Increased sensitivity to platinum drugs of cancer cells with acquired resistance to trabectedin.

BACKGROUND: In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo. METHODS: Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts. RESULTS: 402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G2/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks. CONCLUSIONS: Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.

p53 protein aggregation promotes platinum resistance in ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC), the most lethal gynecological cancer, often leads to chemoresistant diseases. The p53 protein is a key transcriptional factor regulating cellular homeostasis. A majority of HGSOCs have inactive p53 because of genetic mutations. However, genetic mutation is not the only cause of p53 inactivation. The aggregation of p53 protein has been discovered in different types of cancers and may be responsible for impairing the normal transcriptional activation and pro-apoptotic functions of p53. We demonstrated that in a unique population of HGSOC cancer cells with cancer stem cell properties, p53 protein aggregation is associated with p53 inactivation and platinum resistance. When these cancer stem cells differentiated into their chemosensitive progeny, they lost tumor-initiating capacity and p53 aggregates. In addition to the association of p53 aggregation and chemoresistance in HGSOC cells, we further demonstrated that the overexpression of a p53-positive regulator, p14ARF, inhibited MDM2-mediated p53 degradation and led to the imbalance of p53 turnover that promoted the formation of p53 aggregates. With in vitro and in vivo models, we demonstrated that the inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment. Moreover, by two-dimensional gel electrophoresis and mass spectrometry we discovered that the aggregated p53 may function uniquely by interacting with proteins that are critical for cancer cell survival and tumor progression. Our findings help us understand the poor chemoresponse of a subset of HGSOC patients and suggest p53 aggregation as a new marker for chemoresistance. Our findings also suggest that inhibiting p53 aggregation can reactivate p53 pro-apoptotic function. Therefore, p53 aggregation is a potential therapeutic target for reversing chemoresistance. This is paramount for improving ovarian cancer patients' responses to chemotherapy, and thus increasing their survival rate.

A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus.

Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.

Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

Mongoose rabies in southern Africa: a re-evaluation based on molecular epidemiology.

Relative to the developed world, rabies has been poorly studied in the vast African continent. The southern African countries of Zimbabwe and South Africa, however, are known to sustain a great diversity of lyssaviruses, with large biological variations amongst genotype 1 (rabies viruses) at present more apparent here than elsewhere on the continent. One recognized biotype of rabies virus in the subcontinent appears to be specifically adapted to a variety of mongooses, belonging to the Viverrinae subfamily (family Herpestidae) and are commonly referred to as viverrid viruses, although the term mongoose rabies would be more correct, considering the taxonomic status of the host species involved. It was our objective to study the genetic relationships of 77 rabies virus isolates of this mongoose biotype, isolated in South Africa and Zimbabwe, towards elucidation of the molecular epidemiology of this interesting group of African viruses. In our study of a 592 nucleotide sequence encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region of the viral genomes, we provide the first comprehensive data on the molecular epidemiology of these viruses and indicate a history of extended evolutionary adaptation in this geographical domain. The molecular epidemiological observations reported here are highly unlikely to be limited to the small geographical areas of South Africa and Zimbabwe and illustrate the need for lyssavirus surveillance in the rest of sub-Saharan Africa and throughout the entire continent.

Preventing reservoir calculi after augmentation cystoplasty and continent urinary diversion: the influence of an irrigation protocol.

OBJECTIVE: To evaluate the influence of an irrigation protocol in preventing reservoir calculi forming after augmentation cystoplasty and continent urinary diversion. PATIENTS AND METHODS: Between 1985 and 1995, 91 patients had an augmentation cystoplasty and/or continent urinary diversion (group 1; 54 females and 37 males, mean age 11.1 years, range 1-31); these patients were not routinely instructed to use irrigation after surgery. The segments used included ileum (44), colon (36), stomach (eight) and ureter (three). Between 1995 and 2000, 42 patients (group 2) underwent urinary reconstruction (22 females and 20 males, mean age 14.8 years, range 4-27), the segment used being ileum (30), colon (five), ureter (five) and stomach (two) but in contrast to group 1 they then were placed on a standard prophylactic irrigation protocol. The occurrence of stones in the reservoir was then assessed. RESULTS: Thirty-nine of the 91 patients (42.8%) in group 1 presented with reservoir calculi after reconstruction and 22 had several episodes. The mean time to presentation was 30 months. The incidence of stone formation by underlying diagnosis included: myelomeningocele, 32/48 (66%), exstrophy five/25 (25%), posterior urethral valves two/20 (10%) and rhabdomyosarcoma, none of three. Fifty of the 91 patients had an abdominal stoma, with stone formation in 33 (66%), while 41 used the native urethra, with stone formation in six (15%). Three (7%) of the 42 patients in group 2 developed reservoir calculi after reconstruction, two in patients with myelomeningocele and one in a trauma patient who had residual bone spicules in the bladder; the mean time to presentation was 26.5 months. CONCLUSIONS: These data suggest that the irrigation protocol used in group 2 significantly reduced the number of reservoir calculi after urinary tract reconstruction when bowel was used as part of the reconstruction (43% vs. 7%). The most calculi in both groups were in immobile patients with sensory impairment. Also, patients with an abdominal stoma had a greater risk of reservoir calculi (66%) than those using the native urethra (15%).

The urological care and outcome of pregnancy after urinary tract reconstruction.

OBJECTIVE: To assess the obstetric and urological outcomes during and after pregnancy following urinary tract reconstruction, as pregnancies after such surgery can have a significant effect on the function of the reconstructed urinary tract, and the reconstruction can significantly affect the delivery of the fetus. PATIENTS AND METHODS: We retrospectively reviewed the obstetric and urological history of 11 patients (12 pregnancies; 10 singletons and one twin) with previous urinary reconstruction, delivered between 1989 and 2003. Antepartum and postpartum urological function and obstetric outcomes were investigated. RESULTS: All the patients had some difficulty with clean intermittent catheterization (CIC) during pregnancy, and four needed continuous indwelling catheters. During pregnancy 10 women had several bladder infections and all received antibiotic suppression. There were eight Caesarean sections, two vaginal deliveries and one combined delivery. Six Caesareans were elective and three were emergent. The use of CIC returned to normal in all patients after delivery. CONCLUSIONS: Women with a urinary reconstruction can have successful pregnancies. The complexity of the surgery and the concern for possible emergency Caesarean section resulted in most patients having an elective Caesarean delivery before term. Antibiotic prophylaxis is recommended and patients may require indwelling dwelling catheters while pregnant but normal CIC can be resumed after delivery.

Can FDG PET be used to successfully direct preoperative biopsy of soft tissue tumours?

Magnetic resonance imaging (MRI) has been the most useful tool in the anatomical definition of soft tissue sarcoma, although there remains the problem of defining the lesions as benign or malignant. The management of such lesions requires biopsy prior to surgical resection. If the most malignant area could be defined more accurately, then this area could be targeted for biopsy. Fluorodeoxyglucose positron emission tomography (FDG PET) has been found to be useful in identifying malignancy and variations in grade in soft tissue masses. The aim of this study was to assess the use of FDG PET scanning with or without co-registered MRI to indicate the most appropriate biopsy site. Twenty consecutive patients presented with soft tissue masses with clinical signs of malignancy. All patients underwent MRI and FDG PET scanning and the two images were co-registered. A biopsy site that was the most likely to be malignant was defined on the PET scan. All patients underwent an initial biopsy and then complete surgical resection of the mass. The histological results from the mass were compared with those from the biopsy specimen obtained from the site suggested by the PET scan. In malignant masses the biopsy site suggested by the FDG PET scan was found to be representative of the most malignant site on the whole mass histology. Benign lesions had low or no FDG uptake. In no case did the co-registered image add significantly to the appropriate biopsy site. FDG PET can be used to appropriately direct biopsy in soft tissue sarcoma and potentially may lead to computed tomography/MRI directed outpatient biopsy prior to definitive treatment.

Does GH replacement therapy in adult GH-deficient patients result in recurrence or increase in size of pituitary tumours?

OBJECTIVE: Hypopituitary GH-deficient patients have an increased cardiovascular mortality and GH replacement in this population has resulted in considerable therapeutic benefit. GH replacement involves administration of a potentially mitogenic substance to patients with a previous or residual pituitary tumour. Our objective was to evaluate whether GH replacement results in an increase in the size of pituitary tumours. METHODS: This was a non-randomised observational study on patients recruited from the endocrine clinic. All subjects had GH deficiency, proven on an insulin tolerance test and were divided into those who were or were not receiving long-term GH replacement. Comparison of change in pituitary size was made with interval radiological imaging of the pituitary. RESULTS: Seventy-five patients (40 men and 35 women) were in the study, 47 were on long-term GH replacement and there were 28 controls. The average length of treatment for the treated group was 3.6 patient years. Thirty-nine patients in the treated group had at least 2 years of GH treatment between imaging studies of the pituitary. Two patients in the treated group had an increase in pituitary size (non-functioning adenomas) and two in the control group (one functioning and one non-functioning adenoma adenoma). None of these four patients required further treatment. There was no statistically significant difference between the two groups. CONCLUSION: Using a representative cohort of hypopituitary patients attending an endocrine clinic, GH replacement was not associated with an increased pituitary tumour recurrence rate. Although the results are not conclusive, in the period of observation GH had little adverse effect but longer studies are required to be certain.

Lymphocytic hypophysitis: spontaneous resolution on MRI with progression of endocrine defect.

Lymphocytic hypophysitis is an uncommon condition that typically occurs during the last trimester of pregnancy or in the postpartum period. Presentation is of an anterior pituitary mass with varying degrees of pituitary dysfunction. We present a case in which there was dramatic resolution of the pituitary lesion on sequential MRI scanning. Despite this apparent resolution, however, the patient continues to have significant pituitary dysfunction.

Lower limb and back pain in Guillain-Barré syndrome and associated contrast enhancement in MRI of the cauda equina.

UNLABELLED: This study assesses the frequency of lower limb and back pain in children with Guillain-Barré syndrome and reviews the magnetic resonance imaging results of those undergoing spinal imaging. Over an 8-y period, nine children presented with various combinations of severe back pain, leg pains, impairment of gait and bladder dysfunction. Guillain-Barré syndrome was confirmed on clinical examination and peripheral electrophysiology (n = 8). Magnetic resonance imaging in four patients, following contrast injection, showed enhancement of the cauda equine and, additionally, of the cervical nerve roots in one of the patients. A further patient, who was not scanned with contrast, had abnormal thickening of the lumbar roots. Carbamazepine and steroids were effectively used for analgesia in three cases. All the patients recovered. CONCLUSION: Guillain-Barré syndrome should be considered in the differential diagnosis of children presenting with back and/or leg pain. Early diagnosis ensures prompt monitoring for autonomic dysfunction and respiratory compromise.

Biliary complications associated with laparoscopic cholecystectomy: analysis of common misconceptions

AIMS: General surgeons often express the view that the majority of biliary complications following laparoscopic cholecystectomy are caused by trainee surgeons; complications occur most often in the presence of difficult anatomy or pathology; biliary injuries occur more proximally in the biliary tree than at open cholecystectomy; most injuries are recognized at the time of surgery; and most complications can be managed non-operatively. The aim of this study was to determine if these views are substantiated in clinical practice. METHODS: Thirty consecutive patients were referred to a specialist hepatobiliary unit over a 7-year period with biliary complications following laparoscopic cholecystectomy. The mode of presentation, management and outcome of these patients were analysed. RESULTS: In 21 cases the initial operator was a consultant. Four of the 30 complications occurred in the presence of an anatomical variation or unusually difficult pathology. Only patients in whom a previous attempt at repair had been made had injuries at or proximal to the bile duct confluence; the only two deaths occurred in this group. Only 41 per cent of injuries were detected at the time of surgery and 89 per cent required further surgical intervention, hepaticojejunostomy being the most common procedure performed (75 per cent). CONCLUSIONS: The majority of bile duct injuries are not caused by trainees, do not occur because of unusual anatomy or pathology, do not occur in the proximal biliary tree, are not recognized at the time of the initial operation and often require major reconstructive procedures for their management.

Evaluation of (18)fluorodeoxyglucose positron emission tomography ((18)FDG PET) in the detection of malignant peripheral nerve sheath tumours arising from within plexiform neurofibromas in neurofibromatosis 1.

OBJECTIVES: The ability of (18)fluorodeoxyglucose positron emission tomography ((18)FDG PET) to detect malignant change in plexiform neurofibromas from patients with neurofibromatosis 1 (NF1) was evaluated. METHODS: Eighteen NF1 patients who presented with pain, increase in size, or neurological deficit associated with a plexiform neurofibroma were assessed. Magnetic resonance imaging determined the site and extent of the lesion. Qualitative(18)FDG PET was performed and the standard uptake value (SUV) measured the regional glucose metabolism. Histological confirmation of the diagnosis was obtained in 10 patients. RESULTS: Twenty three plexiform neurofibromas were detected in 18 patients. Seven malignant peripheral nerve sheath tumours, four high grade and three low grade tumours, occurred in five patients. In one patient the clinical and radiological characteristics of the tumour suggested malignancy, but histology was inconclusive. Fifteen benign plexiform neurofibromas were identified in 12 patients and these findings were confirmed histologically in five lesions from four patients. Ten plexiform neurofibromas occurring in eight patients were considered benign on(18)FDG PET and the patients did not undergo surgery. They remained stable or their symptoms improved on clinical follow up (median 9 months). The results of qualitative (18)FDG PET were interpreted as indicating that 13 plexiform neurofibromas were benign and 10 were malignant. No malignant tumours were classified as benign, but two benign tumours were reported as malignant. The SUV was calculated for 20 tumours and was significantly higher in five malignant tumours 5.4 (SD 2.4), than in 15 benign tumours 1.54 (SD 0.7), p=0.002. There was an overlap between benign and malignant tumours in the SUV range 2.7-3.3. CONCLUSIONS: (18)FDG PET is helpful in determining malignant change in plexiform neurofibromas in NF1. Increased separation between benign and malignant lesions could be obtained by calculating the SUV at about 200 minutes after injection of (18)FDG, when the peak activity concentration is obtained in malignant tumours.

Biliary complications associated with laparoscopic cholecystectomy--an analysis of common misconceptions.

BACKGROUND: Several views are expressed by surgeons on biliary complications following laparoscopic cholecystectomy as follow: most are caused by trainees; complications occur in the presence of difficult anatomy/pathology; injuries occur more proximally than at open cholecystectomy; most injuries are recognised immediately and most can be managed non-operatively. The aim of our study was to determine if these views are substantiated in clinical practice. METHODS: The mode of presentation, management and outcome of thirty-two patients referred to a hepatobiliary unit over a seven year period were analysed. RESULTS: In 72% of cases the initial operator was a consultant. Five of the 32 complications (16%) occurred in the presence of difficult anatomy/pathology. Two patients had proximal biliary tree injuries, the only mortalities (two) occurring in this group. Only 41% of injuries were detected immediately; 87% required surgical intervention, hepaticojejunostomy being the most common procedure performed (75%). CONCLUSION: Our study shows that the majority of bile duct injuries are not caused by trainees, do not occur because of unusual anatomy/pathology, do not occur in the proximal biliary tree and are not recognised at the time of operation. Most injuries ultimately require major reconstructive surgery for definitive management.

Heterotopic pancreas as lead point in intussusception: new variant of vitellointestinal tract malformation.

Two cases of intussusception are reported with heterotopic pancreatic tissue attached to and draining into the ileum. The first patient, a boy aged 16 months, presented with ileoileal intussusception. The diagnosis was confirmed on ultrasound scan. Laparotomy and resection were performed. A 12-mm nodule of heterotopic pancreatic tissue was identified in the ileal serosa at the apex of the intussusceptum, fully formed with acinar tissue, islets, and draining duct. The second patient, also a boy aged 16 months, presented with obstructed ileocolic intussusception in which the lead point at surgery resembled a Meckel's diverticulum. Histopathology revealed a similar 10-mm nodule of fully formed pancreatic tissue in the ileal serosal tissues, with some acinar tissue extending through the wall of the intestine alongside ductal structures. In both cases there was ectopic gastric mucosa either in the distal part of the draining duct or in the small intestine itself at the opening. Heterotopic pancreas is a rare cause of intussusception. We propose that this lesion is of vitellointestinal tract origin, conceptually similar to a Meckel's diverticulum but without a diverticulum as such. Heterotopic pancreatic tissue occurring alone is more common in the proximal small intestine, duodenum, and stomach than in the ileum, and it is often asymptomatic.

Self-regulated polymerization of the actin-related protein Arp1.

The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic dynein motor machinery [1] [2] [3]. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to conventional actin [4] [5] [6] and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize. In vivo, all cytosolic Arp1 sediments at 20S [7] suggesting that it assembles into oligomers, most likely dynactin - a multiprotein complex known to contain eight or nine Arp1 monomers in a 37 nm filament [8]. The uniform length of Arp1 polymers suggests a novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping protein at one end and a heterotetrameric protein complex containing the p62 subunit (D.M. Eckley, S.R. Gill, J.B.B., J.E. Heuser, T.A.S., unpublished observations) at the other [8]. In the present study, we analyzed the behavior of highly purified, native Arp1. Arp1 was found to polymerize rapidly into short filaments that were similar, but not identical, in length to those in dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional actin filaments.

Presence of antibodies to canine distemper virus, canine parvovirus and canine adenovirus type 1 in free-ranging jackals (Canis adustus and Canis mesomelas) in Zimbabwe.

A survey of free-ranging jackals (Canis adustus and Canis mesomelas) in Zimbabwe was conducted to determine the prevalence of serum antibodies to canine distemper virus (CDV), canine parvovirus (CPV) and canine adenovirus type 1 (CAV-1). Sera from 16 Canis adustus and 22 Canis mesomelas were collected from 1990 to 1993 from various regions of Zimbabwe and assayed by means of immunofluorescent techniques. Seroprevalence in C. adustus and C. mesomelas respectively were 50% and 63.6% for CDV, 12.5% and 18.2% for CPV and 37.5 and 9.1 for CAV-1. These results demonstrate that jackals are infected with these viruses and may act as reservoirs of them, although their susceptibility to the viruses is not known.