Studies on the mechanism of accumulation of cholesterol in the gallbladder mucosa. Evidence that sterol 27-hydroxylase is not a pathogenetic factor

BACKGROUND/AIMS: Cholesterolosis is characterized by accumulation of esterified cholesterol in human gallbladder mucosa. The present study aimed at investigating possible pathogenetic factors for cholesterolosis. The hypothesis was tested that a reduced sterol 27-hydroxylase or an increased amount of ACAT-1 enzyme may be of importance. METHODS: Gall bladder mucosa and bile were obtained from patients with cholesterol gallstones undergoing cholecystectomy (30 with and 43 without cholesterolosis). RESULTS: In cholesterolosis, the gall bladder mucosa was characterized by a several-fold increase in esterified cholesterol and normal content of free cholesterol. The amount of ACAT-1 protein, measured by immunoblotting, was similar in patients with and without cholesterolosis. The level of 27-hydroxycholesterol in gallbladder mucosa was elevated sevenfold as compared with cholesterol in patients with cholesterolosis. Most (87%) of this oxysterol was esterified and the accumulation is most probably secondary to the higher total amount of cholesterol in the cells. Patients with cholesterolosis had normal levels of both sterol 27-hydroxylase mRNA (real time polymerase chain reaction) and protein (immunoblotting). The enzymatic activity of the sterol 27-hydroxylase in gallbladder mucosa was normal or increased in cholesterolosis. CONCLUSIONS: The pathogenesis of cholesterolosis may be multifactorial, but is not caused by reduced efflux of cholesterol due to a defect sterol 27-hydroxylase mechanism.     

MDR1 gene expression and drug resistance of AML cells

We investigated the cellular drug resistance to aclarubicin (Acla), cytosine arabinoside (Ara-C), daunorubicin (Dau), doxorubicin (Dox), etoposide (Etop) and mitoxantrone (Mitox) using the MTT assay at time of disease presentation in 93 cases of acute myeloid leukaemia (AML). In 31 cases we concomitantly investigated MDR1 (multiple drug resistance 1 gene) expression (semi-quantitative competitive RT-PCR) of the leukaemic cells. Drug resistance towards Dau, Dox and Etop was correlated to the MDR1 expression of the AML cells ( P  < 0.05) with high MDR1 expression being associated with high drug resistance towards these drugs. Although the data did not allow firm conclusions to be drawn on the correlation between MDR1 expression and drug resistance towards Ara-C and Mitox, the drug resistance towards Acla clearly was not correlated to, or dependent on, the MDR1 expression level of the AML blast cells. In addition, when examining the cross-activities among the six drugs distinct patterns emerged. Thus, high to very high degrees of cross-activity were found to exist between Dau, Dox, Etop and Mitox, whereas Ara-C had moderate cross-activity with the other drugs except Acla, which showed absent to moderate cross-activity with the other drugs. We conclude that MDR1 gene expression is of significance for cellular drug resistance towards specific (MDR1-related) drugs in AML, whereas it is not of significance regarding drug resistance towards other drugs, which is the case with the anthracycline Acla. We suggest that in the place of other more or less complicated ways to circumvent MDR1-mediated drug resistance, Acla may be used to replace Dau, Dox and other MDR1-related drugs if proven as potent as the drug it is to substitute.     

Web-Based Immersive Virtual Patient Simulators: Positive Effect on Clinical Reasoning in Medical Education

Background

Clinical reasoning is based on the declarative and procedural knowledge of workflows in clinical medicine. Educational approaches such as problem-based learning or mannequin simulators support learning of procedural knowledge. Immersive patient simulators (IPSs) go one step further as they allow an illusionary immersion into a synthetic world. Students can freely navigate an avatar through a three-dimensional environment, interact with the virtual surroundings, and treat virtual patients. By playful learning with IPS, medical workflows can be repetitively trained and internalized. As there are only a few university-driven IPS with a profound amount of medical knowledge available, we developed a university-based IPS framework. Our simulator is free to use and combines a high degree of immersion with in-depth medical content. By adding disease-specific content modules, the simulator framework can be expanded depending on the curricular demands. However, these new educational tools compete with the traditional teaching

Objective

It was our aim to develop an educational content module that teaches clinical and therapeutic workflows in surgical oncology. Furthermore, we wanted to examine how the use of this module affects student performance.

Methods

The new module was based on the declarative and procedural learning targets of the official German medical examination regulations. The module was added to our custom-made IPS named ALICE (Artificial Learning Interface for Clinical Education). ALICE was evaluated on 62 third-year students.

Results

Students showed a high degree of motivation when using the simulator as most of them had fun using it. ALICE showed positive impact on clinical reasoning as there was a significant improvement in determining the correct therapy after using the simulator. ALICE positively impacted the rise in declarative knowledge as there was improvement in answering multiple-choice questions before and after simulator use.

Conclusions

ALICE has a positive effect on knowledge gain and raises students’ motivation. It is a suitable tool for supporting clinical education in the blended learning context.     

Topiramate‐induced nephrolithiasis

Nephrolithiasis is a less common side effect of the antiepileptic drug topiramate. We report the case of a 3‐year‐old boy who presented to the emergency department with abdominal pain; examinations revealed a large calcification in the left kidney. Regular ultrasound examinations are recommended in children using topiramate.     

Identification of lymphogranuloma venereum‐associated Chlamydia trachomatis serovars by fluorescence in situ hybridisation – a proof‐of‐principle analysis

We describe a proof‐of‐principle evaluation of a fluorescence in situ hybridisation (FISH) procedure to identify Chlamydia trachomatis serovars L1–L3, the causative agents of lymphogranuloma venereum, in cell cultures based on newly designed DNA probes. Rapid and easy‐to‐perform FISH could facilitate the diagnosis of lymphogranuloma venereum without nucleic acid amplification or serotyping, but requires broader evaluation studies, for example, in tropical high‐endemicity regions.     

A splice variant of the two-pore domain potassium channel TREK-1 with only one pore domain reduces the surface expression of full-length TREK-1 channels

We have identified a novel splice variant of the human and rat two-pore domain potassium (K_2P) channel TREK-1. The splice variant TREK-1e results from skipping of exon 5, which causes a frame shift in exon 6. The frame shift produces a novel C-terminal amino acid sequence and a premature termination of translation, which leads to a loss of transmembrane domains M3 and M4 and of the second pore domain. RT-PCR experiments revealed a preferential expression of TREK-1e in kidney, adrenal gland, and amygdala. TREK-1e was nonfunctional when expressed in Xenopus oocytes. However, both the surface expression and the current density of full-length TREK-1 were reduced by co-expression of TREK-1e. Live cell imaging in COS-7 cells transfected with GFP-tagged TREK-1e showed that this splice variant was retained in the endoplasmic reticulum (ER). Attachment of the C-terminus of TREK-1e to two different reporter proteins (Kir2.1 and CD8) led to a strong reduction in the surface expression of these fusion proteins. Progressive truncation of the C-terminus of TREK-1e in these reporter constructs revealed a critical region (amino acids 198 to 205) responsible for the intracellular retention. Mutagenesis experiments indicated that amino acids I204 and W205 are key residues mediating the ER retention of TREK-1e. Our results suggest that the TREK-1e splice variant may interfere with the vesicular traffic of full-length TREK-1 channels from the ER to the plasma membrane. Thus, TREK-1e might modulate the copy number of functional TREK-1 channels at the cell surface, providing a novel mechanism for fine tuning of TREK-1 currents.