Effect of preoperative biliary drainage on cholestasis-associated inflammatory and fibrotic gene signatures in perihilar cholangiocarcinoma.

Preoperative biliary drainage (PBD) is used routinely in the evaluation of patients with potentially resectable perihilar cholangiocarcinoma to relieve cholestasis and improve the liver's resilience to surgery. Little preclinical or translatational data are, however, currently available to guide the use of PBD in this patient group. The effect of PBD on hepatic gene expression profiles was therefore studied by microarray analysis. Drainage affects inflammatory and fibrotic gene signatures.

Port wine stain treatment outcomes have not improved over the past three decades.

BACKGROUND: Since the early '80s, the pulsed dye laser has been the standard treatment tool for non-invasive port wine stain (PWS) removal. In the last three decades, a considerable amount of research has been conducted to improve clinical outcomes, given that a fraction of PWS patients proved recalcitrant to laser treatment. Whether this research actually led to increased therapeutic efficacy has not been systematically investigated. OBJECTIVE: To analyse therapeutic efficacy in PWS patients globally from 1986 to date. METHODS: PubMed was searched for all available PWS trials. Studies with a quartile percentage improvement scale were included, analysed and plotted chronologically. Treatment and patient characteristics were extracted. A mean clearance per study was calculated and plotted. A 5-study simple moving average was co-plotted to portray the trend in mean clearance over time. The data were separately analysed for multiple treatment sessions in previously untreated patients. RESULTS: Sixty-five studies were included (24.3% of eligible studies) comprising 6207 PWS patients. Of all patients, 21% achieved 75-100% clearance. Although a few studies reported remarkably good outcomes in a subset of carefully selected patients, there was no upward trend over time in mean clearance. CONCLUSION: The efficacy of PWS therapy has not improved in the past decades, despite numerous technical innovations and pharmacological interventions. With an unwavering patient demand for better outcomes, the need for development and implementation of novel therapeutic strategies to clear all PWS is as valid today as it was 30 years ago.

Effect of obeticholic acid on liver regeneration following portal vein embolization in an experimental model.

BACKGROUND: The bile salt-activated transcription factor farnesoid X receptor (FXR) is a key mediator of proliferative bile salt signalling, which is assumed to play a role in the early phase of compensatory liver growth. The aim of this study was to evaluate the effect of a potent FXR agonist (obeticholic acid, OCA) on liver growth following portal vein embolization (PVE). METHODS: Rabbits were allocated to receive daily oral gavage with OCA (10 mg/kg) or vehicle (control group) starting 7 days before PVE (n = 18 per group), and continued until 7 days after PVE. PVE of the cranial liver lobes was performed using polyvinyl alcohol particles and coils on day 0. Caudal liver volume (CLV) was analysed by CT volumetry on days -7, -1, +3 and +7. Liver function was determined by measuring mebrofenin uptake using hepatobiliary scintigraphy. Additional parameters analysed were plasma aminotransferase levels, and histological scoring of haematoxylin and eosin- and Ki-67-stained liver sections. RESULTS: Three days after PVE of the cranial lobes, the increase in CLV was 2·2-fold greater in the OCA group than in controls (mean(s.d.) 56·1(20·3) versus 26·1(15·4) per cent respectively; P < 0·001). This increase remained greater 7 days after PVE (+1·5-fold; P = 0·020). The increase in caudal liver function at day +3 was greater in OCA-treated animals (+1·2-fold; P = 0·017). The number of Ki-67-positive hepatocytes was 1·6-fold higher in OCA-treated animals 3 days after PVE (P = 0·045). Plasma aminotransferase levels and histology did not differ significantly between groups. CONCLUSION: OCA accelerated liver regeneration after PVE in a rabbit model. OCA treatment might increase the efficacy of PVE and, thereby, resectability. Surgical relevance Liver failure is the most feared complication after liver surgery, with no effective treatment options. Liver regeneration is essential to avoid liver failure, and recently bile acid signalling was implicated in the initiation of liver regeneration through the nuclear bile acid receptor farnesoid X receptor (FXR). In this study, the potent FXR agonist obeticholic acid accelerated liver regeneration following portal vein embolization in a rabbit model, in terms of liver volume, liver function and proliferation. Obeticholic acid treatment could enhance the efficacy of portal vein embolization, thereby increasing resectability, and could reduce the interval to surgery. In addition, obeticholic acid might have a place in the prevention of liver failure after liver surgery.

Use of an absorbable embolization material for reversible portal vein embolization in an experimental model.

BACKGROUND: Portal vein embolization (PVE) is used to increase future remnant liver size in patients requiring major hepatic resection. PVE using permanent embolization, however, predisposes to complications and excludes the use of PVE in living donor liver transplantation. In the present study, an absorbable embolization material containing fibrin glue and different concentrations of the fibrinolysis inhibitor aprotinin was used in an experimental animal model. METHODS: PVE of the cranial liver lobes was performed in 30 New Zealand White rabbits, which were divided into five groups, fibrin glue + 1000, 700, 500, 300 or 150 kunits/ml aprotinin, and were compared with a previous series of permanent embolization using the same experimental set-up. Caudal liver lobe hypertrophy was determined by CT volumetry, and portal recanalization was identified on contrast-enhanced CT images. Animals were killed after 7 or 42 days, and the results were compared with those of permanent embolization. RESULTS: PVE using fibrin glue with aprotinin as embolic material was effective, with 500 kunits/ml providing the optimal hypertrophic response. Lower concentrations of aprotinin (150 and 300 kunits/ml) led to reduced hypertrophy owing to early recanalization of the embolized segments. The regeneration rate over the first 3 days was higher in the group with 500 kunits/ml aprotinin than in the groups with 300 or 150 kunits/ml or permanent embolization. In the 500-kunits/ml group, four of five animals showed recanalization 42 days after embolization, with minimal histological changes in the cranial lobes following recanalization. CONCLUSION: Fibrin glue combined with 500 kunits/ml aprotinin resulted in reversible PVE in 80 per cent of animals, with a hypertrophy response comparable to that achieved with permanent embolization material. Surgical relevance Portal vein embolization (PVE) is used to increase future remnant liver volume in patients scheduled for major liver resection who have insufficient future remnant liver size to perform a safe resection. The current standard is PVE with permanent embolization materials, which renders patients found to have unresectable disease prone to complications owing to the permanently deportalized liver segments. Absorbable embolization might prevent the PVE-associated morbidity and lower the threshold for its application. In this study, PVE using fibrin glue and aprotinin resulted in an adequate hypertrophy response with 80 per cent recanalization after 42 days. Considering the minor histological changes following recanalization of embolized segments and potentially preserved function, reversible PVE might also be applied in living donor liver transplantation.

The donor OH stretching-libration dynamics of hydrogen-bonded methanol dimers in cryogenic matrices.

FTIR spectra of the methanol dimer trapped in neon matrices are presented. The fundamental, overtone and combination bands involving the donor OH libration and stretching motions were observed in order to extract relevant anharmonicity constants. We find a stretching-libration coupling constant of +43(5) cm(-1) and a diagonal librational anharmonicity constant of -71(5) cm(-1). The spectra are compared to a number of VPT2 calculations and a torsionally localized monomer model in order to enhance previous explanations of the observable OH stretching red-shift upon dimerization.

[Guideline (S2k, AWMF) of the Deutsche Gesellschaft für Pneumologie und Beatmungsmedizin and the Deutsche Gesellschaft für Arbeitsmedizin und Umweltmedizin "Diagnostics and Expert Opinion in the Occupational Disease No. 4101 Silicosis (Including Coal Worker's Pneumoconiosis)"]. / S2k-Leitlinie nach AWMF-Schema der Deutschen Gesellschaft für Pneumologie und Beatmungsmedizin und der Deutschen Gesellschaft für Arbeitsmedizin und Umweltmedizin "Diagnostik und Begutachtung der Berufskrankheit Nr. 4101 Quarzstaublungenerkrankung (Silikose) der Berufskrankheitenverordnung".

During the last 1.5 years an update of the guideline on silicosis was made by an interdisciplinary working group. New medical and scientific knowledge and the experience in expert opinion practice were taken into account.By preparing the initial guideline in 2010 standardization of diagnostics and adaption of the "Moers convention" which was not based on medical knowledge was in the focus, whereas the current update deals with fine emendation and extension, especially of the compensation rate (adaption with the Reichenhall recommendation).The diagnosis of silicosis (including mixed dust pneumoconiosis) is based on a detailed occupational history, and predominantly on the typical radiological findings. However, at initial diagnosis the standardized LD-HRCT takes an important role because of its high sensitivity and specificity. Exceptional cases are those with characteristic findings in chest X-ray follow-up. Correspondingly, it is mentioned in the guideline: "The standardized appraisal of the Low-Dose-Volume HRCT requires application of the CT classification (ICOERD, International Classification of Occupational and Environmental Respiratory diseases). In order to diagnose silicosis in CT scan opacities with sharp borders in both central upper lung fields and their circumferencies have to be documented. By comparing with ILO standard radiographs at least profusion category 1 in the right and left upper lung fields has to be reached (total profusion category 2)."The pathologic minimal requirement for the diagnosis of silicosis which has undergone controversial discussion has now also been defined. Corresponding to Hnizdo et al. 2000 it is now mentioned: "Finding of less than 5 silicotic granuloma per lung lobe by palpation is regarded as insignificant." This is a convention and not a threshold based on detailed medical scientific and statistical studies; it is based on extended experience in the South African gold mines.This guideline also deals with silicotic hilar (and sometimes mediastinial) lymph nodes; according to the guideline working group they do not closely correlate with the degree of pulmonary involvement. Extended conglomerating and enduring lymph-node processes may lead to dislocation of the hili with impairment of large bronchi and vessels. Shell-like calcifications dominating in the periphery of lymph nodes produce so-called egg-shell hili.The paragraph on exercise testing is now extended: if neither ergometry nor spiroergometry can be performed a 6 minute walking test by measuring oxygen saturation should be done.Furthermore, in individual expert opinion examinations right heart catheterization (the patient is not obliged to give informed consent) may be recommended, if echo cardiography gives evidence for pulmonary hypertension or if it is difficult to differentiate between right and left heart failure. The presence of pulmonary hypertension which is of prognostic relevance has to be considered when grading reduction in earning capacity.For interpretation of spirometry values the new GLI reference values has to be applied. Grading of impairment is due to the recommendation of the DGP.According to current medical scientific knowledge it is unclear, whether certain disorders of the rheumatic group such is scleroderma or Caplan syndrome which are sometimes associated with silicosis (or coal workers' pneumoconiosis) belong in toto to the occupational disease number 4101 (silicosis). Within this context, additional studies are needed to clarify the role of occupational quartz exposure and other risk factors.The guideline working group hopes that this update will help to optimize diagnostics and expert opinion of silicotic patients.

The effect of hydrogen bonding on torsional dynamics: a combined far-infrared jet and matrix isolation study of methanol dimer.

The effect of strong intermolecular hydrogen bonding on torsional degrees of freedom is investigated by far-infrared absorption spectroscopy for different methanol dimer isotopologues isolated in supersonic jet expansions or embedded in inert neon matrices at low temperatures. For the vacuum-isolated and Ne-embedded methanol dimer, the hydrogen bond OH librational mode of the donor subunit is finally observed at ~560 cm(-1), blue-shifted by more than 300 cm(-1) relative to the OH torsional fundamental of the free methanol monomer. The OH torsional mode of the acceptor embedded in neon is observed at ~286 cm(-1). The experimental findings are held against harmonic predictions from local coupled-cluster methods with single and double excitations and a perturbative treatment of triple excitations [LCCSD(T)] and anharmonic. VPT2 corrections at canonical MP2 and density functional theory (DFT) levels in order to quantify the contribution of vibrational anharmonicity for this important class of intermolecular hydrogen bond vibrational motion.

Hepatic regeneration and functional recovery following partial liver resection in an experimental model of hepatic steatosis treated with omega-3 fatty acids.

BACKGROUND: Omega-3 fatty acids (FAs) have been shown to reduce experimental hepatic steatosis and protect the liver from ischaemia-reperfusion injury. The aim of this study was to examine the effects of omega-3 FAs on regeneration of steatotic liver. METHODS: Steatosis was induced in rats by a 3-week methionine/choline-deficient diet, which was continued for an additional 2 weeks in conjunction with oral administration of omega-3 FAs or saline solution. Steatosis was graded histologically and quantified by proton magnetic resonance spectroscopy ((1) H-MRS) before and after the diet/treatment. Liver function was determined by (99m) Tc-labelled mebrofenin hepatobiliary scintigraphy (HBS). In separate experiments, the hepatic regenerative capacity and functional recovery of omega-3 FA-treated, saline-treated or non-steatotic (control) rats were investigated 1, 2, 3 and 5 days after partial (70 per cent) liver resection by measurement of liver weight change and hepatocyte proliferation (Ki-67) and HBS. RESULTS: Severe steatosis (over 66 per cent) in the saline group was reduced by omega-3 FAs to mild steatosis (less than 33 per cent), and hepatic fat content as assessed by (1) H-MRS decreased 2·2-fold. (99m) Tc-mebrofenin uptake in the saline group was more than 50 per cent lower than in the control group, confirming the functional effects of steatosis. (99m) Tc-mebrofenin uptake and regenerated liver mass were significantly greater in the omega-3 group compared with the saline group on days 1 and 3. The posthepatectomy proliferation peak response was delayed until day 2 in saline-treated rats, compared with day 1 in the omega-3 and control groups. CONCLUSION: Omega-3 FAs effectively reduced severe hepatic steatosis, which was associated with improved liver regeneration and functional recovery following partial hepatectomy.

Noninvasive quantification of hepatic steatosis in rats using 3.0 T 1H-magnetic resonance spectroscopy.

PURPOSE: To assess the accuracy of noninvasive 3.0 T (1)H-magnetic resonance spectroscopy ((1)H-MRS) in an experimental steatosis model for the discrimination of clinically relevant macrovesicular steatosis degrees and to evaluate three different (1)H-MR spectrum-based fat quantification methods. MATERIALS AND METHODS: Steatosis was induced in rats by a methionine/choline-deficient diet for 0-5 weeks. (1)H-MRS measurements of hepatic fat content were compared with histopathological and biochemical steatosis degree. In (1)H-MR spectra, areas under the curve (AUC) of fat (1.3 ppm), water (4.7 ppm), total fat (0.5-5.3 ppm), and total spectrum peaks (0.5-5.3 ppm) were determined and hepatic fat content calculated as follows: [AUC(total fat peaks)/AUC(total peaks)], [AUC(fat)/AUC(fat) + (AUC(water)/0.7)], and [AUC(fat)/AUC(water)]. RESULTS: A significant correlation was found between (1)H-MRS and macrovesicular steatosis (r = 0.932, P < 0.0001) and between (1)H-MRS and total fatty acids (r = 0.935, P < 0.0001). (1)H-MRS accurately distinguished mild from moderate and moderate from severe steatosis. Calculations using [AUC(fat)/AUC(water)] ratio in severe steatotic livers resulted in higher hepatic fat percentages as compared to the other methods due to a decrease in hepatic water content. CONCLUSION: (1)H-MRS quantification of hepatic fat content showed high correlations with histological and biochemical steatosis determination in an experimental steatosis rat model and accurately discriminated between clinically relevant steatosis degrees. These results encourage further application of (1)H-MRS in patients for accurate steatosis assessment.

Crossing borders: A systematic review with quantitative analysis of genetic mutations of carcinomas of the biliary tract.

Biliary tract carcinoma (BTC) comprises gallbladder and intra-/extrahepatic cholangiocarcinoma (GBC, ICC, EHC), which are currently classified by anatomical origin. Better understanding of the mutational profile of BTCs might refine classification and improve treatment. We performed a systematic review of studies reporting on mutational profiling of BTC. We included articles reporting on whole-exome/whole-genome-sequencing (WES/WGS) and targeted sequencing (TS) of BTC, published between 2000-2017. Pooled mutation proportions were calculated, stratified by anatomical region and sequencing technique. A total of 25 studies with 1806 patients were included. Overall, TP53 was the most commonly mutated gene in BTC. GBC was associated with mutations in PFKFB3, PLXN2 and PGAP1. Mutations in IDH1, IDH2 and FGFR fusions almost exclusively occurred in ICC patients. Mutations in APC, GNAS and TGFBR2 occurred exclusively in EHC patients. In conclusion, subtypes of BTCs exhibit minor differences in mutational profile, which is likely influenced by the cell of origin.

Laser-induced (endo)vascular photothermal effects studied by combined brightfield and fluorescence microscopy in hamster dorsal skin fold venules.

The putative features of the (endo)vascular photothermal response, characterized by laser-induced thermal denaturation of blood and vessel wall constituents, have been elucidated individually, but not simultaneously in dynamic, isolated in vivo systems. A hamster dorsal skin fold model in combination with brightfield/fluorescence intravital microscopy was used to examine the effect of laser pulse duration and blood flow velocity on the size of the thermal coagulum, its attachment behavior, and laser-mediated vasomotion. The size of the coagulum and the extent of vasoconstriction and latent vasodilation were proportional to the laser pulse duration, but pulse duration had no effect on coagulum attachment/dislodgement. Blood flow velocity exhibited no significant effect on the studied parameters. The (endo)vascular photothermal response is governed predominantly by laser energy deposition and to a marginal extent by blood flow velocity.

In vitro detection of cholangiocarcinoma cells using a fluorescent protein-expressing oncolytic herpes virus.

Pathological confirmation is desired prior to high-risk surgery for suspected perihilar cholangiocarcinoma (PHC), but preoperative tissue diagnosis is limited by poor sensitivity of available techniques. This study aimed to validate whether a tumor-specific enhanced green fluorescent protein (eGFP)-expressing oncolytic virus could be used for cholangiocarcinoma (CC) cell detection. Extrahepatic CC cell lines SK-ChA-1, EGI-1, TFK-1 and control cells (primary human liver cells) were exposed to the oncolytic herpes simplex type 1 virus NV1066 for up to 24 h in adherent culture. The technique was validated for cells in suspension and cultured cells that had been exposed to crude patient bile. Optimal incubation time of the CC cells with NV1066 at a multiplicity of infection of 0.1 was determined at 6-8 h, yielding 15% eGFP-expressing cells, as measured by flow cytometry. Cells were able to survive 2-h crude bile exposure and remained capable of producing eGFP following NV1066 infection. Detection of malignant cells was possible at the highest dilution tested (10 CC cells among 2 × 105 control cells), though hampered by non-target cell autofluorescence. The technique was not applicable to cells in suspension due to insufficient eGFP production. Accordingly, as yet the technique is not suitable for standardized clinical diagnostics in PHC.

Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy.

Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested.

Urinary excretion of frameshift mutagens in rats caused by passive smoking.

Several male Wistar rats were individually placed in a chamber resembling a room provided with minimal air flow. They were exposed separately to the main- and sidestream smoke of a commercial brand of cigarettes smoked by a smoking machine. Exposure to both sidestream and mainstream smoke of at least two cigarettes resulted in significant excretions of frameshift mutagens in urine within 24 h, detected by the bacterial microtiter fluctuation test with Salmonella typhimurium TA1538. Doubling exposure to the mainstream smoke resulted in similar quantitative mutagenic activities. Doubling exposure to the sidestream smoke resulted in reduced water intake by the animals and thus toxic effects of the urine concentrates on the test bacteria.

Investigations on metabolism, genotoxic effects and carcinogenicity of 2,2'-dichlorodiethylether.

Either 40 mumole or 160 mumole 2,2'-DDE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in the 24-h urine specimens. Comparative investigations were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. In a further step, inhalation experiments were performed to determine urinary excretion of the two metabolites after an 8-h exposure of male Wistar rats to 10, 50, 100, and 500 ppm 2,2'-DDE and to 50, 200, und 1,000 ppm vinyl chloride. A long-term study was conducted to investigate the possible carcinogenicity of 2,2'-DDE in male and female Sprague-Dawley rats following s.c. injections of 4.36 mumole and 13.1 mumole 2,2'-DDE in DMSO per week. The evaluation of tumor development in treated groups and controls were based on macroscopic inspection and histological examinations of the suspect organs and tissues. Analysis of the metabolites showed that HEMA excretion was much lower than the excretion of TdGA following the uptake of 2,2'-DDE, 2-chloroethanol and 2-chloroacetaldehyde diethyl acetal. Contrary to these, vinyl chloride uptake resulted in a higher urinary excretion of HEMA than TdGA. There was no appreciable increase in the number of tumors detected in 2,2'-DDE-treated animals when compared with untreated or DMSO-treated groups. Since irradiation of 2,2'-DDE with UV did not elevate mutagenic activity of the compound against Salmonella typhimurium TA100, the high mutagenicity of the compound found in a desiccator cannot be due to the liberation of mutagenic compounds produced under the influence of UV light.

Investigations on metabolism and carcinogenicity of 1,1,2-trichloroethane.

Two groups of male and female Sprague-Dawley rats (50 animals/group per sex) were treated with either 15.37 or 46.77 mumole of 1,1,2-TCE in DMSO/rat for 2 years. The animals were treated once a week by s.c. injection of test compound in the skin of neck. Two groups of controls received either DMSO or no treatment at all. The incidence of benign mesenchymal and epithelial tumors was not significant when compared with either DMSO-treated or untreated controls. The animals treated with 46.77 mumole 1,1,2-TCE significantly developed sarcomas when compared with the untreated controls. In a further experiment, either 40 mumole or 160 mumole 1,1,2-TCE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in 24-h urine samples. Comparative studies were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. Analysis of the metabolites showed that no detectable HEMA was excreted in urine after treatment of rats with 1,1,2-TCE or chloroethanol. TdGA was excreted in urine much more among chloroacetaldehyde-treated animals than among 1,1,2-TCE- or chloroethanol-treated rats.

Afferent connections of the laterodorsal thalamic nucleus in the rat.

The laterodorsal thalamic afferent connections have been studied by means of the retrograde transport of horseradish peroxidase (HRP). The following new sources of projections to the laterodorsal thalamic nucleus (LD) were observed: the zona incerta, the lateral dorsal tegmental nucleus (bilaterally), the lateral hypothalamus and the precentral agranular cortex.

Effect of urokinase on the proliferation of primary cultures of human prostatic cells.

The effects of exogenously added urokinase type plasminogen activator, tissue type plasminogen activator, plasmin and thrombin on the proliferation of primary cultures of cells derived from prostatic hyperplasia or prostatic carcinomas were investigated by measuring the incorporation of 3H-thymidine into the cultures. Addition of urokinase type plasminogen activator (1.35 x 10(-9) M) or thrombin (10(-7) M) to the culture medium caused a two-fold increase of 3H-thymidine incorporation, regardless of the origin of the prostatic cells. Tissue type plasminogen activator did not alter the rate of 3H-thymidine incorporation, whereas plasmin caused a 25% decrease of 3H-thymidine incorporation in all cultures.

Can homeopathically prepared mercury cause symptoms in healthy volunteers? A randomized, double-blind placebo-controlled trial.

OBJECTIVE: To pilot a method for determining whether homeopathically prepared mercury causes more symptoms (a "drug proving") in healthy volunteers than placebo. METHODS: One hundred and eighteen (118) healthy volunteers ages 18 to 65 were recruited by local advertising. Subjects unfamiliar with homeopathy undertook a 1-week single-blind placebo run-in, a 1-week of double-blind, randomized treatment on either homeopathically prepared mercury 12C or placebo, and a third week of placebo run-out. Each day, symptoms were recorded on a checklist that included both true mercury symptoms and symptoms not expected to be caused by mercury (false symptoms). Additional symptoms were assessed by open reporting. Outcome was assessed by calculating a score for each day as the number of true symptoms minus the number of false symptoms. The mean score during placebo was then subtracted from the mean score for weeks two and three of the trial. RESULTS: Fourteen (14) subjects dropped out during placebo run-in. The remaining 104 completed the trial. Baseline comparability was good. Mean difference score was -0.125 (SD 3.47) for mercury and -0.221 (SD 3.01) for placebo (p > 0.2). No significant differences between groups were found for the number of subjects meeting predefined criteria for a drug-proving reaction. CONCLUSION: This pilot study failed to find evidence that mercury 12C causes significantly more symptoms in healthy volunteers than placebo. Questionnaires with a limited number of gross symptoms do not seem to be an appropriate methodological technique in drug proving research. If drug-proving phenomena exist, they appear to be rare.