation of SLPI as well as tissue AngII and mediators of inflammation and matrix/LV remodeling including SPARC, OPN, and ADAMs. Whether increasing SLPI on top of background AngII inhibition or therapy such as CN or OMA might produce added remodeling benefit needs study.