In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells.

Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.

Clinical Profile of Patients With Diabetes Mellitus and Liver Transplantation: Results After a Multidisciplinary Team Intervention.

BACKGROUND: Over the years, survival after liver transplantation has increased and metabolic complications are becoming more common, contributing to patients' morbidity and mortality. The objectives of this study were to describe a population of patients with hepatic transplantation and diabetes mellitus (DM), evaluate the frequency of metabolic complications, and assess the impact of a multidisciplinary team on DM management. MATERIALS AND METHODS: This was a retrospective study involving interview and medical record analysis of 46 consecutive patients followed at the diabetes mellitus and liver transplantation unit of a tertiary university hospital, all evaluated by a multidisciplinary team. RESULTS: Of all patients, 76.1% were men, with a median age 60 years old (interquartile range: 56 to 65 years) and liver transplantation time of 5 years (interquartile range: 0.6-9 years). Hypertension, hypercholesterolemia, hypertriglyceridemia, alcoholism, and smoking were present in 47.8%, 34.8%, 23.9%, 34.8%, and 30.4% of the patients, respectively. The most frequent immunosuppressant in use was tacrolimus (71.1%). Regarding nutritional status, 37.9% of patients were classified as overweight according to body mass index, and 41.2% were considered overweight according to the triceps skin fold. The median glycosylated hemoglobin and weight before and after intervention of the multidisciplinary team in all 46 patients were, respectively, 7.6% (5.7% to 8.8%) versus 6.5% (5.7% to 7.7%); P = .022 and 70.5 kg (64.7 to 82.0 kg) versus 71.6 kg (65.0 to 85.0 kg); P = .18. CONCLUSIONS: Hypertension and dyslipidemia were common in transplanted patients with DM. Intervention of the multidisciplinary team resulted in a significant improvement in glycosylated hemoglobin without significant weight gain.

Frequency of Thyroid Dysfunction in Patients With Diabetes Mellitus Before and After Liver Transplantation.

BACKGROUND: There is mutual influence between the liver and thyroid hormone metabolism. Patients with diabetes mellitus (DM) also have an increased prevalence of thyroid disorders (TDs). The objectives of this study were to evaluate the frequency of TD before and after liver transplantation (LT) in a population of patients with DM as a whole and when categorized by sex. MATERIALS AND METHODS: This was a retrospective study involving interview and medical record analysis of 46 consecutive patients followed at the diabetes mellitus and liver transplantation unit of a tertiary university hospital. RESULTS: Of all patients, 76.1% were men with a median age of 60 years old (interquartile range: 56 to 65 years) and time since LT of 5 years (range, 0.6 to 9 years). Hypertension, hypercholesterolemia, hypertriglyceridemia, alcoholism, and smoking were present in 47.8%, 34.8%, 23.9%, 34.8%, and 30.4% of the patients, respectively. The most frequent immunosuppressant in use was tacrolimus (71.1%). TD was present in 4.3% and 13% before and after LT, respectively (P = .058). In women and men, these frequencies were 9.1% and 18.2% (P = .563), and 2.9% and 11.8% (P = .045), respectively. CONCLUSIONS: Frequency of TD was high both before and after LT. After transplantation, prevalence of TD increased in men and differences between males and females almost disappeared. Further studies are needed to assess if screening for TD before and after LT in patients with DM might be beneficial, especially in men.

Acute Liver Failure-25 Years at a Single Center: Role of Liver Transplantation in the Survival of Adult Patients.

BACKGROUND: Acute liver failure (ALF) leads to high morbidity and mortality and is characterized by an accelerated deterioration of hepatic function in patients without prior liver disease. The survival rate is <15% without liver transplantation (LT). The aim of this study was to describe the population of patients with ALF in the Unit of Liver Transplantation of the University of Campinas, Brazil, from 1991 to 2017, comparing those submitted and not submitted to LT. METHODS: The patients were divided into 2 groups: 1, listed but not transplanted; and 2, transplanted. RESULTS: There were 73 patients with ALF listed for LT, with a mean age of 33.6 years, 49 (67.1%) female and 24 (32.9%) male. Group 1, with 32 patients, had a mean age of 29.3 years; 26 (81.25%) died on the waiting list; 6 (8.45%), with a mean age of 12.33 years, were removed from the list because of recovery of liver function. Considering only adult patients, the mortality without LT was 96.29%. Group 2 had 41 patients, with a mean age of 37.1 years, and a 30-day survival of 41.02%. Thus, LT led to a significant improvement in the survival of adult patients with ALF. The time of surgery, packed red blood cells, and intraoperative plasma, were associated with LT survival after logistic regression study, whereas age, body mass index, bilirubin, international normalized ratio, creatinine, sodium, and Model for End-Stage Liver Disease score were not. CONCLUSIONS: ALF affects an active age range, and LT decreases mortality; there was no good preoperative prognostic indicator to assess which patients would benefit from transplantation.

Acute Liver Failure Induced by Carthamus tinctorius Oil: Case Reports and Literature Review.

BACKGROUND: Acute liver failure (ALF) is a clinical syndrome that results from the abrupt loss of liver function in a patient without previous liver disease. The most frequent causes are viral hepatitis, drug induced, and autoimmune disease, but in 20% of cases no cause is identified. Carthamus tinctorius (safflower) oil is used as a dietary supplement for weight loss and antioxidant. There are 4 cases described in the literature of ALF induced by the use of this substance. The objective of this study was to report 3 cases of ALF treated at the Clinical Hospital of the State University of Campinas that suggest the use of C tinctorius oil as a probable etiologic factor. CASE REPORTS: The 3 patients had a diagnosis of ALF according to the King's College criteria. All had a history of ingestion of this oil for weight loss. During etiologic evaluation, viral hepatitis, autoimmune diseases, or any other drug cause were excluded, thus pointing to C tinctorius oil as the triggering factor. All 3 patients underwent liver transplantation: 2 had good postoperative evolution, and 1 died 12 days after the procedure. CONCLUSIONS: Two cases are described in which the hepatic insufficiency induced by C tinctorius oil was successfully treated through liver transplantation. This highlights the risk of misuse of this substance for weight loss.

Immunomodulation, Acute Renal Failure, and Complications of Basiliximab Use After Liver Transplantation: Analysis of 114 Patients and Literature Review.

Basiliximab is considered to be effective in preventing cellular rejection (CR) in solid organ transplantation and is commonly used for renal transplants. The aim of this study was describe the population of patients undergoing orthotopic liver transplantation (LT) receiving basiliximab in the period 2012-2016 in the liver transplant service at the State University of Campinas, São Paulo, Brazil. We analyzed 114 patients who underwent LT and received basiliximab; 83 (72.8%) were male and 31 (27.2%) female, with an overall mean age of 54.3 years. Immunosuppression was performed with corticosteroids during anesthetic induction, and postoperatively with tacrolimus in 85.5%, sodium mycophenolate in 81.7%, cyclosporine in 12.7%, and everolimus in 15.5% of patients. CR was observed in 25.43% of patients, confirmed by biopsy in 15 patients: 50% acute CR, 21.42% late acute CR, and 28.57% chronic CR. Thus, the data are consistent with the literature regarding the benefit of using basiliximab as induction therapy while reducing the incidence of CR after LT, but on univariate analysis to evaluate factors associated with the occurrence of CR, the analyzed variables did not present statistical significance. There was acute renal failure (ARF) in 46.84% of patients and hemodialysis was performed in 20% of cases. In a previous series in our service, there was an ARF rate of 50%, so the incidence reduction of ARF after basiliximab use was 3.16%. Moreover, there was 6.95% hepatic artery thrombosis, 2.6% portal vein thrombosis, 2.6% biliary fistulas, 17.4% pneumonia, and 3.4% sepsis, which did not differ from the literature or from our earlier study without the use of basiliximab, suggesting the safety of this medication. In conclusion, in this series, basiliximab influenced the decrease of the CR incidence with no proven benefit on improvement in the ARF.

Immunoexpression of Heat Shock Protein 70, Glypican 3, Glutamine Synthetase, and Beta-Catenin in Hepatocellular Carcinoma After Liver Transplantation: Association Between Positive Glypican 3 and Beta-Catenin With the Presence of Larger Nodules.

BACKGROUND: Hepatocellular carcinoma (HCC) is the 6th leading cause of cancer worldwide. Its recurrence ranges from 6% to 26%. In the literature, many factors are associated with higher risk of recurrence, without a clear definition of the best method that could predict this highly lethal event. OBJECTIVE: The aim of this study was to evaluate the immunoexpression of immunohistochemical markers: HSP70, glypican 3, glutamine synthetase, and beta-catenin, as well as studying their association with tumor characteristics and prognosis of patients undergoing liver transplantation for HCC. METHODS: We studied 90 patients who underwent liver transplantation from 1998 to 2012. Afterwards we evaluated factors related to survival, tumor recurrence, and the correlation of expression of the immunohistochemical markers. RESULTS: Immunohistochemical marker glutamine synthetase showed a positive trend toward better survival. HSP70-positive patients had a higher prevalence of histologic grade III. Patients with positive glypican 3 showed larger lesions and a higher number with AFP >200 ng/mL. Patients with positive beta-catenin showed larger nodules and more with histologic grade III. The association between beta-catenin and glypican 3 showed positive association with larger nodules. CONCLUSIONS: Most of the markers studied had a correlation with at least one of the variables studied, confirming our hypothesis that these markers can indeed assist in assessing the prognosis of patients undergoing liver transplantation for HCC.

Truncated RUNX1 protein generated by a novel t(1;21)(p32;q22) chromosomal translocation impairs the proliferation and differentiation of human hematopoietic progenitors.

We have identified a new t(1;21)(p32;q22) chromosomal translocation in a MDS/AML patient that results in expression of an aberrant C-terminally truncated RUNX1 protein lacking several regulatory domains. As similar truncated RUNX1 proteins are generated by genetic aberrations including chromosomal translocations and point mutations, we used the t(1;21)(p32;q22) chromosomal translocation as a model to explore whether C-terminally truncated RUNX1 proteins trigger effects similar to those induced by well-characterized leukemogenic RUNX1 fusion genes. In vitro analysis of transduced human hematopoietic/progenitor stem cells showed that truncated RUNX1 proteins increase proliferation and self-renewal and disrupt the differentiation program by interfering with RUNX1b. These effects are similar to but milder than those induced by the RUNX1/ETO fusion protein. GSEA analysis confirmed similar altered gene expression patterns in the truncated RUNX1 and RUNX1/ETO models, with both models showing alterations in genes involved in self-renewal and leukemogenesis, including homeobox genes, primitive erythroid genes and leukemogenic transcription factors. We propose that C-terminally truncated RUNX1 proteins can contribute to leukemogenesis in a similar way to RUNX1 fusion genes but through a milder phenotype.

Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system.

Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. Here we present a strategy for generating cancer-related human chromosomal translocations in vitro based on the ability of the RNA-guided CRISPR-Cas9 system to induce DSBs at defined positions. Using this approach we generate human cell lines and primary cells bearing chromosomal translocations resembling those described in acute myeloid leukaemia and Ewing's sarcoma at high frequencies. FISH and molecular analysis at the mRNA and protein levels of the fusion genes involved in these engineered cells reveal the reliability and accuracy of the CRISPR-Cas9 approach, providing a powerful tool for cancer studies.

Downregulation of specific miRNAs in hyperdiploid multiple myeloma mimics the oncogenic effect of IgH translocations occurring in the non-hyperdiploid subtype.

Currently, multiple myeloma (MM) patients are broadly grouped into a non-hyperdiploid (nh-MM) group, highly enriched for IgH translocations, or into a hyperdiploid (h-MM) group, which is typically characterized by trisomies of some odd-numbered chromosomes. We compared the micro RNA (miRNA) expression profiles of these two groups and we identified 16 miRNAs that were downregulated in the h-MM group, relative to the nh-MM group. We found that target genes of the most differentially expressed miRNAs are directly involved in the pathogenesis of MM; specifically, the inhibition of hsa-miR-425, hsa-miR-152 and hsa-miR-24, which are all downregulated in h-MM, leads to the overexpression of CCND1, TACC3, MAFB, FGFR3 and MYC, which are the also the oncogenes upregulated by the most frequent IgH chromosomal translocations occurring in nh-MM. Importantly, we showed that the downregulation of these specific miRNAs and the upregulation of their targets also occur simultaneously in primary cases of h-MM. These data provide further evidence on the unifying role of cyclin D pathways deregulation as the key mechanism involved in the development of both groups of MM. Finally, they establish the importance of miRNA deregulation in the context of MM, thereby opening up the potential for future therapeutic approaches based on this molecular mechanism.

Chromatin modifications induced by the AML1-ETO fusion protein reversibly silence its genomic targets through AML1 and Sp1 binding motifs.

The AML1-ETO fusion protein, which is present in 10-15% of cases of acute myeloid leukemia, is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene enabled us to identify 1168 AML1-ETO target genes, 103 of which were co-occupied by histone deacetylase 1 (HDAC1) and had lost the hyperacetylation mark at histone H4, and 264 showed a K9 trimethylation at histone H3. Enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed in the presence of these epigenetic modifications associated with an 'inactive' chromatin status. Furthermore, AML1-ETO target genes had a significant correlation between the chromatin marks studied and transcriptional silencing. Interestingly, AML1 binding sites were absent on a large number of selected AML1-ETO promoters and an Sp1 binding site was found in over 50% of them. Reversible silencing induced by the fusion protein in the presence of AML1 and/or Sp1 transcription factor binding site was confirmed. Therefore, this study provides a global analysis of AML1-ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA binding pattern of AML1-ETO, suggesting a role for Sp1-targeted therapy in this leukemia subtype.

Influencia de la analgesia epidural en el parto / Influence of epidural analgesia during labor

Introducción La analgesia epidural (AE) es la técnica más efectiva para aliviar el dolor durante el trabajo de parto, pero puede tener algunos efectos secundarios. El objetivo de este trabajo es estudiar la influencia de la AE en la tasa de partos instrumentales, cesáreas y fiebre intraparto. Material y método Realizamos un estudio de cohorte retrospectivo de los 10.115 partos atendidos entre el 1 de enero de 2000 y el 31 de julio de 2008. La variable predictora es AE y las variables resultados son la forma de terminación del parto (eutócico, parto instrumental y cesárea) y la presencia de fiebre materna durante el parto. Posteriormente realizamos una regresión logística binaria multivariante (odds ratio [OR] ajustadas), para comprobar la relación entre AE y las variables resultados, evitando el sesgo de las variables confusoras. Resultados En el análisis descriptivo observamos cómo hay mayor incidencia de primíparas (68,3–37,4%), gestantes con cesárea anterior (10,1–4,6%), tasa de inducciones (35–18,9%), partos instrumentales (15,6–3,8%), cesáreas (12,3–5,1%) y fiebre intraparto (10,7–0,9%) en las gestantes con AE, siendo las diferencias significativas. En el análisis multivariante observamos que en las gestantes con AE, las OR ajustadas de parto instrumental, de cesárea y de fiebre intraparto son, respectivamente, 2,63 ((intervalo de confianza [IC] del 95%: 2,19–3,17), 1,41 (IC: 1,18–1,68) y 8,45 (IC: 6,89–10,35).Conclusiones En nuestra población, la aplicación de AE durante el parto es un factor predisponente para la realización de partos instrumentales y de cesáreas. Así mismo, hay mayor incidencia de fiebre materna durante el parto (AU)

Introduction Epidural analgesia (EA) is the most effective procedure for pain relief during labor but can cause adverse effects. The aim of this study was to analyze the influence of EA on the rate of instrumental deliveries, cesarean sections and intrapartum fever. Material and method We conducted a retrospective cohort study of 10,115 deliveries between January 1, 2000 and July 31, 2008. The predictor variable was epidural analgesia and the outcome variables were the mode of delivery (eutocic, instrumental vaginal delivery and cesarean section) and the presence of maternal fever during labor. A multivariate binary logistic regression (adjusted odds ratio [OR]) was performed to verify the relationship between EA and the results of these variables, avoiding the bias of confounding variables. Results The descriptive analysis showed a significantly higher incidence of primiparity (68.3% vs 37.4%), previous cesarean section (10.1% vs 4.6%), induction (35% vs 18.9%), instrumental delivery (15.6% vs 3.8%), cesarean section (12.3% vs 5.1%) and intrapartum fever (10.7% vs 0.9%) in women with EA. The multivariate analysis showed that women with EA had adjusted ORs of instrumental delivery, cesarean section and intrapartum fever of 2.63 (95% confidence interval [CI]: 2.19–3.17), 1.41 (CI: 1.18–1.68) and 8.45 (CI: 6.89–10.35), respectively. Conclusions In our population, application of EA during labor is a predisposing factor for instrumental vaginal delivery and cesarean section. Likewise, there is an increased incidence of maternal fever during labor (AU)

Antioxidant capacity of infant fruit beverages; influence of storage and in vitro gastrointestinal digestion / Capacidad antioxidante de las bebidas infantiles de frutas; influencia del almacenamiento y de la digestión gastrointestinal in vitro

Aim: The total antioxidant capacity of three beverages based on fruit juice, milk and cereals, intended for infants and young children up to 3 years of age was evaluated by two methods Trolox Equivalent Antioxidant Capacity and Oxygen Radical Absorption Capacity. Results: According to the total antioxidant values obtained by both methods, the beverages can be ranked as follows: grape-orange-banana > peach-apple > pineapple-banana. Ascorbic acid was the main contributor (60%) to the total antioxidant capacity, while the contribution of skimmed milk was less than 1.2%. After one month of storage at -20 ºC, significant losses (p < 0.05) in total antioxidant capacity were found, though these were lower than 3% and therefore lacked nutritional significance. The bioaccessible fractions (maximum soluble fraction in simulated gastrointestinal media) of the beverages, obtained by in vitro gastrointestinal digestion, had antioxidant activities significantly lower (p < 0.05) than the original beverages, though the loss of antioxidant activity was always lower than 19% -thus indicating the stability of the total antioxidant capacity under the applied conditions. Conclusions: The total antioxidant capacity values of the bioaccessible fraction show that most antioxidants are available for absorption after digestion, and might contribute to the beneficial effects attributed to antioxidants (AU)

Objetivo: Se evaluó la capacidad antioxidante de tres bebidas basadas en zumo de frutas, leche y cereales, indicadas para lactantes y niños de hasta 3 años de edad, mediante dos métodos: Capacidad Antioxidante Equivalente Trolox y Capacidad de Absorción del Oxígeno Reactivo. Resultados: de acuerdo con los valores antioxidantes totales obtenidos con ambos métodos, se pudo clasificar las bebidas de la siguiente manera: uva-naranja-plátano > melocotón-manzana > piña-plátano. El ácido ascórbico fue el principal contribuyente (60%) a la capacidad antioxidante total, mientras que la contribución de la leche desnatada fue menor del 1,2%. Tras un mes de almacenamiento a -20 ºC, se encontraron unas pérdidas significativas de la capacidad antioxidante total (p < 0,05), aunque éstas fueron menores del 3% y, por lo tanto, carecían de significación nutricional. Las fracciones bioaccesibles (fracción soluble máxima en medios gastrointestinales simulados) de las bebidas, obtenidas mediante digestión gastrointestinal in vitro, tuvieron actividades antioxidantes significativamente menores (p< 0,05) que las bebidas originales, si bien la pérdida de actividad antioxidante fue siempre menor del 19% - indicando así la estabilidad de la capacidad antioxidante bajo las condiciones aplicadas. Conclusiones: Los valores de la capacidad antioxidante de la fracción bioaccesible muestran que la mayoría de los antioxidantes están disponibles para ser absorbidos tras la digestión y podrían contribuir a los efectos beneficiosos atribuidos a los antioxidantes (AU)

MLL gene fusions in human leukaemias: in vivo modelling to recapitulate these primary tumourigenic events.

Recurrent reciprocal chromosomal translocations are frequently found in leukaemias and sarcomas as initiating events in these cancers. Mouse models of chromosomal translocations are not only important for the elucidation of the mechanism of these factors underlying the disease but are also important pre-clinical models for assessing new drug combinations, developing new rational therapeutic strategies based on new drugs and testing novel macromolecular drugs. We describe three technologies for creating chromosomal translocation mimics in mice, applied specifically to understand how the MLL-fusions contribute to leukaemia. An important finding of this work is that the lineage of the tumours can be controlled by the MLL-protein fusion. The translocation mimic methods can be applied to any human reciprocal chromosomal translocation.

Antioxidant capacity of infant fruit beverages: influence of storage and in vitro gastrointestinal digestion.

AIM: The total antioxidant capacity of three beverages based on fruit juice, milk and cereals, intended for infants and young children up to 3 years of age was evaluated by two methods Trolox Equivalent Antioxidant Capacity and Oxygen Radical Absorption Capacity. RESULTS: According to the total antioxidant values obtained by both methods, the beverages can be ranked as follows: grape-orange-banana > peach-apple > pineapple-banana. Ascorbic acid was the main contributor (60%) to the total antioxidant capacity, while the contribution of skimmed milk was less than 1.2%. After one month of storage at -20 degrees C, significant losses (p < 0.05) in total antioxidant capacity were found, though these were lower than 3% and therefore lacked nutritional significance. The bioaccessible fractions (maximum soluble fraction in simulated gastrointestinal media) of the beverages, obtained by in vitro gastrointestinal digestion, had antioxidant activities significantly lower (p < 0.05) than the original beverages, though the loss of antioxidant activity was always lower than 19%--thus indicating the stability of the total antioxidant capacity under the applied conditions. CONCLUSIONS: The total antioxidant capacity values of the bioaccessible fraction show that most antioxidants are available for absorption after digestion, and might contribute to the beneficial effects attributed to antioxidants.

Nutritional control, gene regulation, and transformation of vascular smooth muscle cells in atherosclerosis.

Contractile-state smooth muscle cells (SMC), the only cell type in the arterial media, undergoes migration to the intima, proliferation, and abundant extracellular matrix production during the early stages of atherosclerosis. This involves the ingestion of low-density lipoprotein (LDL) and modified or oxidised LDL by macrophages together with SMC by several pathways including a scavenger pathway leading to accumulation of cholesterol esters and formation of foam cells. High-plasma cholesterol levels constitute a major causative risk for atherosclerosis. The membrane-bound transcription factor called sterol regulatory element binding protein (SREBP) activates gene-encoding enzymes of cholesterol and fatty acid biosynthesis. The SREBP expression, in response to diet, shows that are involved in both lipogenesis and cholesterol homeostasis, moreover SREBPs are regulated directly by cholesterol. Animal models were used in trials of atherosclerosis, and cholesterol feeding has been described elsewhere as producing atherosclerotic lesions. We have examined the morphological, molecular and proliferative change in arterial SMC mimicking such a cholesterol diet, this transformed SMC is a good model to study the alterations of the differentiated state of SMC, and the transformation into foam cell, caused by cholesterol-rich diet. Despite the complexity of the interactions in atherosclerosis, there are many opportunities to affect the homeostatic balance of the artery wall at SMC levels. We have considered here some of the possible targets for intervention with promising strategies for the nutritional control of the genes, and, in a general way, the possibilities for modulating the expression of genes influencing atherosclerosis.

Simultaneous detection of the immunophenotypic markers and genetic aberrations on routinely processed paraffin sections of lymphoma samples by means of the FICTION technique.

Disciplines such as morphology, immunophenotyping and genetics widely contributed over decades to the understanding of the cellular mechanisms of cancer. To obtain a greater insight into the complex processes of tumorigenesis, scientists have joined their efforts to combine many of the available techniques. Here, we report on the development of a FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a tool for the Investigation of Neoplasms) technique that allows a simultaneous detection of immunophenotypic markers and genetic aberrations on routinely processed lymphoma samples. As the antigen retrieval method seems to play an important role in the final results, we tested the pressure-cooking method at different times (2, 4 and 8 min) using three different buffers (EDTA, Tris-EDTA and citrate), resulting in improved sensitivity for the detection of both immunophenotypic markers and genetic aberrations. We also applied this method to different types of lymphoma using double immunofluorescence assays (including CD30, CD20, CD8 monoclonal antibodies) and several fluorescence in situ hybridization probes to demonstrate that the FICTION technique could be easily applied on paraffin sections in different combinations for the diagnosis and research of cancer.

Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques.

The A673 cell line was established from a patient with a primary rhabdomyosarcoma (RMS), which is referred to in the literature either as a Ewing tumor (ET) or as RMS. Although the two tumoral types are associated with specific and well-characterized translocations, no cytogenetic report on this cell line has been published. We characterized the A673 cell line using a combination of spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR), which revealed the presence of a complex karyotype and a translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes, both events being specific to ET. Neither cytogenetics nor molecular alterations specific to RMS were found.

Membrane permeability transition as induced by dysfunction of the electron transport chain.

The results in this paper indicate that mitochondrial permeability transition is activated by dysfunction of the respiratory chain caused by anaerobiosis, exhaustion of the oxidative substrate, and antimycin A, in the presence of 100 microM Ca2+. Membrane damage coincides with the collapse of the electric gradient. The opening of the non-selective pore is prevented by cyclosporin A.