Low Frequency (≪100Hz) applied electric fields have been shown to modulate neuronal activity both In Vitro and in acute whole animal studies [1–3]. We have been working to apply this technology for seizure control in chronically implanted animals. We have developed electronics for simultaneously recording neural activity while stimulating with low frequency fields. We have observed transient entrainment of spike and wave activity during spontaneous seizures with open loop sinusoidal stimulation with frequencies between 9–15 Hz. This is the first demonstration of low frequency field modulation of neural activity in chronically implanted mammalian brain.
Modern management of leukemia and selection of optimal treatment approaches entails the analysis of multiple recurrent cytogenetic abnormalities with independent diagnostic or prognostic value. We report the first multicenter validation of a multiplex molecular assay for 12 relevant fusion transcripts relative to cytogenetic methods. Performance was evaluated using a set of 280 adult and pediatric acute or chronic leukemias representative of the variety of presentations and pre-analytical parameters encountered in the clinical setting. The positive, negative and overall agreements were >98.5% with high concordance at each of the four sites. Positive detection of cases with low blast count or at relapse was consistent with a method sensitivity of 1%. There was 98.7% qualitative agreement with independent reference molecular tests. Apparent false negatives corresponded to rare alternative splicing isoforms not included in the panel. We further demonstrate that clinical sensitivity can be increased by adding those rare variants and other relevant transcripts or submicroscopic abnormalities. We conclude that multiplex RT-PCR followed by liquid bead array detection is a rapid and flexible method attuned to the clinical laboratory workflow, complementing standard cytogenetic methods and generating additional information valuable for the accurate diagnosis, prognosis and subsequent molecular monitoring of leukemia.
leukemia; diagnosis; prognosis; molecular classification; RT-PCR; multiplex
Adrenocortical carcinoma is an uncommon malignancy and feminizing symptoms secondary to adrenal estrogen-secretion are extremely rare. The direct secretion of estradiol by adrenocortical tumors requires, in addition to the expression of aromatase (CYP19), the expression of one or more of the reductive 17β-hydroxysteroid dehydrogenases. The expression of CYP19 transcripts and protein were markedly induced in the H295 adrenocortical carcinoma cell line after treatment with either forskolin or vasoactive intestinal peptide (VIP). Western immunoblotting demonstrated a marked induction of the CYP19 protein of characteristic size after only a short (6 h) treatment period with VIP or forskolin. The CYP19 mRNA transcripts were derived from both promoters PII (Ic) and I.3 (Id) after treatment with both agents. The reductive type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) was also constitutively expressed in the H295 cells but neither its mRNA transcript nor protein levels were altered after forskolin or VIP treatment. Western immunoblotting of an estrogen-secreting adrenal carcinoma revealed notable levels of both aromatase and AKR1C3 expression while an aldosterone-producing adrenal adenoma lacked aromatase expression and showed a reduced level of AKR1C3 expression. Immunohistochemistry of the carcinoma-bearing adrenal revealed localization of AKR1C3 not only in the tumor but also principally in the zona reticularis of the normal adrenal tissue. Adrenal aromatase and AKR1C3 expression therefore appear to be features of adrenocortical malignancies that are associated with biosynthesis of active estrogen.
human adrenocortical H295 cells; estrogen biosynthesis; aromatase; CYP19; 17β-hydroxysteroid dehydrogenase type 5; AKR1C3; human adrenal cortex
Recent studies have shown that the adrenal cortex expresses high levels of farnesoid X receptor (FXR), but its function remains not known. Herein, using microarray technology, we tried to identify candidate FXR targeting genes in the adrenal glands, and showed that FXR regulates 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) expression in human adrenocortical cells. We further demonstrated that FXR stimulated HSD3B2 promoter activity and have defined the cis-element responsible for FXR regulation of HSD3B2 transcription. Transfection of H295R adrenocortical cells with FXR expression vector effectively increased FXR expression levels and additional treatment with chenodeoxycholic acid (CDCA) caused a 25-fold increase in the mRNA for organic solute transporter alpha (OSTα), a known FXR target gene. HSD3B2 mRNA levels also increased following CDCA treatment in a concentration-dependent manner. Cells transfected with a HSD3B2 promoter construct and FXR expression vector responded to CDCA with a 20-fold increase in reporter activity compared to control. Analysis of constructs containing sequential deletions of the HSD3B2 promoter suggested a putative regulatory element between -166 and -101. Mutation of an inverted repeat between -137 and -124 completely blocked CDCA/FXR induced reporter activity. Chromatin immunoprecipitation assays further confirmed the presence of a FXR response element in the HSD3B2 promoter. In view of the emerging role of FXR agonists as therapeutic treatment of diabetes and certain liver diseases, the effects of such agonists on other FXR expressing tissues should be considered. Our findings suggest that in human adrenal cells, FXR increases transcription and expression of HSD3B2. Alterations in this enzyme would influence the capacity of the adrenal gland to produce corticosteroids.
FXR; HSD3B2; Adrenal gland; Bile acids
Redox properties of the photosynthetic gene repressor PpsR and the blue-light photoreceptor/antirepressor AppA from Rhodobacter sphaeroides have been characterized. Redox titrations of PpsR reveal the presence of a two-electron couple, with an Em value of −320 mV at pH 7.0, which is likely to arise from the reversible conversion of two cysteine thiols to a disulfide. This Em value is very much more negative than the Em = −180 mV value measured previously at pH 7.0 for the disulfide/dithiol couple in CrtJ, the homolog for PpsR in the closely related bacterium Rhodobacter capsulatus. AppA, a flavin-containing blue-light receptor that is also involved in the regulation of gene expression in R. sphaeroides, contains multiple cysteines in its C-terminal region, two of which function as a redox-active dithiol/disulfide couple with an Em value of −325 mV at pH 7.0 in the dark. Titrations of this dithiol/disulfide couple in illuminated samples of AppA indicate that the Em value of this disulfide/dithiol couple is −315 mV at pH 7.0, identical to the value obtained for AppA in the dark within the combined experimental uncertainties of the two measurements. The Em values of AppA and PpsR demonstrate that these proteins are thermodynamically capable of electron transfer for their activity as an antirepressor/repressor in R. sphaeroides.
AppA; CrtJ; Disulfide; PpsR; Redox; Regulation of photosynthetic gene expression; Rhodobacter sphaeroides; Transcriptional regulation
Historians have the opportunity of viewing events, people, and their epoch through an aperture in time. With retrospective clarity, change and the forces effecting change can be appropriately categorized, emphasized, and interpreted. Sociologists see change in a forward-focused manner. When we examine our patients today, it is clear our current patients having total joint arthroplasty are different from those in years past. The sociologic influences effecting this change are many and include the revolutionary explosion of, access to, and dissemination of information; increased wealth, life activity expectation, and life expectancy; and an aging workforce. Concurrent with these forces registering change in our patient population is an erosion in respect for professionalism and vertically oriented authoritarian structure throughout society. Our patients are citizens of our modern age. Our public has come to expect miracles in medicine as the norm, yet these miracles are not without inherent risk. The trap implicit in allowing an incompletely informed populace to drive the decisions we make may be bridged by a more complete understanding of who our patients are and what their needs include. This discussion attempts to offer some insight into the forces at play. It focuses on how the changes in society, population, and technology have affected patients’ knowledge and attitude toward medicine and what our response as physicians should be.
Objectives: Takayasu's arteritis (TA) is a rare disease, in which early diagnosis and assessment of treatment efficacy remain a problem. Signs and symptoms may be non-specific and conventional blood tests unreliable, with vascular inflammation often persisting in the face of a normal acute phase response. The current "gold standard" investigation, x ray angiography, is invasive and only identifies late, structural changes in vessels. Recently, non-invasive imaging methods have shown promise in the assessment of patients with TA.
Methods: The invasive and non-invasive imaging performed on all patients in the rheumatology department at the Hammersmith Hospital between May 1996 and May 2002 who fulfilled the ACR criteria for TA were reviewed. All patients were clinically active at diagnosis and were treated with high dose oral prednisolone and additional oral or intravenous immunosuppression.
Results: Non-invasive imaging methods ([18F]fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) and magnetic resonance imaging (MRI)) provided important additional information about disease activity ([18F]FDG-PET) and progression of vessel wall thickening (MRI) when compared with x ray angiography.
Conclusions: Non-invasive imaging methods provide useful additional information towards the diagnosis and management of TA. Such techniques may allow earlier diagnosis and more accurate assessment of response to treatment than conventional clinical assessment and/or angiography. Non-invasive imaging is likely to be useful in the management of other large vessel vasculitides.
In humans, sexual differentiation of the external genitalia is established at 7–12 weeks post conception (wpc). During this period, maintaining the appropriate intrauterine hormone environment is critical. In contrast to other species, this regulation extends to the human fetal adrenal cortex, as evidenced by the virilization that is associated with various forms of congenital adrenal hyperplasia. The mechanism underlying these clinical findings has remained elusive. Here we show that the human fetal adrenal cortex synthesized cortisol much earlier than previously documented, an effect associated with transient expression of the orphan nuclear receptor nerve growth factor IB-like (NGFI-B) and its regulatory target, the steroidogenic enzyme type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). This cortisol biosynthesis was maximal at 8–9 wpc under the regulation of ACTH. Negative feedback was apparent at the anterior pituitary corticotrophs. ACTH also stimulated the adrenal gland to secrete androstenedione and testosterone. In concert, these data promote a distinctive mechanism for normal human development whereby cortisol production, determined by transient NGFI-B and HSD3B2 expression, provides feedback at the anterior pituitary to modulate androgen biosynthesis and safeguard normal female sexual differentiation.
Takayasu arteritis is a chronic vasculitis involving the aorta and its main branches, the pulmonary arteries, and the coronary tree, and needs to be considered in a young patient with angina, in particular when pulses are absent. This case illustrates the limitations of exercise testing in diagnosing the extent of coronary artery disease and the risks associated with coronary angiography in patients with inflammatory disease in the left main stem coronary artery. It also highlights the novel use of non-invasive scanning with positron emission tomography using 18-fluorodeoxyglucose in assessing remission from this disease. Revascularisation was performed with percutaneous transluminal coronary angioplasty and stenting as an emergency procedure, but treatment of the restenosis with directional atherectomy was based on a review of the available literature. The lymphocytic alveolitis seen in this patient has not been previously described in Takayasu’s disease.
Takayasu’s arteritis; left main stem stenosis
The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.
folate status appears to promote colonic carcinogenesis by, as of yet,
undefined mechanisms. Impaired DNA repair plays a significant role in
the evolution of many colon cancers. Since folate is essential for the
de novo synthesis of nucleotides and since
folate depletion has previously been associated with excessive DNA
strand breaks, it was hypothesised that folate depletion may impair DNA
repair. Studies were therefore performed to examine whether folate
depletion affects the two major categories of DNA repair.
Methods—Study 1: eight
weanling male Sprague-Dawley rats were fed on diets containing either 0 or 8 mg folate/kg diet with 1% succinylsulphathiazole for four weeks.
After viable colonocytes had been harvested, DNA excision repair was
evaluated by a single cell gel electrophoresis assay. Study 2: eighteen
animals were fed on similar diets for five weeks. Also in study 2, 18 additional rats were fed on the same defined diet without
succinylsulphathiazole for 15 weeks. Weekly injections with the
procarcinogen, 1,2-dimethylhydrazine (20 mg base/kg), were administered
to the latter group of animals. Five microsatellite loci from different
chromosomes were investigated for instability in hepatic and colonic DNA.
Results—In study 1, a
significantly retarded rate of DNA excision repair was observed in the
folate deficient colonocytes compared with controls (p<0.05). In study
2, there was no evidence of instability at the five microsatellite loci
associated with either short or long term folate depletion.
deficiency impairs DNA excision repair in rat colonic mucosa; a similar
degree of deficiency, even when administered in conjunction with a
colonic carcinogen, did not produce evidence of a widespread defect in
folate; colon cancer; DNA repair; single cell gel
electrophoresis; microsatellite instability; rat
BACKGROUND: New selective serotonin reuptake inhibitors (SSRIs) are perceived to be much safer in use than older tricyclic antidepressants (TCAs). However, previous assessments of association with fatal toxicity were made too soon after the introduction of the new drugs to permit accurate estimation. AIM: To determine the level of association of antidepressant drugs with fatal poisoning in the treatment of depression. METHOD: National data for England and Wales for three years (1993 to 1995) for fatal poisonings associated with antidepressants were obtained and, together with national primary care data on prescribing, were used to calculate fatality association by antidepressant drug. RESULTS: There were substantial variations between drugs in the level of association with fatal poisoning. Assuming an average treatment episode lasted three months, one fatality is associated with 11,800 treatment episodes of antidepressant use (95% CI = 11,120 to 12,580) when only single substance fatalities are considered. For SSRIs as a group the association was one in 411,800 (95% CI = 243,300 to 1.34 million) and for TCAs one in 8130 (95% CI = 7650 to 8670). However, for one of the newer TCAs, lofepramine, the single substance fatality rate associated with its use was one in 233,700 (95% CI = 124,500 to 1.89 million), which is not statistically significantly different from the SSRIs (P = 0.35). CONCLUSIONS: Estimated death rates associated with specific antidepressants should be compared with caution because drugs may be used selectively in patients with differing severity of depression. The proportion of these fatalities that could be prevented by switching to safer antidepressants is unclear when so few deaths are recorded as accidental; when there is intent to do self-harm the potential for switching to other means is unknown. However, this approach to relative toxicity may remain the best available since it is unlikely that a randomised trial will ever be conducted with a large enough sample size to obtain experimental data. Fatalities from antidepressant poisoning are very rare but if safety is paramount then lofepramine or an SSRI are justifiable treatment choices.
We report two cases of facial haematoma following streptokinase therapy. This is an important complication as it can have fatal consequences. Early recognition and close monitoring are essential as emergency intervention may be required.
The methods available for analysis of the chromatin of Schizosaccharomyces pombe are time consuming (>8 h) and/or result in some degradation of the chromatin. Here we report an optimised method for the preparation of spheroplasts and the isolation of nuclei which takes <25 min and is suitable for analysis of chromatin structure by micrococcal nuclease, restriction endonuclease or by immunoprecipitation.
BACKGROUND AND AIMS: Diminished folate status is associated with enhanced colorectal carcinogenesis. This study investigated the potential chemopreventive role of dietary folate in the dimethylhydrazine colorectal cancer model. SUBJECTS AND METHODS: Sprague-Dawley rats were fed diets containing either 0, 2 (daily dietary requirement), 8 or 40 mg folate/kg diet for 20 weeks. After five weeks of diet, rats were injected with dimethyl-hydrazine (44 mg/kg) weekly for 15 weeks. Fifteen weeks after the first injection of dimethylhydrazine, all rats were killed. Folate status was determined, and the entire colorectum from each rat was analysed for macroscopic and microscopic neoplasms. RESULTS: Plasma and colonic folate concentrations correlated directly with dietary folate levels (p < 0.005). The incidence of microscopic neoplasms was similar among the four groups. However, the incidence and the average number of macroscopic tumours per rat decreased progressively with increasing dietary folate levels up to 8 mg/kg diet (p < 0.05). In the strongly procarcinogenic milieu used in this study, folate supplementation at 20 times the basal requirement was associated with rates of macroscopic tumour development that were intermediate, and not statistically distinct, from rates observed at either 0 or 8 mg/kg diet. CONCLUSIONS: These data indicate that in this rat model, (a) increasing dietary folate up to four times the basal requirement leads to a progressive reduction in the evolution of macroscopic neoplasms from microscopic foci; and (b) folate supplementation beyond four times the requirement does not convey further benefit.
A previously well 84 year old man without prior history of abdominal trauma, instrumentation or surgery presented with localized biliary peritonitis (biloma). An associated bile duct stone was detected by abdominal ultrasound and endoscopic retrograde cholangiopancreatography and was successfully treated with systemic antibiotics and percutaneous and endoscopic stenting. The case is unusual in that the presentation was spontaneous.