Targeted next generation sequencing application in cardiac channelopathies: Analysis of a cohort of autopsy-negative sudden unexplained deaths.

Genetic testing for cardiac channelopathies in sudden unexplained death (SUD) has developed substantially over the last years. The Next Generation Sequencing (NGS) technology provides an unprecedented opportunity to screen for genetic variations underlying arrhythmogenic genes in a short period of time at a low cost. The present study aimed to perform genetic testing with NGS technologies on the Ion Torrent Personal Genome Machine™ (Ion PGM™) sequencer, in targeting a total of 23 genes reported to be associated with inherited cardiac channelopathies in order to identify the possible cause of death in a cohort of post-mortem cases. The molecular analyses focused on 16 cases of SUD, aged less than 35 years old. In all cases, the cause of death could not be determined after a rigorous autopsy associated with histopathological and toxicological analyses according to the guidelines of the Association for European Cardiovascular Pathology. DNA was extracted from fresh frozen tissue. An average of 200 variants was identified per case. However, after the prioritization process using a new scoring program (VaRank) and after the conjunction of clinical data and molecular findings, four "likely pathogenic" variants (including two undescribed variants), were identified in three cases (18.75%) of our cohort in the genes KCNH2, ANK2, SCN5A and RYR2. One case, who died during psychiatric hospitalization after administration of a QT prolonging drug, showed a double "likely pathogenic" variant in Long QT genes (ANK2 and SCN5A) which may have predisposed to drug-induced cardiac arrhythmias. Our study illustrates that the NGS approach based on AmpliSeq™ libraries and Ion Torrent PGM™ sequencing may be an efficient approach, integrated to post-mortem examination. Given the massive amount of information generated by NGS, a rigorous filtration strategy of variants coupled with multidisciplinary collaboration is crucial to determine the potential pathogenic role of identified variants in the cause of death.

RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 µl saliva, 5-0.01 µl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 µl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.

Testing for kavain in human hair using gas chromatography-tandem mass spectrometry.

A sensitive, specific and reproducible method for the quantitative determination of kavain in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 50 mg) was incubated in 1 ml of methanol for 1 h, in an ultrasonic bath, in presence of 20 ng of methaqualone-d7 used as internal standard. The methanolic solution was evaporated to dryness, and the residue reconstituted by adding 30 microl of methanol. A 2 microl aliquot of the extract was injected onto the column (Optima5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.25 mm i.d. x 0.25 mm film thickness) of a Hewlett-Packard (Palo Alto, CA) gas chromatograph (5890). Kavain was detected by its parent ion at m/z 230 and daughter ions at m/z 111 and 202 through a Finnigan TSQ 700 MS/MS system. The assay was capable of detecting 30 pg/mg of kavain (limit of detection (LOD)). Linearity was observed for kavain concentrations ranging from 100 to 2000 pg/mg with a correlation coefficient of 0.998. Intra-day precision at 400 pg/mg was 13.7%. The analysis of a segment of hair, obtained from an occasional consumer, revealed the presence of kavain at the concentration of 418 pg/mg. A higher concentration (1708 pg/mg) was detected in the corresponding pubic hair.

[Crime under the influence of psychoactive drugs: the problem of the duration of detection]. / Usage criminel de substances psycho-actives: le problème de la durée de détection.

On a regular basis, the media presents the potential risks of the use of psycho-active compounds, including misused pharmaceuticals (flunitrazepam, GHB) or drugs of abuse (cannabis, LSD, ecstasy). Ethanol is also frequently encountered. These drugs can be used for recreational purposes by addicts or can be observed after sexual assaults (drugs spiked in food). Forensic toxicology can be involved in several situations to document impairment, such as: crime under influence, date rape, driving under influence, psychiatric disorders, determination of the cause of death... In some particular situations, it can be very cautious to investigate exposure to psycho-active drugs, due to late sampling of biological specimens. To enhance the window of detection of 3 specific drugs, the authors propose the following: 1. Use of an ultra-sensitive technique, such as GC/MS/MS/NCI for 7-aminoflunitrazepam; 2. Use of a cumulative specimen, such as sweat for GHB and 3. Use of a metabolite with a long half-life, such as ethyl glucuronide for ethanol.

Doping control for methenolone using hair analysis by gas chromatography-tandem mass spectrometry.

A sensitive, specific and reproducible method for the quantitative determination of methenolone in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 100 mg) was solubilized in 1 ml 1 M NaOH, 15 min at 95 degrees C, in presence of 1 ng testosterone-d3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C18 eluted with methanol) and a liquid-liquid (pentane) extraction. The residue was derivatized by adding 50 microl MSTFA-NH4I-2-mercaptoethanol (1000:2:5, v/v/v), then incubated for 20 ml at 60 degrees C. A 1.5-microl aliquot of the derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.25 mm I.D., 0.25 microm film thickness) of a Hewlett-Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Methenolone was detected by its parent ion at m/z 446 and daughter ions at m/z 208 and 195 through a Finnigan TSQ 700 MS-MS system. The assay was capable of detecting 1 pg/mg of methenolone when approximately 100 mg hair material was processed. Linearity was observed for methenolone concentrations ranging from 2 to 100 pg/mg with a correlation coefficients of 0.965-0.981. Intra-day and between-day precisions at 2, 10 and 25 pg/mg were 10.9-14.1% and 13.7-16.8%, respectively, with an extraction recovery of 97.6%. The analysis of a strand of hair obtained from two bodybuilders, revealed the presence of methenolone at the concentrations of 7.3 and 8.8 pg/mg.

Usage criminel de substances psycho-actives : le problème de la durée de détection.

On a regular basis, the media presents the potential risks of the use of psycho-active compounds, including misused pharmaceuticals (flunitrazepam, GHB) or drugs of abuse (cannabis, LSD, ecstasy). Ethanol is also frequently encountered. These drugs can be used for recreational purposes by addicts or can be observed after sexual assaults (drugs spiked in food). Forensic toxicology can be involved in several situations to document impairment, such as : crime under influence, date rape, driving under influence, phsychiatric disorders, determination of the cause of death … In some particular situations, it can be very cautious to investigate exposure to psycho-active drugs, due to late sampling of biological specimens. To enhance the window of detection of 3 specific drugs, the authors propose the following : 1. Use of an ultra-sensitive technique, such as GC/MS/MS/NCI for 7-aminoflunitrazepam; 2. Use of a cumulative specimen, such as sweat for GHB and 3. Use of a metabolite with a long half-life, such as ethyl glucuronide for ethanol.

Detection of cannabis in oral fluid (saliva) and forehead wipes (sweat) from impaired drivers.

Saliva and sweat have been presented as two alternative matrices for the establishment of drug abuse. The noninvasive collection of a saliva or sweat sample, which is relatively easy to perform and can be achieved under close supervision, is one of the most important benefits in a driving-under-the-influence situation. Moreover, the presence of certain analytes in saliva is a better indication of recent use than when the drug is detected in urine, so there is a higher probability that the subject is experiencing pharmacological effects at the time of sampling. We developed an original procedure using gas chromatography-mass spectrometry to test for delta9-tetrahydrocannabinol (THC), the psychoactive ingredient of cannabis, in oral fluid and forehead wipes, collected with Sarstedt Salivettes and cosmetic pads, respectively. Blood, urine, oral fluid, and forehead wipes were simultaneously collected from 198 injured drivers admitted to an Emergency Hospital in Strasbourg, France. Of the 22 subjects positive for 11-nor-9-carboxy-THC (THCCOOH) in urine, 14 and 16 were positive for THC in oral fluid (1 to 103 ng/Salivette) and forehead wipe (4 to 152 ng/pad), respectively. 11-Hydroxy-THC and THCCOOH were not detected in these body fluids. Two main limitations of saliva and sweat are apparent: the amount of matrix collected is smaller when compared to urine, and the levels of drugs are higher in urine than in saliva and sweat. A current limitation in the use of these specimens for roadside testing is the absence of a suitable immunoassay that detects the parent compound in sufficiently low concentrations.

Testing of the anabolic stanozolol in human hair by gas chromatography-negative ion chemical ionization mass spectrometry.

A sensitive, specific and reproducible method for the quantitative determination of stanozolol in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride and the sonication in methanol of 100 mg of powdered hair for 2 h. After elimination of the solvent, the hair sample was solubilized in 1 ml 1 M NaOH, 15 min at 95 degrees C, in the presence of 10 ng stanozolol-d3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C18) and a liquid-liquid (pentane) extraction. After evaporation of the final organic phase, the dry extract was derivatized using 40 microl MBHFA-TMSI (1000:20, v/v), incubated for 5 min at 80 degrees C, followed by 10 microl of MBHFBA, incubated for 30 min at 80 degrees C. The derivatized extract was analyzed by a Hewlett-Packard GC-MS system with a 5989 B Engine operating in the negative chemical ionization mode of detection. Linearity of the detector response was observed for stanozolol concentrations ranging from 5 to 200 pg/mg with a correlation coefficient of 0.998. The assay was capable of detecting 2 pg of stanozolol per mg of hair when approximately 100 mg hair material was processed, with a quantification limit set at 5 pg/mg. Intra-day precision was 5.9% at 50 pg/mg and 7.8% at 25 pg/mg with extraction recoveries of 79.8 and 75.1%, respectively. The analysis of a 3-cm long hair strand, obtained from a young bodybuilder (27 year old) assuming to be a regular user of Winstrol (stanozolol, 2 mg), revealed the presence of stanozolol at the concentration of 15 pg/mg.

[Evidence of pesticide exposure by hair analysis]. / Mise en évidence de l'exposition aux pesticides par analyse des cheveux.

The current report summarizes the development of an analytical method for the identification and the quantification of pesticides in hair by gas chromatography/mass spectrometry and its application to 75 real samples. Hair strands [table: see text] were obtained from wine workers exposed to one or more pesticides. After decontamination, hair were cut into small pieces and incubated overnight at 45 degrees C in methanol. The solvent was evaporated to dryness, the dry extract was redissolved in methanol and injected in a gas chromatography/mass spectrometry system. The detector was operated in electronic impact and in negative chemical ionization mode of detection (reactant gas: methane). In the first series of 75 hair specimens, obtained before the period of pesticide use, none of the 15 target compounds was detected. In the second series of 75 specimens, obtained from the same subjects but after the use of pesticides, 14 tested positive for 9 different pesticides.

Evaluation of acetylcodeine as a specific marker of illicit heroin in human hair.

In addition to acetylmorphine (6-AM), acetylcodeine (AC) has been suggested as a marker for the use of illicit heroin. Because no procedure was available for AC testing in hair, a new method was developed for the simultaneous identification and quantitation of morphine (MOR), codeine (COD), 6-AM, and AC. After decontamination, each hair specimen was cut into 1-mm pieces. A 50-mg aliquot was incubated overnight at 50 degrees C in 1 mL Soerensen buffer (pH 7.6) in presence of 200 ng of MOR-d3, COD-d3, 6-AM-d3, and AC-d3. After pH adjustment to 8.4, the analytes were extracted in 5 mL of chloroform/isopropanol/n-heptane (25:10:65, v/v/v). The organic phase was removed and evaporated to dryness, and the residue was derivatized by silylation (BSTFA + 1% TMCS). Drugs were analyzed by gas chromatography-mass spectrometry in electron impact mode. Limits of quantitation were set to 0.1 ng/mg. Fifty hair specimens obtained from subjects who died from fatal opiate overdose were analyzed. AC was detected in 22 samples in concentrations ranging from 0.17 to 5.60 ng/mg with a mean value of 1.04 ng/mg. 6-AM was also present in these samples at concentrations ranging from 1.35 to 41.10 ng/mg with a mean value of 7.79 ng/mg. Of the 28 specimens negative for AC, 21 were positive for 6-AM at concentrations ranging from 0.18 to 7.13 ng/mg. When detected, the AC concentrations were an average of 15.5% (2.8 to 32.6%) of the 6-AM concentrations. There was a positive relationship between AC concentrations and 6-AM concentrations (r = 0.915, p = 0.001). Neither AC nor COD was identified in hair specimens collected from 20 subjects taking part in a heroin-maintenance program in Switzerland and receiving pure pharmaceutical heroin hydrochloride daily. Although it is indicative of illicit heroin use, AC would not make a suitable biomarker in place of 6-AM because of its low concentration in hair compared with that of 6-AM and its absence in about 50% of the specimens that tested positive for 6-AM.

Dose-concentration relationships in hair from subjects in a controlled heroin-maintenance program.

Hair specimens were collected from the vertex area of 20 subjects taking part in a heroin-maintenance program. Subjects were administered, under controlled conditions, heroin hydrochloride in 2 or 3 doses/day intravenously. Heroin doses ranged from 30 to 800 mg/day, and were self-administered. In all cases, a 4-cm segment from the proximal zone (root) was analyzed, which corresponded to about 100 days of hair growth. During that period, total heroin administered ranged from 14,100 to 71,540 mg. All special features of hair such as coloring, bleaching, etc. were noted. Each sample was washed twice with dichloromethane (5 mL, 2 min) and, after drying, cut into small pieces of approximately 1 mm. A 30-35-mg aliquot was incubated overnight at 45 degrees C in 1 mL methanol in the presence of 200 ng of heroin-d9, 6-acetylmorphine-d3, and morphine-d3. The methanolic extract was then evaporated to dryness, and the residue was derivatized by silylation (BSTFA + 1% TMCS). Drugs were analyzed by gas chromatography-mass spectrometry in electron impact mode. Limits of quantitation were set to 0.1 ng/mg. Concentrations ranged from 0 to 4.53, 0.38 to 10.11, and 0.71 to 5.20 ng/mg for heroin, 6-acetylmorphine, and morphine, respectively. 6-Acetylmorphine was the major analyte present in hair in all but five cases. Heroin was present in the highest concentration in three cases, and morphine was the major metabolite in two cases, probably because of hydrolysis. Subjects tested positive for heroin in all but two cases. No correlation between the doses of administered heroin and the concentrations of total opiates in hair was observed (r = 0.346). However, when considering a single analyte, it was observed that the correlation coefficient seemed to be linked to its plasma half-life. A weak correlation coefficient corresponds to a drug with a short plasma half-life, and the correlation coefficient increases when plasma half-life increases, as r = 0.12, 0.25, and 0.64 for heroin, 6-acetylmorphine, and morphine, respectively. These results suggest that using quantitative drug measurements in hair to determine the amount of drug ingested will remain inapplicable until more is known about the factors that may influence the incorporation of drugs into hair and a way to reduce the observed variability.

Nicotine monitoring in sweat with a sweat patch.

In recent years, remarkable advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional samples, such as sweat. In a study conducted with cigarettes smokers and nonsmokers, PharmChek sweat patches were applied to 29 subjects for 72 h. Nicotine was extracted in 5 ml methanol in the presence of 200 ng nicotine-d4, used as internal standard. After 20 min agitation, the methanolic solution was evaporated to dryness in the presence of 10 microl octanol to ensure nonvolatility of nicotine. Nicotine was determined using gas chromatography coupled to mass spectrometry after separation on a 30-m capillary HP5 MS column. The assay was linear in the range 50-2500 ng/patch, with an extraction recovery of 76+/-5%. Limit of detection was 10 ng/patch. Nicotine concentrations in sweat were not detected for the nonexposed nonsmokers (n = 8), 87 to 266 ng/patch for the passive smokers (n = 6) and 150 to 2498 ng/patch for the smokers (n = 15). This study demonstrated a useful application of the sweat patch for monitoring tobacco exposure.

Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry.

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepam, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n=42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n=14), flunitrazepam (19-148 pg/mg, n=31), lorazepam (31-49 pg/mg, n=4) and alprazolam (0.3-1.24 ng/mg, n=2). Bromazepam, diazepam and triazolam were not detected.

[Patient information and obtaining informed consent in laparoscopic surgery]. / L'information du patient et l'obtention du consentement en chirurgie laparoscopique.

Since the advent of laparoscopic surgery, the number of suits against surgeons has risen. One of the most frequent complaints is the lack of sufficient information. Physicians in France have a formal obligation to provide information in the contractual legal context established since 1936. This notion has been confirmed in several court cases. The requirement for patient informed consent has been confirmed by several decisions of the Appeals Court and is stated in the code of deontology. The value of classical oral information has been recently questioned in certain court cases. We analyse the current legal situation in France and try to define the content of information required in the case of laparoscopic surgery in addition to the way this information is provided and the means of obtaining informed consent. The information provided must be personalised. The patient must informed that laparoscopy remains a surgical operation. It is licit to warn the patient of predictable risks according to statistical probabilities, of the team's experience and of the patients own status including past history and psychological factors. A written statement may be prepared but must remain a document complementary to personalised oral information. The surgeon must obtain and assure good patient comprehension. The surgical community should publish risk rates in order for surgeons to have reliable references which can be used to define the notion of exceptional risk.

Influence of cholesterol derivatives on cytoskeletal organization of human carcinoma cells.

Recent developments of immunotherapeutic approaches have shown that artificial ordering of tumor cell membranes with cholesterol hemisuccinate (CHS) or 25-hydroxycholesterol (25-OH) may significantly enhance the immunogenicity of human renal adenocarcinoma cells. To gain further insight into the molecular mechanism of these sterols, we investigated cytoskeletal modification, which is related to the cell membrane. After treatment of human renal carcinoma cells with these cholesterol (at 10(-6) and 10(-7) M) for 5 days, we observed a disorganization of the submembrane end of the cytoplasmic actin stress fibers by cytofluorescence. The microtubule network was not affected. Thus, in the present study, we found that changes in membrane physicochemical properties impaired the anchorage of actin microfilaments in the plasma membrane of human renal cancer cells. Under the same experimental conditions, such modifications were not observed in normal cells (human fibroblasts) or in human hepatoma cells. We suggest that incubation of cancer cells with these sterols induced a redistribution of the cholesterol-rich membrane microdomains which are linked to the cytoskeleton through submembrane proteins.

Sex determination of forensic samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence.

The detection of restriction fragment length polymorphisms (RFLP) (1) in DNA extracted from forensic samples remains impossible in a significant number of cases due to deterioration and contamination of the biological material and the extremely low quantities of DNA isolated. The polymerase chain reaction (PCR) is a recent and particularly convenient method for analysing and typing very small amounts (10-20 ng) of degraded human DNA. DNA analysis at the level of a few cells present in forensic samples such as bloodstains, semen stains, vaginal swabs and head hair bulbs now appears possible using DNA amplification. A PCR protocol was adapted to simultaneously amplify a Y-specific DNA repeat sequence from the DYZ1 locus and an X-specific DNA repeat sequence from the DXS424 locus. The co-amplified Y-specific DNA fragment (102 bp) and X-specific DNA fragments (181-199 bp) were visualized on an ethidium bromide-stained 4% agarose gel. The male or female type of the amplified DNA extracted from blood samples, bloodstains, semen stains, vaginal swabs, brain tissue and 1, 2, 5, or 10 head hair bulbs was determined.

Detection of drugs in human hair using Abbott ADx, with confirmation by gas chromatography/mass spectrometry (GC/MS).

The authors suggest use of the fluorescence polarization immunoassay (FPIA) technique in evaluation of chronic drug abuse using human hair. Hair was decontaminated in 5 mL of ethanol for 15 min at 37 degrees C and then incubated in 3 mL of 1M sodium hydroxide (NaOH) for 1 h at 100 degrees C. Afterwards, the aliquots were neutralized and analyzed using Abbott ADx for a negative or positive response for the following drugs: benzodiazepines, barbiturates, antidepressants, opiates, cocaine, amphetamine, and cannabis. All the positive samples were confirmed by gas chromatography/mass spectrometry (GC/MS). Only one false positive was detected (caused by interference of a phenothiazine with the antidepressants kit), clearly demonstrating the capability of ADx for toxicological screening of human hair.

Evaluation of nicotine and cotinine in human hair.

To validate data on tobacco use, the authors investigated the use of hair samples for quantifying nicotine and cotinine by gas chromatography/mass spectrometry. Hair was taken from 22 nonsmokers and 42 smokers, cut close to the scalp at the back of the head. The hair (about 100 mg from each subject) was incubated in 3 mL of 1N NaOH at 100 degrees C for 1 h. After this, the samples were extracted by diethyl ether. The drugs were separated on a 12-m BP-5 capillary column and detected using selected ion monitoring (nicotine, m/z 84; cotinine, m/z 98). Hair from nonsmokers and smokers contains nicotine and cotinine. Although it is difficult to determine an absolute cutoff level, an amount greater than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from nonsmokers. In the population of nonsmokers, the influence of environmental smoke exposure was noted.

Increased immunogenicity of human renal carcinoma cells following treatment with cholesterol derivatives.

Tumor cells isolated from human renal cell carcinoma biopsies were treated with cholesteryl hemisuccinate or 25-hydroxycholesterol and the subsequent changes in their membrane fluidity and capacity to induce skin reactivity in the homologous patient were investigated. Both cholesterol derivatives were found equally efficient in decreasing membrane fluidity when measured by fluorescence polarization of diphenylhexatriene. Using trimethylammonium-diphenylhexatriene, a specific cell surface probe, 25-hydroxycholesterol, appeared much more efficient than cholesteryl hemisuccinate in inducing a membrane rigidification in the carcinoma cells. Cells treated with cholesteryl hemisuccinate induced a strong positive skin reaction compatible with delayed-type hypersensitivity in 54% of the patients, whereas 25-hydroxycholesterol-treated cells were less potent (36% positive skin reactions). Thus, manipulation of the physicochemical state of the membrane of human renal carcinoma cells could increase their immunogenicity in the autologous patient, although this seemed not to be related only to membrane rigidification.

[Stimulation of antigenicity of renal adenocarcinoma cells]. / Stimulation de l'antigénicité des cellules d'adénocarcinome rénal.

It was hypothesized that membrane rigidification of tumor cells could enhance the expression of tumor associated antigens (6) For this purpose, we treated in vitro hypernephroma cell with cholesterol hemisuccinate (CHS) or 25-hydroxysterol (250HC) and we evaluated their immunogenicity by skin-tests in 26 patients after radical nephrectomy. The skin-tests were positive in 50% cases with CHS treated cells, and in 35% with 250HC treated cells. The immune responses were characterized as delayed hypersensitivity. However determinations of all membrane fluidity suggested that membrane rigidification was not the unique factor involved. These results could therefore be of clinical interest in the treatment of renal carcinoma by active immunotherapy.