Donor or recipient origin of posttransplant lymphoproliferative disorders following solid organ transplantation.

Previous studies of donor or recipient origin of posttransplant lymphoproliferative disorders (PTLDs) following solid organ transplantation (SOT) have either been small or with selected patient groups. We studied tumor origin in a population-based cohort of 93 patients with PTLD following SOT. Tumor origin of PTLD tissue was analyzed by fluorescence in situ hybridization of the sex chromosomes in cases of sex mismatch between donor and recipient (n = 41), or HLA genotyping in cases of identical sex but different HLA type (n = 52). Tumor origin of PTLD could be determined in 67 of the 93 cases. All 67 PTLDs were of recipient origin. They were found in recipients of kidney (n = 38), liver (n = 12), heart (n = 10) and lung (n = 7). The most common recipient-derived lymphomas were monomorphic B-cell PTLDs (n = 45), monomorphic T cell PTLDs (n = 9), indolent lymphomas (n = 6), and polymorphic PTLD (n = 4). Half of the recipient-derived PTLDs were Epstein-Barr virus-positive. Twelve of the recipient-derived PTLDs were located in the grafts: in four cases exclusively and in eight cases in combination with disseminated disease outside the graft. Tumor origin was indeterminable in 26 cases, probably due to low DNA quality. We conclude that the vast majority of PTLDs after SOT was of recipient origin.

El impacto de la cirugía robótica en Urología / The Impact of Robotic Surgery in Urology

Introducción: Hace más de una década la cirugía robótica llego a la Urología. Desde entonces, la comunidad urológica comenzó a observar la cirugía desde un ángulo diferente. Los cambios son evidentes en el presente, el futuro y la forma en que vemos nuestra pasada experiencia quirúrgica. Métodos: Se revisó la literatura médica publicada entre el año 2000 y el 2011 usando la base de datos de la National Library of Medicine y las siguientes palabras clave: cirugía robótica, asistencia robótica en cirugía, prostatectomía radical. Se hizo énfasis en el impacto de la cirugía robótica en Urología. Analizamos además las series más representativas (curva de aprendizaje finalizada) en cada uno de los abordajes robóticos en referencia a la morbilidad perioperatoria y los resultados oncológicos. Resultados: Este artículo analiza el impacto de la robótica en Urología a partir de las aplicaciones quirúrgicas y no quirúrgicas previas, la forma en que se introdujo a la Urología, sus primeros pasos, la situación actual y las expectativas futuras. Al narrar este viaje hemos intentado resaltar las modificaciones importantes que ayudaron a la cirugía robótica a recorrer su camino hasta su posición actual. Se hace énfasis también en los importantes cambios que trajo la cirugía robótica al campo del entrenamiento quirúrgico y sus consecuencias en su curva de aprendizaje. Conclusión: Los principios básicos de la cirugía aún se ponen en práctica en la cirugía robótica: la experiencia del cirujano y la prolongada práctica dan conocimiento y habilidades. Siguiendo esto, las ventajas potenciales provenientes de la tecnología pueden ser bien explotadas y verse reflejadas en mejores resultados para los pacientes (AU)

Introduction: More than a decade ago, robotic surgery was introduced into urology. Since then, the urological community started to look at surgery from a different angle. The present, the future hopes, and the way we looked at our past experience have all changed. Methods: Between 2000 and 2011, the published literature was reviewed using the National Library of Medicine database and the following key words: robotic surgery, robot-assisted, and radical prostatectomy. Special emphasis was given to the impact of the robotic surgery in urology. We analyzed the most representative series (finished learning curve) in each one of the robotic approaches regarding perioperative morbidity and oncological outcomes. Results: This article looks into the impact of robotics in urology, starting from its background applications before urology, the way it was introduced into urology, its first steps, current status, and future expectations. By narrating this journey, we tried to highlight important modifications that helped robotic surgery make its way to its position today. We looked as well into the dramatic changes that robotic surgery introduced to the field of surgical training and its consequence on its learning curve. Conclusion: Basic surgical principles still apply in Robotics: experience counts, and prolonged practice provides knowledge and skills. In this way, the potential advantages delivered by technology will be better exploited, and this will be reflected in better outcomes for patients (AU)

The impact of robotic surgery in urology.

INTRODUCTION: More than a decade ago, robotic surgery was introduced into urology. Since then, the urological community started to look at surgery from a different angle. The present, the future hopes, and the way we looked at our past experience have all changed. METHODS: Between 2000 and 2011, the published literature was reviewed using the National Library of Medicine database and the following key words: robotic surgery, robot-assisted, and radical prostatectomy. Special emphasis was given to the impact of the robotic surgery in urology. We analyzed the most representative series (finished learning curve) in each one of the robotic approaches regarding perioperative morbidity and oncological outcomes. RESULTS: This article looks into the impact of robotics in urology, starting from its background applications before urology, the way it was introduced into urology, its first steps, current status, and future expectations. By narrating this journey, we tried to highlight important modifications that helped robotic surgery make its way to its position today. We looked as well into the dramatic changes that robotic surgery introduced to the field of surgical training and its consequence on its learning curve. CONCLUSION: Basic surgical principles still apply in Robotics: experience counts, and prolonged practice provides knowledge and skills. In this way, the potential advantages delivered by technology will be better exploited, and this will be reflected in better outcomes for patients.

The frequency and prognostic impact of dic(9;20)(p13.2;q11.2) in childhood B-cell precursor acute lymphoblastic leukemia: results from the NOPHO ALL-2000 trial.

The dic(9;20)(p13.2;q11.2) is reported to be present in ∼2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL). However, it easily escapes detection by G-banding analysis and its true prevalence is hence unknown. We performed interphase fluorescence in situ hybridization analyses-in a three-step manner-using probes for: (i) CDKN2A at 9p21, (ii) 20p and 20q subtelomeres and (iii) cen9 and cen20. Out of 1033 BCP ALLs diagnosed from 2001 to 2006, 533 were analyzed; 16% (84/533) displayed 9p21 deletions, of which 30% (25/84) had dic(9;20). Thus, dic(9;20)-positivity was found in 4.7% (25/533), making it the third most common genetic subgroup after high hyperdiploidy and t(12;21)(p13;q22). The dic(9;20) was associated with a female predominance and an age peak at 3 years; 18/25 (72%) were allocated to non-standard risk treatment at diagnosis. Including cases detected by G-banding alone, 29 dic(9;20)-positive cases were treated according to the NOPHO ALL 2000 protocol. Relapses occurred in 24% (7/29) resulting in a 5-year event-free survival of 0.69, which was significantly worse than for t(12;21) (0.87; P=0.002) and high hyperdiploidy (0.82; P=0.04). We conclude that dic(9;20) is twice as common as previously surmised, with many cases going undetected by G-banding analysis, and that dic(9;20) should be considered a non-standard risk abnormality.

A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line.

Ethidium bromide (EtBr) is widely used to deplete mitochondrial DNA (mtDNA) and produce mitochondrial DNA-less cell lines. However, it frequently fails to deplete mtDNA in mouse cells. In this study we show by using a highly sensitive real-time PCR, that low doses of EtBr (10 microM) did lead to a three-fold increase of the total amount of mitochondrial DNA in a human neuronal cell line (Ntera 2). A higher dose of EtBr (25 microM) led to the expected decrease of mtDNA until day 22 when the cells almost died. Cell growth and mtDNA content could be restored after additional 22 days of non-EtBr treatment. The highest concentration of 50 microM also led to a significant increase of mtDNA. The cells died when they had only about 10% of mtDNA left, indicating a mtDNA threshold for cell survival. Additionally, the so-called common 4977 bp deletion could be induced by prolonged exposure to ethidium bromide. Whereas the higher doses led to significant higher amounts of deleted mtDNA.

Human and mouse ABCA1 comparative sequencing and transgenesis studies revealing novel regulatory sequences.

The expression of ABCA1, a major participant in apolipoprotein-mediated cholesterol efflux, is regulated by a variety of factors, including intracellular cholesterol concentration. To identify sequences involved in its regulation, we sequenced and compared approximately 200 kb of mouse and human DNA containing the ABCA1 gene. Furthermore, expression of the human gene containing different 5' ends was examined in transgenic mice. Sequence comparison revealed multiple conserved noncoding sequences. The two most highly conserved noncoding elements (CNS1, 88% identity over 498 bp; CNS2, 81% identity over 214 bp) were also highly conserved in other organisms. Mice containing the human ABCA1 gene, 70 kb of upstream DNA, and 35 kb of downstream DNA expressed the transgene similarly to endogenous Abca1. A second transgene beginning 3' to exon 1 was expressed only in liver, providing strong evidence of an unsuspected liver-specific promoter. The identified conserved noncoding sequences invite further investigation to elucidate ABCA1 regulation.

Regulation and activity of the human ABCA1 gene in transgenic mice.

The ABCA1 transporter is one of the limiting steps in cellular cholesterol efflux. To study the expression and activity of the human ABCA1 gene in vivo we have examined mice containing two human BAC transgenes with different 5' ends. Mice containing a 255-kilobase (kb) BAC transgene, including 70 kb upstream of the previously defined exon 1, demonstrated a pattern of tissue-specific expression mimicking that of the endogenous mouse gene. Compared with macrophages from control mice, macrophages from these transgenics had increases in apoA-I cholesterol efflux heightened in response to increases in cell cholesterol content. The observed increase in macrophage apoA-I-mediated cholesterol efflux was not accompanied by alterations in plasma high density lipoprotein in the transgenics. Although mice containing a smaller 171-kb human BAC transgene, lacking the previously described exon 1 and ABCA1 promoter, did not express human ABCA1 in macrophages, they did express the human transgene in liver at levels comparable with those of the orthologous mouse gene. Analysis by 5' rapid amplification of cDNA ends of liver mRNA from these animals revealed a new ABCA1 exon 1 (exon 1A) and a previously unrecognized promoter. Analysis of human tissue revealed that exon 1A containing transcripts accounted for a high proportion of the ABCA1 mRNAs present in human liver. This analysis of ABCA1 transgenics showed that the expression of human ABCA1 transgenes can result in increased cholesterol efflux from macrophages, unaccompanied by changes in plasma high density lipoprotein, and identified a new ABCA1 promoter in humans.

MtDNA mutations in maternally inherited diabetes: presence of the 3397 ND1 mutation previously associated with Alzheimer's and Parkinson's disease.

Mutations in the mitochondrial tRNA(leu) (UUR) gene have been associated with diabetes mellitus and deafness. We screened for the presence of mtDNA mutations in the tRNA(leu) (UUR) gene and adjacent ND1 sequences in 12 diabetes mellitus pedigrees with a possible maternal inheritance of the disease. One patient carried a G to A substitution at nt 3243 (tRNA(leu) (UUR) gene) in heteroplasmic state. In a second pedigree a patient had an A to G substitution at nt 3397 in the ND1 gene. All maternal relatives of the proband had the 3397 substitution in homoplasmic state. This substitution was not present in 246 nonsymptomatic Caucasian controls. The 3397 substitution changes a highly conserved methionine to a valine at aa 31 and has previously been found in Alzheimer's (AD) and Parkinson's (PD) disease patients. Substitutions in the mitochondrial ND1 gene at aa 30 and 31 have associated with a number of different diseases (e.g. AD/PD, MELAS, cardiomyopathy and diabetes mellitus, LHON, Wolfram-syndrome and maternal inherited diabetes) suggesting that changes at these two codons may be associated with very diverse pathogenic processes. In a further attempt to search for mtDNA mutations outside the tRNAleu gene associated with diabetes, the whole mtDNA genome sequence was determined for two patients with maternally inherited diabetes and deafness. Except for substitutions previously reported as polymorphisms, none of the two patients showed any non-synonymous substitutions either in homoplasmic or heteroplasmic state. These results imply that the maternal inherited diabetes and deafness in these patients must result from alterations of nuclear genes and/or environmental factors.

MtDNA substitution rate and segregation of heteroplasmy in coding and noncoding regions.

The mitochondrial DNA (mtDNA) substitution rate and segregation of heteroplasmy were studied for the non-coding control region (D-loop) and 500 bp of the coding region between nucleotide positions 5550 and 6050, by sequence analysis of blood samples from 194 individuals, representing 33 maternal lineages. No homoplasmic nucleotide substitutions were detected in a total of 292 transmissions. The estimated substitution rate per nucleotide per million years for the control region (micro>0.21, 95% CI 0-0.6) was not significantly different from that for the coding region (micro>0.54, 95% CI 0-1.0). Variation in the length of homopolymeric C streches was observed at three sites in the control region (positions 65, 309 and 16,189), all of which were in the heteroplasmic state. Segregation of heteroplasmic genotypes between generations was observed in several maternal pedigrees. At position 309, a longer poly C tract length was strongly associated with a higher probability for heteroplasmy and rapid segregation between generations. The length heteroplasmy at positions 65 and 16,189 was found at low frequency and was confined to a few families.

Analysis of mtDNA copy number and composition of single mitochondrial particles using flow cytometry and PCR.

Mitochondrial DNA (mtDNA) is a multicopy, maternally inherited, genome. Individuals frequently carry a mixture of genetically distinct mtDNA molecules whose proportions may vary between sexual generations or among tissues from the same individual. Analyses of the genetic composition of mitochondria have previously relied on electron microscopy and have not permitted the genotype of single mitochondria to be determined. We have developed flow cytometry techniques to isolate single mitochondrial particles and PCR-based assays to determine the mtDNA copy number and composition of individual particles. In a first application of this method, we studied mitochondrial particles from fibroblast cells heteroplasmic for the tRNA lys(8344) point mutation, associated with myoclonus epilepsy and ragged red fiber (MERRF). Individual mitochondrial particles contained between 0 and 11 mtDNA molecules with a mean of 2.0 (95% CI 1.6-2.4). The majority (75%) of the mitochondrial particles from which a PCR product was obtained contained only one type of mtDNA, consistent with the low mean mtDNA copy number. The method developed may be applied to studies of the copy number and distribution of mtDNA genomes in different cell types.

Mitochondrial sequence variants in patients with schizophrenia.

To investigate whether mitochondrial mutations underly susceptibility to schizophrenia, we sequenced the mtDNAs of two unrelated Swedish patients with schizophrenia and low cytochrome oxidase activity and two maternally related Scottish patients from a family with suspected maternal inheritance of the disease. We found five substitutions in coding regions that have not previously been described as polymorphisms. These new substitutions were studied in 81 schizophrenic patients and five control groups from Sweden and Scotland and found to differ in frequency between populations, emphasizing the importance of using large and well-defined control materials for evaluating the association of mtDNA mutations with disease. The results do not lend strong support to the association of a particular mtDNA substitution with increased risk for schizophrenia. However, the trend towards a higher frequency of substitutions in the patients deserves further attention.

Formation of GSH-derivatives as a pathway for inactive intermediates in vinylidene chloride-treated rats.

The two conjugates, S-[N-(2-hydroxyethyl)carbamoylmethyl]glutathione (GSAAE), and its corresponding mercapturic derivative N-acetyl-S-[N-(2-hydroxyethyl)carbamoylmethyl]cysteine (NCySAAE) were administered to fasted Sprague-Dawley rats as putative metabolites of vinylidene chloride (VDC). Methylthioacetylaminoethanol (MAAE) was identified in the urine of GSAAE- or NCySAAE-treated rats (0.5-2.0 mmol/kg, i.p.), as well as in the urine of VDC-treated rats (0.5-2.0 mmol/kg, p.o.). The effects of VDC, GSAAE and NCySAAE on the kidney and liver were also examined using aspartate aminotransferase (ASAT). N-acetyl-beta-D-glucosaminidase (NAG) and beta 2-microglobulin (beta 2-m) as urinary parameters of nephrotoxicity, and glutamate dehydrogenase (GLDH), sorbitol dehydrogenase (SDH) and alanine aminotransferase (ALAT) as serum parameters of hepatotoxicity. Unlike treatment with VDC, treatment with both GSAAE and NCySAAE failed to cause kidney and liver toxicity. The results support the hypothesis that MAAE originates from the formation of GSAAE and further metabolization to NCySAAE, and that MAAE excretion does not reveal a pathway of reactive intermediates.

Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA.

We have analyzed the level of intraindividual sequence variability (heteroplasmy) of mtDNA in human brain by denaturing gradient gel electrophoresis and sequencing. Single base substitutions, as well as insertions or deletions of single bases, were numerous in the noncoding control region (D-loop), and 35-45% of the molecules from a single tissue showed sequence differences. By contrast, heteroplasmy in coding regions was not detected. The lower level of heteroplasmy in the coding regions is indicative of selection against deleterious mutations. Similar levels of heteroplasmy were found in two brain regions from the same individual, while no heteroplasmy was detected in blood. Thus, heteroplasmy seems to be more frequent in nonmitotic tissues. We observed a 7.7-fold increase in the frequency of deletions/insertions and a 2.2-fold increase in the overall frequency of heteroplasmic mutations in two individuals aged 96 and 99, relative to an individual aged 28. Our results show that intraindividual sequence variability occurs at a high frequency in the noncoding regions of normal human brain and indicate that small insertions and deletions might accumulate with age at a lower rate than large rearrangements.

Role of cysteine conjugation in vinylidene chloride-induced nephrotoxicity and hepatotoxicity in fasted rats.

Pretreatment of fasted rats with aminooxyacetic acid (AOAA, 0.25 mmol kg-1, i.p.), methimazole (MTZ, 0.35 mmol kg-1, i.p.) and acivicin (AT-125, 56 mumol kg-1, i.p.) 30 min prior to a 4-h inhalation exposure to 180-200 ppm or 150-180 ppm vinylidene chloride (VDC) was used to study the role of cysteine beta-lyase, cysteine conjugate S-oxidase and gamma-glutamyltranspeptidase (gamma-GT) in VDC-induced liver and kidney toxicity. Pretreatment with AOAA reduced by 65-95% those increases in serum alanine aminotransferase (ALAT), glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) caused by exposure to 180-200 ppm VDC. This pretreatment also prevented VDC-induced increases in aspartate aminotransferase (ASAT) and N-acetyl-beta-d-glucosaminidase (NAG) activities and in the concentration of beta 2-microglobulin (beta 2-m) in 24-h urine samples. There was only a slight potentiation of VDC-induced liver and renal toxicities by MTZ given before exposure to 180-200 ppm VDC, but potentiation became significant (40-80%) when MTZ was administered before a slightly lower level of exposure (150-180 ppm). Pretreatment with AT-125 did not significantly change the liver and renal effects of exposure to 180-200 ppm VDC. These results suggest that the formation of a cysteine conjugate may be involved in the renal and liver toxicity of VDC in fasted rats.

Decreased cytochrome-c oxidase activity and lack of age-related accumulation of mitochondrial DNA deletions in the brains of schizophrenics.

Defects in mitochondrial energy production have been implicated in several neurodegenerative disorders, such as Parkinson disease and amyotrophic lateral sclerosis. To study the contribution of mitochondrial defects to Alzheimer disease and schizophrenia, cytochrome-c oxidase (COX) activity and levels of the mtDNA4977 deletion in postmortem brain tissue specimens of patients were compared with those of asymptomatic age-matched controls. No difference in COX activity was observed between Alzheimer patients and controls in any of five brain regions investigated. In contrast, schizophrenic patients had a 63% reduction of the COX activity in the nucleus caudatus (P < 0.0001) and a 43% reduction in the cortex gyrus frontalis (P < 0.05) as compared to controls. The average levels of the mtDNA4977 deletion did not differ significantly between Alzheimer patients and controls, and the deletion followed similar modes of accumulation with age in the two groups. In contrast, no age-related accumulation of mtDNA deletions was found in schizophrenic patients. The reduction in COX activity in schizophrenic patients did not correlate with changes in the total amount of mtDNA or levels of the mtDNA4977 deletion. The lack of age-related accumulation of the mtDNA4977 deletion and reduction in COX activity suggest that a mitochondrial dysfunction may be involved in the pathogenesis of schizophrenia.

Nephrotoxicity mechanism of 1,1-dichloroethylene in mice.

Male Swiss OF1 mice were administered orally with a single dose (200 mg/kg) of 1,1-dichloroethylene (DCE). Examination of cryostat kidney sections stained for alkaline phosphatase (APP) revealed damage to about 50% of the proximal tubules at 8 h following DCE administration. Pretreatment with the anionic transport inhibitor probenecid by i.p., (0.75 mmol/kg, 30 min prior to and 10 min and 5 h following DCE administration) and with the gamma-glutamyltranspeptidase (GGT) inactivator acivicin by gavage and i.p. (50 mg/kg, 1 h and 30 min prior to DCE administration) failed to prevent DCE-induced renal toxicity. Pretreatment with the beta-lyase inactivator amino-oxyacetic acid (AOAA) by gavage (100 mg/kg, 30 min prior to and 10 min and 5 h following DCE administration), and with the renal cysteine conjugate S-oxidase inhibitor methimazole by i.p. (40 mg/kg, 30 min prior to DCE administration) reduced the number of damaged tubules by approximately 50 and 60%, respectively in mice treated with DCE. The results suggest that the DCE undergoes biotransformation by NADPH-cytochrome P450 to several reactive species which conjugate with glutathione (GSH). After arriving in the kidneys, the resulting conjugates reach the renal cells by a mechanism which depends on neither GGT, nor on an anionic transport system which is sensitive to probenecid. Once in the cells, the presumed GSH conjugates and/or their derivatives undergo secondary modification by beta-lyase and cysteine conjugate S-oxidase to reactive metabolite(s).

Plasmodium falciparum: the immune response in rabbits to the clustered asparagine-rich protein (CARP) after immunization in Freund's adjuvant or immunostimulating complexes (ISCOMs).

The Plasmodium falciparum clustered asparagine-rich protein (CARP) is a merozoite-associated antigen which contains approximately 30% asparagine. Analysis of the DNA sequences located 5' of the cloned 1.4-kb CARP gene in the P. falciparum genome suggests that this gene fragment may encode the complete CARP and that the gene product is a protein of M(r) 50,000. To analyze the immunogenicity of CARP, the gene was expressed as a fusion protein with staphylococcal protein A (SpA-CARP). Immunization of rabbits with SpA-CARP in Freund's complete adjuvant (FCA) resulted in a strong antibody response against CARP as measured in ELISA. This response was efficiently boosted and sustained over a long time while that induced by two immunizations with SpA-CARP in ISCOMs was weak and of shorter duration. In both instances, the antibody levels against CARP were further increased by a second booster injection consisting of either SpA-CARP or CARP fused to the serum albumin-binding region (BB) of streptococcal protein G (BB-CARP) in PBS, indicating that immunizations with SpA-CARP in FCA or ISCOMs had induced a CARP-specific immunological memory. Boosting with BB-CARP in PBS was more efficient than boosting with SpA-CARP in PBS. In all rabbits, the antibodies obtained after the booster with CARP in PBS were the most efficient inhibitors of merozoite invasion in vitro. The antisera reacted with the intracellular parasite in immunofluorescence and with a band of M(r) 50,000 in immunoblotting while several high-molecular-weight components as well as the one of M(r) 50,000 were immunoprecipitated. The specificity of the antibody responses varied between the different rabbits as indicated in ELISA, with short synthetic peptides representing different CARP sequences. Taken together, the results suggest that a previously cloned genomic DNA fragment may encode the complete P. falciparum blood-stage antigen CARP and that CARP is immunogenic in rabbits both when administered in FCA or ISCOMs.