Porcine dermal collagen mesh (Permacol™) as a bioprosthesis in the ligation of intersphincteric tract (BioLIFT) procedure.

BACKGROUND: Ligation of intersphincteric tract (LIFT) is a sphincter-saving technique used to treat anal fistulas. Incorporation of a bioprosthesis in LIFT (BioLIFT) aims to improve healing. The use of cross-linked porcine dermal collagen mesh Permacol™ in BioLIFT has never been investigated. The aim of this study was to compare the healing rates and outcome of LIFT and BioLIFT for complex anal fistulas using the Permacol™ biological mesh. METHODS: A retrospective analysis of all patients having LIFT or BioLIFT for complex fistulas from January 2010 to November 2019 was performed in a tertiary referral centre. Patient data from a prospectively collected database of all patients having LIFT or BioLIFT were analyzed. RESULTS: LIFT and BioLIFT were performed in 48 (82.8%) and 10 (17.2%) patients, respectively. All BioLIFT patients had previous interventions for their fistulas compared to 30 (62.5%) of patients who had LIFT, p = 0.023. The primary healing rate for LIFT was 87.5% (42/48) compared to 80% (8/10) in BioLIFT, (p = 0.42). Eight (13.8%) patients developed complications, 6 (12.5%) in the LIFT group vs 2 (20%) in the BioLIFT group (p = 0.62). On univariate analysis, the number of previous operations was predictive of complications (p = 0.03). BioLIFT was not associated with complication (OR = 1.75, 95% CI: 0.30-10.3, p = 0.54) or primary healing (OR = 0.57, 95% CI: 0.97-3.36, p = 0.54). There was no significant difference in recurrence (LIFT 12.5% vs BioLIFT 0%, p = 0.58). Kaplan-Meier analysis found no difference in time to recurrence between the two groups (p = 0.65). CONCLUSION: Permacol™ mesh in BioLIFT is feasible and achieves a high primary healing rate of 80%. Prospective evidence is needed to establish the benefits of BioLIFT and determine whether Permacol™ is superior to the non-cross-linked porcine submucosal mesh.

cIAP1 and cIAP2 limit macrophage necroptosis by inhibiting Rip1 and Rip3 activation.

Cellular inhibitor of apoptosis proteins (cIAPs) have emerged as important anti-cell death mediators, particularly in cancer. Although they are known to be expressed in immune tissue, their specific immune function remains unclear. We observed that degradation of cIAPs with SMAC mimetic (SM) results in death of primary bone-marrow-derived macrophages. SM-induced death of macrophages occurred by programmed necrosis (necroptosis), which was dependent on TNF receptor expression. Consistent with necroptosis, SM-induced death of macrophages was abrogated by inhibition of receptor interacting protein 1 (Rip1) kinase signaling or by receptor interacting protein 3 (Rip3) knockdown. SM-induced necroptosis was also dependent on inhibition of SM-induced apoptosis due to the expression of the endogenous caspase inhibitor, xIAP. We found that cIAPs limit Rip3, and to a lesser extent Rip1, expression via post-transcriptional mechanisms, leading to inhibition of the Rip1-Rip3 death complex (necrosome). Reduced cIAP activity in vivo, via SM treatment or specific knockout of either cIAP, resulted in elevated macrophage cell death and compromised control of an intracellular bacterium, Listeria monocytogenes. These results show that cIAPs have an important role in limiting programmed necrosis of macrophages, which facilitates effective control of a pathogen.

SMG1 and NIK regulate apoptosis induced by Smac mimetic compounds.

Smac mimetic compounds (SMCs) are experimental small molecules that induce tumour necrosis factor alpha (TNFα)-dependent cancer cell death by targeting the inhibitor of apoptosis proteins. However, many cancer cell lines are resistant to SMC-mediated apoptosis despite the presence of TNFα. To add insight into the mechanism of SMC-resistance, we used functional siRNA-based kinomic and focused chemical screens and identified suppressor of morphogenesis in genitalia-1 (SMG1) and NF-κB-inducing kinase (NIK) as novel protective factors. Both SMG1 and NIK prevent SMC-mediated apoptosis likely by maintaining FLICE inhibitory protein (c-FLIP) levels to suppress caspase-8 activation. In SMC-resistant cells, the accumulation of NIK upon SMC treatment enhanced the activity of both the classical and alternative nuclear factor-κB pathways, and increased c-FLIP mRNA levels. In parallel, persistent SMG1 expression in SMC-resistant cells repressed SMC-mediated TNFα-induced JNK activation and c-FLIP levels were sustained. Importantly, SMC-resistance is overcome by depleting NIK and SMG1, which appear to facilitate the downregulation of c-FLIP in response to SMC and TNFα treatment, leading to caspase-8-dependent apoptosis. Collectively, these data show that SMG1 and NIK function as critical repressors of SMC-mediated apoptosis by potentially converging on the regulation of c-FLIP metabolism.

Methylation of an intronic region regulates miR-199a in testicular tumor malignancy.

In the testicular cancer cell line, NT2, we previously demonstrated that differentially methylated regions were located in introns or intergenic regions, and postulated these might regulate non-coding RNAs. Three microRNAs and three small nucleolar RNAs were differentially methylated; one, miR-199a, was associated with the progression and prognosis of gastric and ovarian cancers. In this report we document, by epigenomic profiling of testicular tissue, that miR-199a is transcribed as antisense of dynamin 3 (chromosome 1q24.3), and hypermethylation of this region is correlated with miR-199a-5p/3p repression and tumor malignancy. Re-expression of miR-199a in testicular cancer cells led to suppression of cell growth, cancer migration, invasion and metastasis. The miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with tumor malignancy. We further identified the embryonal carcinoma antigen podocalyxin-like protein 1 (PODXL), an anti-adhesive protein expressed in aggressive tumors, as a target of miR-199a-5p. We demonstrated PODXL is overexpressed in malignant testicular tumor, and cellular depletion of PODXL resulted in suppression of cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests PODXL is a downstream effector mediating the action of miR199a-5p. This report identifies DNA methylation, miR-199a dysregulation and PODXL as critical factors in tumor malignancy.

Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer.

BACKGROUND: Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples. METHODS: In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs). RESULTS: We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system. CONCLUSION: This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.

Digital fabrication of multi-material biomedical objects.

This paper describes a multi-material virtual prototyping (MMVP) system for modelling and digital fabrication of discrete and functionally graded multi-material objects for biomedical applications. The MMVP system consists of a DMMVP module, an FGMVP module and a virtual reality (VR) simulation module. The DMMVP module is used to model discrete multi-material (DMM) objects, while the FGMVP module is for functionally graded multi-material (FGM) objects. The VR simulation module integrates these two modules to perform digital fabrication of multi-material objects, which can be subsequently visualized and analysed in a virtual environment to optimize MMLM processes for fabrication of product prototypes. Using the MMVP system, two biomedical objects, including a DMM human spine and an FGM intervertebral disc spacer are modelled and digitally fabricated for visualization and analysis in a VR environment. These studies show that the MMVP system is a practical tool for modelling, visualization, and subsequent fabrication of biomedical objects of discrete and functionally graded multi-materials for biomedical applications. The system may be adapted to control MMLM machines with appropriate hardware for physical fabrication of biomedical objects.

IAP-targeted therapies for cancer.

DNA damage, chromosomal abnormalities, oncogene activation, viral infection, substrate detachment and hypoxia can all trigger apoptosis in normal cells. However, cancer cells acquire mutations that allow them to survive these threats that are part and parcel of the transformation process or that may affect the growth and dissemination of the tumor. Eventually, cancer cells accumulate further mutations that make them resistant to apoptosis mediated by standard cytotoxic chemotherapy or radiotherapy. The inhibitor of apoptosis (IAP) family members, defined by the presence of a baculovirus IAP repeat (BIR) protein domain, are key regulators of cytokinesis, apoptosis and signal transduction. Specific IAPs regulate either cell division, caspase activity or survival pathways mediated through binding to their BIR domains, and/or through their ubiquitin-ligase RING domain activity. These protein-protein interactions and post-translational modifications are the subject of intense investigations that shed light on how these proteins contribute to oncogenesis and resistance to therapy. In the past several years, we have seen multiple approaches of IAP antagonism enter the clinic, and the rewards of such strategies are about to reap benefit. Significantly, small molecule pan-IAP antagonists that mimic an endogenous inhibitor of the IAPs, called Smac, have demonstrated an unexpected ability to sensitize cancer cells to tumor necrosis factor-alpha and to promote autocrine or paracrine production of this cytokine by the tumor cell and possibly, other cells too. This review will focus on these and other developmental therapeutics that target the IAPs in cancer.

Both cIAP1 and cIAP2 regulate TNFalpha-mediated NF-kappaB activation.

The cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2) proteins have been implicated in the activation of NF-kappaB by TNFalpha; however, genetic deletion of either cIAP1 or 2 did not support a physiologically relevant role, perhaps because of functional redundancy. To address this, we used combined genetic and siRNA knockdown approaches and report that cIAP1 and 2 are indeed critical, yet redundant, regulators of NF-kappaB activation upon TNFalpha treatment. Whereas NF-kappaB was properly activated by TNFalpha in cultured and primary cells deficient in either cIAP1 or 2, removal of both cIAPs severely blunted its activation. After treatment with TNFalpha, cIAP1 and 2 were rapidly recruited to the TNF receptor 1, along with the adapter protein TNF receptor associated factor 2. Importantly, either cIAP1 or 2 was required for proper TNF receptor 1 signalosome function. In their combined absence, polyubiquitination of receptor interacting protein 1, an upstream event necessary for NF-kappaB signaling, was attenuated. As a result, phosphorylation of the inhibitor of kappaB kinase beta was diminished, and signal transduction was severely blunted. Consequently, cells missing both cIAP1 and 2 were sensitized to TNFalpha-mediated apoptosis. Collectively, these data demonstrate that either cIAP1 or 2 is required for proper Rip1 polyubiquitination and NF-kappaB activation upon TNFalpha treatment.

Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia.

The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases.

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.

Coronary artery fistulas: long-term results of surgical correction.

BACKGROUND: Opinions vary as to whether operation should be offered patients with coronary artery fistula, particularly to those who are asymptomatic. Published studies lacked long-term follow-up data. METHODS: We studied 41 patients with coronary artery fistula operated in our unit in the past 30 years with restudies including coronary angiograms in those who agreed to the investigation. RESULTS: There was no operative mortality and operative morbidity was low. The mean duration of follow-up was 9.1 years and 96.9% of the patients were asymptomatic. Twenty-one patients had a coronary angiogram. The native coronary artery either remained dilated and tortuous, or more frequently had thromboses with a short proximal stump. (None of these patients had evidence of myocardial ischemia.) Four patients had demonstrable recurrence fistula but without hemodynamic disturbance. CONCLUSIONS: We advocate operation for all patients with coronary artery fistulas and demonstrable shunting in view of minimal operative risks. Small asymptomatic fistulas without demonstrable shunting should be left alone. The relatively high incidence of residual or recurrent fistula makes long-term follow-up mandatory.

Altered association of protein tyrosine kinases with postsynaptic densities after transient cerebral ischemia in the rat brain.

Transient cerebral ischemia results in an increase in the tyrosine phosphorylation of proteins associated with postsynaptic densities (PSDs). The authors investigated the possible mechanisms behind this increase by analyzing isolated PSDs for protein tyrosine kinase activity and for the presence of specific tyrosine kinases. Transient (15 minutes) global ischemia was produced in adult rats by four-vessel occlusion, and PSDs were isolated immediately after ischemia or after 20 minutes or 6 hours of reperfusion. Tyrosine phosphorylation of several PSD proteins, including the N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, was enhanced relative to shams after 20 minutes of reperfusion and underwent a further increase between 20 minutes and 6 hours. The ability of intrinsic PSD tyrosine kinase to phosphorylate PSD proteins, including the NMDA receptor, increased threefold after ischemia. Whereas PSD-associated proline-rich tyrosine kinase 2 (PYK2) and gp145TrkB were elevated immediately after the ischemic event, increases in Src and Fyn were not apparent until 6 hours of reperfusion. The level of PSD-associated pp125FAK decreased after ischemia. The results demonstrate that ischemia results in selective changes in the association of protein tyrosine kinases with the PSD which may account for ischemia-induced increases in the tyrosine phosphorylation of PSD proteins.

The effect of transient global ischemia on the interaction of Src and Fyn with the N-methyl-D-aspartate receptor and postsynaptic densities: possible involvement of Src homology 2 domains.

Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase-SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.

An unusual cause of disabling angina following aortic valve replacement.

Severe ostial stenosis of the coronary arteries following aortic valve replacement is a potentially lethal complication. The usual presentations are recent onset of severe angina, ventricular arrhythmias, congestive heart failure, and sudden death. It is generally accepted to arise from injury to the coronary arteries during direct cannulation and continuous perfusion of cardioplegia under high pressure during operation. We report on a patient who developed critical left coronary ostial stenosis after aortic valve replacement. The cause for the stenosis was probably related to the over-sizing and orientation of the prosthesis. The prosthesis was replaced and patch angioplasty of the left coronary ostia performed. The patient was well with normal coronary anatomy three years after surgery.

Left subclavian arterioesophageal fistula induced by a foreign body.

A left subclavian arterioesophageal fistula was diagnosed in a 35-year-old man at exploratory thoracotomy for suspected aortoesophageal fistula. After successful closure of the arterial fistula the patient developed a mediastinal abscess and esophagopleural fistula. The latter was successfully managed by retrosternal jejunal esophagoplasty followed by excision of the thoracic esophagus. This report documents a case of left subclavian arterioesophageal fistula and illustrates the importance of early diagnosis and surgical intervention of arterial perforation secondary to a foreign body in the esophagus.