Corrigendum: Eastern Canadian Gastrointestinal Cancer Consensus Conference 2014.

[This corrects the article DOI: 10.3747/co.22.2603.].

ABO-incompatible heart transplants.

A month-old baby girl with blood type O positive received a donor heart organ from a donor with blood type B. This was the first institutional ABO-incompatible heart transplant. Infants listed for transplantation may be considered for an ABO-incompatible heart transplant based on their antibody levels and age. The United Network of Organ Sharing (UNOS) protocol is infants under 24 months with titers less than or equal to 1:4.(1) This recipient's anti-A and anti-B antibodies were monitored with titer assays to determine their levels; antibody levels less than 1:4 are acceptable pre-transplant in order to proceed with donor and transplant arrangements.1 Immediately prior to initiating cardiopulmonary bypass (CPB), a complete whole body exchange transfusion of at least two-times the patient's circulating blood volume was performed with packed red blood cells (pRBC), fresh frozen plasma (FFP) and 25% albumin. Titer assays were sent two minutes after initiation of full CPB and then hourly until the cross-clamp was removed. Institutionally, reperfusion of the donor heart is not restored until the antibody level from the titer assay is known and reported as less than 1:4; failing to achieve an immulogically tolerant recipient will provide conditions for hyperacute rejection. The blood collected during the transfusion exchange was immediately processed through a cell saver so the pRBC's could be re-infused to the patient during CPB, as necessary. The remainder of the transplant was performed in the same fashion as an ABO-compatible heart transplant. The patient has shown no signs of rejection following transplantation.

Eastern Canadian Gastrointestinal Cancer Consensus Conference 2014.

The annual Eastern Canadian Colorectal Cancer Consensus Conference was held in Montreal, Quebec, 23-25 October 2014. Expert radiation, medical, and surgical oncologists and pathologists involved in the management of patients with gastrointestinal malignancies participated in presentations and discussions resulting in consensus statements on such hot topics as management of neuroendocrine tumours, advanced and metastatic pancreatic cancer, and metastatic colorectal cancer.

Anomalies in the inflammatory response in endometriosis and possible consequences: a review.

Defined by the presence of endometrial-like cells outside the uterus, endometriosis is one of the most diagnosed gynecological disorders, affecting 5 to 10 % of reproductive age women, but the true incidence is unknown. Endometriosis is a major cause of pelvic pain, dysmenorrhea, dyspareunia, infertility and menstrual irregularities, but there is no clear correlation between the symptoms and the extent of the disease. Despite decades of intensive investigations, little is known about the pathogenesis of endometriosis. The disease is often associated with chronic pelvic inflammation. Abnormal levels of immune cells such macrophages, dendritic and natural killer cells were found in the peritoneal cavity of patients. However these cells seem to be unable to detect and eliminate ectopic endometrial cells. Several studies showed that peritoneal immune cells are dysfunctional and may rather contribute to endometriosis development. A review of relevant clinical and scientific studies was carried out. This review sheds light on cellular and immunological pro-inflammatory changes which were observed in patients with endometriosis, their impact on angiogenesis, apoptosis, extracellular matrix remodeling and hormonal production and consequences on fertility.

Incorporating high fidelity simulation into perfusion education.

The new Perfusion Simulation Center at the Medical University of South Carolina provides a new level of high fidelity simulation training for perfusion students. A key component is the Orpheus Perfusion Simulator which is a computer-driven simulator integrated with the mechanical connections of the heart-lung machine to allow for real time operative procedures and perfusion incidents. Due to the ability to consistently reproduce cardiac surgical scenarios, it is possible to develop both basic perfusion skills as well as advanced emergency skills more effectively than with animal models. The purpose of this paper is to provide details about advanced simulation for perfusionists and to illustrate how simulation can be used to promote the assets of good communication, team work, and surgical awareness. Two sets of four cardiac surgical scenarios were recorded in the perfusion simulation operating room. Scenario team member roles included a cardiac surgeon, an anesthesiologist, a perfusionist and an operating room nurse. The scripted surgical scenarios were viewed by a focus group of students charged with identifying key personality traits of different members of the operating team and to characterize them using a list of descriptive words adapted from the Medical University of South Carolina's Peer Review Tool. In the first set of scenarios, initial scores were negative, with irresponsibility, impatience, and carelessness listed as the top behavioral characteristics leading to human error. In the second set of scenarios, logical, clear-thinking, and attentive were the most common personality traits observed of the effective team members. Simulation has become an invaluable tool for perfusion education and the goal of improving patient safety during cardiopulmonary bypass. The opportunities for advanced training in the perfusion simulation environment will certainly expand in the future.

Pathways for the nuclear transport of proteins and RNAs.

The nuclear pore complex catalyses the import and export of both proteins and RNAs. The molecular mechanisms of RNA and protein translocation through the nuclear pore are likely to be similar; however, their signals and targeting apparatus may differ. Recent insights into RNA transport have come from studies of kinetic control mechanisms and the preconditions for translocation that include processing, RNP assembly, and a targeting function for 5' caps.

Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway.

Protein import to the nucleus is a signal-mediated process that exhibits saturation kinetics. We investigated whether signal bearing proteins compete with U2 and U6 snRNPs during import. When injected into Xenopus oocytes, saturating concentrations of P(Lys)-BSA, a protein bearing multiple nuclear localization signals from SV40 large T-antigen, reduce the rate of [125I]P(Lys)-BSA and of [125I]nucleoplasmin import, consistent with their competing for and sharing the same limiting component of the import apparatus. In contrast, saturating concentrations of P(Lys)-BSA do not reduce the rate of HeLa [32P]U2 snRNP assembly or import. The import of U6 snRNP is also competed by P(Lys)-BSA. We conclude that U2 snRNP is imported into oocyte nuclei by a kinetic pathway that is distinct from the one followed by P(Lys)-BSA, nucleoplasmin, and U6 snRNP.

Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways.

The inhibitory effects of wheat germ agglutinin and mAb 414 on the nuclear import of all types of U snRNAs indicate that they cross the nuclear envelope through the nuclear pore complex. However, the import of different U snRNAs occurs by kinetically distinct targeting pathways that can be distinguished from one another by the competitive effects of free trimethylguanosine cap dinucleotide (m3GpppG) and P(Lys)-BSA, an efficient synthetic karyophile based on the nuclear localization signal of SV40 large T antigen. The import of U snRNAs that contain 5' m3GpppN caps and are complexed by Sm proteins (U1, U2, U4, and U5) is competed by coinjection with free m3GpppG, indicating a shared transport factor, but not by P(Lys)-BSA. The import of U6 snRNA, which lacks a m3GpppN cap and is not complexed by the Sm proteins, is competed by P(Lys)-BSA but not by free m3GpppG. Thus, by the criterion of kinetic competition, U6 snRNA import is identical to that of the karyophilic proteins P(Lys)-BSA and nucleoplasmin. Uniquely, the import of U3 snRNA, which contains a m3GpppN cap but does not bind Sm proteins is not competed by either free m3GpppG or P(Lys)-BSA. Thus, U3 snRNA appears to be imported by a novel third kinetic pathway.

An NAD derivative produced during transfer RNA splicing: ADP-ribose 1"-2" cyclic phosphate.

Transfer RNA (tRNA) splicing is essential in Saccharomyces cerevisiae as well as in humans, and many of its features are the same in both. In yeast, the final step of this process is removal of the 2' phosphate generated at the splice junction during ligation. A nicotinamide adenine dinucleotide (NAD)-dependent phosphotransferase catalyzes removal of the 2' phosphate and produces a small molecule. It is shown here that this small molecule is an NAD derivative: adenosine diphosphate (ADP)-ribose 1"-2" cyclic phosphate. Evidence is also presented that this molecule is produced in Xenopus laevis oocytes as a result of dephosphorylation of ligated tRNA.

14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3- and Ras-independent manner.

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induced meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.

Identification of constitutive and ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression.

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.

Light and electron microscopic analysis of tattoos treated by Q-switched ruby laser.

Short-pulse laser exposures can be used to alter pigmented structures in tissue by selective photothermolysis. Potential mechanisms of human tattoo pigment lightening with Q-switched ruby laser were explored by light and electron microscopy. Significant variation existed between and within tattoos. Electron microscopy of untreated tattoos revealed membrane-bound pigment granules, predominantly within fibroblasts and macrophages, and occasionally in mast cells. These granules contained pigment particles ranging from 2-in diameter. Immediately after exposure, dose-related injury was observed in cells containing pigment. Some pigment particles were smaller and lamellated. At fluences greater than or equal to 3 J/cm2, dermal vacuoles and homogenization of collagen bundles immediately adjacent to extracellular pigment were occasionally observed. A brisk neutrophilic infiltrate was apparent by 24 h. Eleven days later, the pigment was again intracellular. Half of the biopsies at 150 d revealed a mild persistent lymphocytic infiltrate. There was no fibrosis except for one case of clinical scarring. These findings confirm that short-pulse radiation can be used to selectively disrupt cells containing tattoo pigments. The physial alteration of pigment granules, redistribution, and elimination appear to account for clinical lightening of the tattoos.

Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to 7 weeks after treatment.

PURPOSE: To study the long-term effects of photodynamic therapy (PDT), using liposomal benzoporphyrin derivative (BPD) or Verteporfin, on experimental choroidal neovascularization (CNV) and on normal retina and choroid (with no CNV) in the cynomolgus monkey eye. METHODS: Photodynamic therapy was performed in 8 cynomolgus monkey eyes with experimental CNV induced by laser injury. The effect of PDT on normal retina and choroid (with no CNV) was studied in 9 monkey eyes. Liposomal BPD was administered intravenously (0.375 mg/kg) either as a bolus, as a slow infusion over 32 minutes, or as a fast infusion over 10 minutes. Photodynamic therapy was performed using light at a wavelength of 689 or 692 nm, with an irradiance of 600 mW/cm2 and fluence of 150 J/cm2. Follow-up studies, including fundus photography and FA, were performed at 24 hours after PDT and then weekly. Indocyanine green and BPD angiography were performed in selected cases. Tissues were examined with light and electron microscopy at the end of follow-up. RESULTS: Twenty-three of the 32 areas of CNV treated with PDT showed absence of angiographic leakage at 24 hours. Twenty-eight areas of CNV were followed for 4 weeks; 22 of 28 showed absence of angiographic leakage at 2 weeks; and 20 of 28 at 4 weeks of follow-up. Forty spots on the normal retina and choroid were treated with PDT and were followed for 4 to 7 weeks. These spots showed pigment-laden cells in the outer retina, variably pigmented retinal pigment epithelium (RPE) in the treated area, intact neurosensory retina, and reperfusion of the choriocapillaris. CONCLUSIONS: Photodynamic therapy leads to absence of angiographic leakage for at least 4 weeks in experimental CNV in the monkey model. In the normal monkey eye the RPE and choriocapillaris show generalized recovery with preservation of the neurosensory retina 7 weeks after PDT.

Response of the retinal pigment epithelium to selective photocoagulation.

Multiple short argon laser pulses can coagulate the retinal pigment epithelium selectively, while sparing the adjacent neural retina and choroid; in contrast, continuous-wave laser irradiation typically damages the neural retina and choroid. The healing response to selective photocoagulation of the retinal pigment epithelium was studied in rabbits during a period of 4 weeks. The lesions were never visible ophthalmoscopically. During the healing period, the epithelium was reformed by a single sheet of hypertrophic retinal pigment epithelial cells. In contrast to continuous-wave photocoagulation, only minimal inflammatory response was found. Retinal pigment epithelial cells showed clear signs of viability, eg, phagocytized outer segments. The local edema in the photoreceptor layer and subretinal space found in the early stage disappeared when the blood-retinal barrier was reestablished. The choriocapillaris remained unaffected. No subsequent damage to the photoreceptors was found. This type of photocoagulation may be useful for retinal pigment epithelium-related diseases, eg, diffuse diabetic macular edema.

Intravenous infusion of liposomal benzoporphyrin derivative for photodynamic therapy of experimental choroidal neovascularization.

OBJECTIVE: To compare the effectiveness of photodynamic therapy to close experimental choroidal neovascularization using an intravenous infusion of liposomal benzoporphyrin derivative (verteporfin) with previous work using a rapid intravenous injection, before initiating clinical trials. METHODS: Choroidal neovascularization was induced in cynomolgus monkey eyes using argon laser. Liposomal benzoporphyrin derivative was delivered by an intravenous infusion pump for 10 or 32 minutes at a dose of 0.375 mg/kg. Irradiation was performed with 689- or 692-nm laser light (600-mW/cm2 irradiance and 150-J/cm2 fluence) in 7 normal eyes and 11 eyes with choroidal neovascularization between 30 and 105 minutes after the start of dye infusion. Findings were documented by fundus photography, fluorescein angiography, and light and electron microscopy. RESULTS: Irradiation within 32 to 50 minutes of the start of the fast (10 minutes) or slow (32 minutes) dye infusion resulted in closure of choroidal neovascularization. In normal eyes, this technique caused choriocapillaris closure and retinal pigment epithelium damage with minimal damage to surrounding tissues. CONCLUSION: Photodynamic therapy using intravenous infusion of liposomal benzoporphyrin derivative selectively closed experimental choroidal neovascularization. This may be a suitable modality for clinical use.

Photodynamic therapy of experimental choroidal neovascularization using lipoprotein-delivered benzoporphyrin.

OBJECTIVE: To investigate photodynamic therapy of experimental choroidal neovascularization using benzoporphyrin derivative monoacid (Verteporfin). METHODS: Photodynamic therapy using benzoporphyrin derivative monoacid was investigated in cynomolgus monkeys. Following intravenous injection of benzoporphyrin derivative monoacid (1 to 2 mg/kg) complexed with low-density lipoprotein, the eyes were irradiated with 692-nm light at a fluence of 50 to 150 J/cm2 and irradiance of 150 to 600 mW/cm2. Choroidal neovascularization was documented before photodynamic therapy and closure was demonstrated by fundus photography, fluorescein angiography, and light and electron microscopic examination. RESULTS: Following photodynamic therapy, vessels within choroidal neovascularization were occluded, and there was damage to the choroidal neovascularization endothelium and the subjacent choriocapillaris. Damage to the retinal pigment epithelium and photoreceptors was also observed. CONCLUSION: Photodynamic therapy with lipoprotein-delivered benzoporphyrin derivative monoacid was effective in this animal model of choroidal neovascularization and may be a promising, potentially selective, therapy for choroidal neovascularization.

Tattoo darkening and nonresponse after laser treatment: a possible role for titanium dioxide.

OBJECTIVE: To examine relationships between chemical composition, biopsy findings, and clinical outcome in laser-treated tattoos. DESIGN: Observational nonblinded retrospective study. SETTINGS: University-based dermatology clinic and private practice. PARTICIPANTS: Twenty patients who underwent biopsy of laser-treated tattoos. MAIN OUTCOME MEASURES: Biopsy specimens were analyzed after laser treatment, and the depths of changed particles were recorded. Ultrastructure of the changed particles was examined by electron microscopy. Presence of inorganic chemicals was determined by x-ray diffraction. Correlation between x-ray diffraction, microscopy, and clinical response was attempted. RESULTS: Of the 20 tattoos, 7 lightened, 9 failed to change, and 4 darkened after laser treatment. There was a significant association between presence of titanium dioxide and poor response to laser therapy. Microscopic studies showed variable changes in the ink particles, but there was a trend toward residual deep green pigment in the resistant tattoos. Also, round dark stippling was observed superficially in the darkened specimens. CONCLUSIONS: Titanium is overrepresented in tattoos that respond poorly to laser treatment. Further studies are necessary to show whether this metal is the primary cause of this poor response.

Comparison of responses of tattoos to picosecond and nanosecond Q-switched neodymium: YAG lasers.

OBJECTIVE: To test the hypothesis that picosecond laser pulses are more effective than nanosecond domain pulses in clearing of tattoos. DESIGN: Intratattoo comparison trial of 2 laser treatment modalities. SETTING: A large interdisciplinary biomedical laser laboratory on the campus of a tertiary medical center. PATIENTS: Consecutive patients with black tattoos were enrolled; all 16 patients completed the study. INTERVENTION: We treated designated parts of the same tattoo with 35-picosecond and 10-nanosecond pulses from 2 neodymium:YAG lasers. Patients received a total of 4 treatments at 4-week intervals. All laser pulse parameters were held constant except pulse duration. Radiation exposure was 0.65 J/cm2 at the skin surface. Biopsies were performed for routine microscopic and electron microscopic analysis at the initial treatment session and 4 weeks after the final treatment in 8 consenting patients. Also, ink samples were irradiated in vitro. MAIN OUTCOME MEASURES: In vivo, on the completion of treatment, a panel of dermatologists not associated with the study (and blinded to the treatment type) evaluated photographs to assess tattoo lightening. Formalin-fixed specimens were examined for qualitative epidermal and dermal changes as well as depth of pigment alteration. Electron micrographs were examined for particle electron density and size changes (in vivo and in vitro). The gross in vitro optical density changes were measured. RESULTS: In 12 of 16 tattoos, there was significant lightening in the picosecond-treated areas compared with those treated with nanosecond pulses. Mean depth of pigment alteration was greater for picosecond pulses, but the difference was not significant. In vivo biopsy specimens showed similar electron-lucent changes for both pulse durations. In vitro results were similar for both pulse durations, showing increases in particle sizes and decreased electron density as well as gross ink lightening. CONCLUSIONS: Picosecond pulses are more efficient than nanosecond pulses in clearing black tattoos. Black tattoos clear principally by laser-induced changes in the intrinsic optical properties of the ink.

Treatment of tattoos by Q-switched ruby laser. A dose-response study.

Tattoo treatment with Q-switched ruby laser pulses (694 nm, 40 to 80 nanoseconds) was studied by clinical assessment and light and electron microscopy. Fifty-seven blue-black tattoos or portions thereof (35 amateur and 22 professional) were irradiated with 1.5 to 8.0 J/cm2 at a mean interval of 3 weeks. Substantial lightening or total clearing occurred in 18 (78%) of 23 amateur tattoos and 3 (23%) of 13 professional tattoos in which the protocol was completed. Response was related to exposure dose. Scarring occurred in one case, and persistent confettilike hypopigmentation was frequent. Optimal fluence was 4 to 8 J/cm2. Clinicohistologic correlation was poor. Q-switched ruby laser pulses can provide an effective treatment for tattoos.

[Histology of retinal lesions after continuous irradiation and selective micro-coagulation of the retinal pigment epithelium]. / Histologie von Netzhautläsionen nach kontinuierlicher Bestrahlung und nach selektiver Mikrokoagulation des retinalen Pigmentepithels.

Mild continuous wave (CW) irradiation (100 ms, 20 mW, 514 nm) and irradiation with 100 repetitive 5 microseconds laser pulses (3 or 6 microJ, 514 nm) at a repetition rate of 500 Hz was performed to the regio macularis of chinchilla rabbits. The angiographically visible lesions were histologically followed up to 4 weeks. With both irradiation modalities the original retinal pigment epithelium (RPE) was replaced by a monolayer of new RPE cells. Only minimal immediate and no subsequent damage to the photoreceptors was found after selective RPE photocoagulation. Only minimal inflammatory response was found after selective RPE photocoagulation in contrast to CW photocoagulation where macrophages, RPE cells and lymphocytes regularly appear in the damaged photoreceptor layer.