Valproic acid downregulates NF-κB p50 activity and IRAK-1 in a progressive thyroid carcinoma cell line.

Histone deacetylase inhibitor (HDACI) valproic acid (VPA) is a promising drug, currently in clinical phase 2, for the therapy of advanced/poorly differentiated thyroid cancer. The nuclear factor-κB (NF-κB) pathway is constitutively activated in most tumors, including thyroid carcinomas; this often contributes to aggressive tumor growth and therapeutic resistance. We hypothesized that VPA could be useful to decrease NF-κB activity in human thyroid cancer cells. To clarify this, we treated the highly progressive thyroid cancer cell line BHT-101 with VPA (1.0-3.0 mM) for 48 h. Real-time polymerase chain reaction (PCR) and Western blot were used to measure expression of NF-κB-regulatory genes and proteins. NF-κB p50 activity was measured using an ELISA-based colorimetric transcription factor assay kit. We found that VPA significantly and dose-dependently impaired NF-κB activity reducing DNA binding activity of NF-κB p50 subunit by 30% at 1 mM, 40% at 1.5 mM, and 70% at 3 mM. Expression of interleukin-1 receptor-associated kinase-1 (IRAK-1) protein, an upstream mediator of NF-κB activation, was reduced by Ì´30% at 1 and 1.5 mM. Furthermore, 3 mM VPA treatment significantly decreased expressions of IRAK-1, phospho-IκBα and NF-κB p50 subunit protein by Ì´ 50%. This is the first study to demonstrate that VPA decreases NF-κB activity in a progressive thyroid cancer cell line. Intriguingly, 1mM of VPA, a clinically safe dose in the therapeutic range for epilepsy, was sufficient to reduce NF-κB activity. Thus, VPA may be a promising agent to overcome chemoresistance in cancer therapy and to improve therapeutic efficiency.

[Diagnosis of pulmonary tuberculosis using Ziehl-Neelsen stain and polymerase chain reaction]. / Diagnostik der Lungentuberkulose mit Ziehl-Neelsen-Färbung und Polymerasekettenreaktion.

BACKGROUND: Definitive diagnosis of unclear pulmonary lesions is mainly based on morphological methods. In addition to a neoplasm, inflammatory reactions, in particular tuberculosis (TB), have to be considered in most cases. Therefore, the aim of this work was to determine whether established methods used in general pathology can be efficiently used with cytological material. MATERIALS AND METHODS: An established polymerase chain reaction (PCR) protocol for the detection of Mycobacterium tuberculosis complex (Mtc) DNA in fixed specimens was conducted on fixed material available as an assay for liquid-based cytology (LBC). CytoLyt®-fixed material of 45 patients with clinically suspected TB or other mycobacteriosis were selected and were initially tested cytologically. In cases of absent tumor cells, PCR for detection of Mtc DNA and Ziehl-Neelsen stain (ZN) were performed. RESULTS: In 9 patients (20 %), Mtc DNA was found by PCR. The following methods were used to obtain material: catheter biopsy (5), needle biopsy (2), transbronchial needle aspiration (1), and bronchoalveolar lavage (1). Cytologically an inflammatory reaction was observed in all cases. In 2 patients, a history of TB, in 2 further cases either silicosis or a posttransplant situation was known. In cases with a positive PCR, 7 patients (78 %) were positive in ZN and 3 patients (33.3 %) in TB culture (15.5 % vs. 6.7 % of the total cohort); however, the material used for investigation was not always from identical sources, respectively. In 36 out of 45 patients, both PCR and ZN were negative for the detection of Mtc DNA. CONCLUSION: The material intended for LBC can be used for detection of TB with ZN and Mtc PCR.

[Glandular papilloma of the right main bronchus. Detection of an exon 2 mutation of the KRAS gene (c.35G>A)]. / Glanduläres Papillom des rechten Hauptbronchus. Nachweis einer Exon-2-Mutation des KRAS-Gens (c.35G>A).

Benign epithelial tumors of the tracheobronchial system and the lungs are exceedingly rare. These entities encompass squamous and glandular papillomas (as well as their mixed forms) and adenomas (alveolar adenoma, papillary adenoma, salivary gland-like pleomorphic and mucinous adenomas and mucinous cystadenomas). These tumors are considered to be biologically benign neoplasms; however, they can pose considerable diagnostic difficulties, especially during frozen section evaluation, as they can mimic malignant tumors and in particular they can resemble well differentiated papillary adenocarcinomas. As a result of the extreme rarity of these tumors only a few descriptive diagnostic series exist and a systematic investigation including molecular data does not exist. This article presents the case of a 64-year-old patient with a glandular papilloma of the right main bronchus including the immunohistochemical and molecular work-up as well as a review of the current literature.

[MicroRNA profiles in fine needle biopsy of the thyroid]. / MicroRNA-Profile in der Feinnadelbiopsie der Schilddrüse.

MicroRNAs (miRNAs) are small (20-24 nucleotides), non-coding ribonucleid acids, which regulate gene expression on the post-transcriptional level, thus influencing physiological processes including cellular growth, differentiation and apoptosis. Several miRNAs (e. g. miRNAs 146b, 221 and 222) have been shown to be consistently over-expressed in papillary thyroid carcinoma. The present overview describes and discusses the utilization and problems of miRNA analysis in material from thyroid nodules obtained by fine needle biopsy. Particularly the analysis of defined sets of miRNAs should improve the diagnostic value of this procedure and contribute to a better management of patients with cold thyroid nodules.

Differential miRNA expression profiles in variants of papillary thyroid carcinoma and encapsulated follicular thyroid tumours.

BACKGROUND: Recent studies showed a significant upregulation of distinct microRNAs (miRNAs) in papillary thyroid carcinoma (PTC). The objective of this study was to explore whether this upregulation could also be assigned to distinct histomorphological variants of PTC, especially the follicular variant and other encapsulated follicular thyroid tumours. METHODS: We used total RNA of 113 formalin-fixed paraffin-embedded tissues of 50 PTCs ((10 conventional type (PTC-CT), 10 tall cell variants (PTC-TCVs), 30 follicular variants (PTC-FVs)), 10 follicular adenomas (FAs), 10 multinodular goitres (MNGs), 21 follicular thyroid carcinomas and 22 well-differentiated tumours of unknown malignant potential (WDT-UMP) to analyse the miRNA expression pattern of five selected miRNAs (146b, 181b, 21, 221 and 222) using RT-PCR TaqMan miRNA assay to explore the diagnostic utility of this method. RESULTS: The mean values of the expression pattern of all miRNAS in PTCs show a statistically significant difference from those in MNG and FA with fold changes up to 90 for miRNA 146b, P<0.001. No differences in expression pattern could be showed between MNG and FA. The PTC-FVs differ significantly from FA in all five miRNAS, from MNG in three and from WDT-UMP in one miRNA with fold changes between 1.7 and 21.2, but failed to be of diagnostic value regarding individual cases with substantial overlaps. CONCLUSION: We conclude that analysis of a set of five selected miRNAS distinguish common variants of PTC from FA/MNG but failed to be a useful diagnostic method in individual and doubtful cases, especially in the differential diagnosis of encapsulated follicular thyroid tumours.

Lack of correlation between BRAF V600E mutational status and the expression profile of a distinct set of miRNAs in papillary thyroid carcinoma.

Recent studies demonstrated a significant upregulation of distinct microRNAs (miRNAs), small endogenous RNAs that regulate gene expression, in papillary thyroid carcinoma (PTC). In the pathogenesis of PTC the T1799A (V600E) BRAF mutation is the most common genetic alteration leading to a constitutive activation of the MAPK pathway. The aim of the present study was to elucidate a possible correlation between BRAF mutational status and a distinct miRNA expression profile. In a series of 221 PTC we determined the BRAF V600E mutational status using DNA-sequencing and correlated the occurrence of the mutation with a variety of clinicopathologcial data. The miRNA expression profile of five selected subtypes (miRNA-146b, -181b, -21, -221, -222) in two matched cohorts of BRAF positive (n=28) and wildtype cases (n=26) was examined by RT-PCR TaqMan miRNA assay. The BRAF V600E mutation was significantly found in PTCs with extrathyroidal extension (p <0.001). Among them, V600E was even significantly associated with smaller tumour size of 1 cm or less (microcarcinomas; p<0.003) and the follicular (p=0.017) and tall cell variant (p=0.015). By calculating relative changes in miRNA gene expression no differences in fold changes could be detected between BRAF positive and wildtype PTC suggesting that BRAF has no regulatory influence on the expression of the five examined miRNAs. However, our study confirmed the diagnostic utility of this distinct set of miRNAs to detect PTC by significant fold changes in at least 3 miRNAs (miRNA-146b, -221, -222) irrespective of its histological variant.

Analysis of deregulated miRNAs is helpful to distinguish poorly differentiated thyroid carcinoma from papillary thyroid carcinoma.

Poorly differentiated thyroid carcinoma (PDTC) is defined as a malignant follicular cell derived neoplasm, both morphologically and biologically intermediate between well differentiated and anaplastic thyroid carcinoma (ATC). In the present study we investigated the expression levels of two distinct sets of miRNAs ('set 1': miRNA-146b, -181b, -21, -221, -222, all shown to be significantly upregulated in papillary thyroid carcinoma [PTC]; 'set 2': miRNA-30d, -125b, -26a, -30a-5p, and let7c, all downregulated in ATC) in a series of 15 PDTC (including 3 mixed PDTC/PTC), 9 'pure' PTC, and 9 ATC. Compared to normal thyroid tissue all 'set 1' miRNAs were significantly upregulated in PTC (p<0.001); in ATC 4/5 miRNAs were upregulated (p<0.001) whereas in PDTC the expression levels of all 5 miRNAs did not differ significantly from normal thyroid. All miRNAs of 'set 2' were significantly upregulated in PTC (p<0.004) and downregulated in ATC (p<0.03); in PDTC only 3/5 were downregulated (p<0.011). All 10 miRNAs investigated differed significantly (p<0.003) between PTC and PDTC. In the histologically differentiated PTC compound of mixed PDTC/PTC cases, however, miRNA expression levels of all 10 miRNAs investigated lacked significant difference from those found in the PDTC compound, whereas 6/10 miRNAs differed significantly from 'pure' PTC. Our results indicate that analysis of distinct sets of miRNAs represent useful tools to distinguish PDTC from 'pure' PTC. Additionally our findings suggest that lack of deregulation of some miRNAs may select a subset of PTC prone to progression to PDTC.

Activation of extracellular regulated kinases (ERK1/2) but not AKT predicts poor prognosis in colorectal carcinoma and is associated with k-ras mutations.

Signal transduction and modulation represent central mechanisms in cellular processes such as cell-cycle regulation, oncogenesis, and apoptosis. The aim of this study was to determine the prognostic relevance of two kinases important in the regulation of cell proliferation and apoptosis in 135 colorectal cancer cases: AKT and extracellular regulated kinases (ERK1/2). We investigated the relationship of phospho-ERK1/2 (pERK1/2) and phospho-AKT (pAKT) with associated parameters (EGFR, COX-2, cyclin-D1), proliferative activity (Ki-67), and apoptosis (TUNEL) using immunohistochemistry. Additionally, the k-ras gene was screened for mutations to determine its putative association with ERK1/2 activation. Activation of ERK1/2 but not AKT correlated statistically with the presence of k-ras mutations (P = 0.015). Survival analysis of phospho-ERK1/2 immunoexpression showed a significant correlation with decreased overall survival (OS). The multivariate Cox regression analysis identified pERK1/2 as an independent prognostic parameter (P = 0.005). Activation of ERK1/2 in colorectal cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor. Furthermore, our data suggest that mutations of the k-ras oncogene may induce activation of ERK1/2. We propose immunohistochemical determination of pERK1/2 status as a promising candidate for the identification of high-risk patients who would benefit from new anticancer drugs targeting the ERK pathway.

ETV6-NTRK3 gene fusion in a secretory carcinoma of the breast of a male-to-female transsexual.

Secretory carcinomas of the breast were first described as "juvenile carcinoma" by McDivitt and Stewart in a cohort of children. This term has been replaced by the term "secretory breast carcinoma", because the entity can occur at any time of life. Carcinoma of the male breast is uncommon and accounts for approximately 1% of all cancers in men. Recently, it has been reported that human secretory breast carcinoma expresses the ETV6-NTRK3 gene fusion that was previously cloned in pediatric mesenchymal cancers. We present the case of a 46-year-old male-to-female transsexual in whom a secretory breast carcinoma was an incidental finding. As confirmation of the histopathological diagnosis we detected the novel ETV6-NTRK3 gene fusion in this tumor.

HER2 expression and markers of phosphoinositide-3-kinase pathway activation define a favorable subgroup of metastatic pulmonary adenocarcinomas.

OBJECTIVES: Pulmonary adenocarcinomas (ADC) can be sub-grouped based on dominant oncogenic drivers. EGFR mutations define an entity of metastatic ADC with favorable prognosis and high susceptibility to EGFR tyrosine kinase inhibition. In contrast, the clinical impact of additional ERBB family members in ADC is less defined. To this end we prospectively studied HER2 expression, gene amplification, and mutation in relation to outcome of patients with advanced or metastatic ADC. MATERIALS AND METHODS: Diagnostic tumor biopsies from 193 sequential patients with stage III/IV ADC were prospectively studied for HER2 expression by immunohistochemistry (IHC). Cases with IHC scores 2+ or 3+ were analyzed by HER2 chromogenic in situ hybridization (CISH), and sequencing of HER2 exons 20 and 23. Additional prospectively determined biomarkers included PTEN, cMET, pAKT, and pERK expression, KRAS, EGFR, BRAF and PIK3CA mutations, and ALK fluorescence ISH (FISH). RESULTS AND CONCLUSION: HER2-IHC was feasible in 176 (91.2%) cases. Of 53 (30%) cases with IHC scores 2+/3+, 45 (85%) could be studied by CISH and 34 (64%) by sequencing. The lower number of HER2-mutational analyses resulted from exhaustion of tumor tissue and DNA following mutational analysis of KRAS, EGFR, BRAF and PIK3CA. HER2 amplification was detected in 4 cases (2.3%), while no mutation was found. HER2 expression correlated with expression of pAKT and cMET. Expression of HER2 and pAKT was associated with favorable overall survival in stage IV disease. HER2-expressing ADC more frequently harbored KRAS mutations, while HER2 expression was absent in all 4 cases with BRAF mutation. HER2-IHC was not predictive of HER2 gene amplification or mutation, which both were rare events in prospectively studied patients with advanced or metastatic ADC. Expression of HER2 and pAKT define a population of patients with stage IV ADC with a distinct disease course, who could benefit from specifically tailored pharmacotherapies.

Reverse transcriptase polymerase chain reaction as a reliable method to detect epidermal growth factor receptor exon 2-7 gene deletion in human glioblastomas.

Epidermal growth factor receptor (EGFR) gene amplification has been reported to occur in diverse carcinoma types such as lung, ovarian, and breast carcinomas and in glioblastomas. A 801-bp in-frame deletion close to the aminoterminus of the receptor protein has been found to occur more or less frequently within at least three of these tumor entities. We studied EGFR gene alterations using the polymerase chain reaction and EGFR gene expression of 65 astrocytic tumors (51 glioblastomas World Health Organization [WHO] IV, five anaplastic astrocytomas WHO III, and nine astrocytomas WHO II). EGFR gene amplification, as determined by Southern blotting using a full-length cDNA probe, was observed in 22 of 51 glioblastomas (43%) but in none of the grade II astrocytomas. Two of five anaplastic astrocytomas at WHO III showed a considerable degree of EGFR amplification but, according to the neuroradiological data, these two tumors had to be considered as glioblastomas. The most frequently found genetic alteration was the 801-bp deletion near the receptor aminoterminus comprising a complete loss of exon 2 to exon 7 (del2-7). We showed that RT-PCR is superior to Southern blot analysis in detection of this type of deletion and can be assigned to 9 of 38 (24%) glioblastomas examined. Expression of a EGF receptor protein was enhanced in most of the tumors with gene amplification. However, 5 of 18 tumors that express a receptor protein in the absence of EGFR gene amplification also showed elevated levels of EGFR gene expression. In addition to the full-length receptor protein, a signal in the 140-kDa range was observed in 17 of 35 glioblastomas (49%). This fragment may correspond to the truncated de12-7 receptor protein or might be due to proteolysis of the full-length receptor protein.

Primary structure of locust opsins: a speculative model which may account for ultraviolet wavelength light detection.

The sequences of two locust opsins have been determined by dideoxy nucleotide sequencing of PCR products from cDNA derived from eyecup tissue. The opsins (Lo1 and Lo2) are encoded by 381 and 380 amino acid residues, respectively, with hydropathy profiles and placement of key amino acid residues suggestive of a typical seven-transmembrane rhodopsin structure. The sequence alignment of Lo1 reveals significant homology to mantid opsin. These opsins contain retinal as their visual chromophore and have similarity to the Rh1 type sequences from Drosophila and Calliphora which use 3-hydroxy retinal. Lo2 is most closely related to the Rh3/4 type of visual pigments from Drosophila. The retinal-based opsins show reduced numbers of charged amino acids in the loop region connecting transmembrane segments V and VI compared to the 3-hydroxy retinal opsins. Sequence alignment of all the known insect visual pigments has shown that only those with maximal sensitivity in the blue/UV spectral range, Lo2 and the Rh3/4 opsins of Drosophila, have three charged amino acids in transmembrane segments II, IV and VII. The charged residue in transmembrane VII is two helical turns away from the positively charged Schiff base and could act directly as a counterion to it. From the secondary structure analysis of opsin, the two charged residues in transmembrane II and IV would be in close proximity to form a dipole. These polar motifs in Lo2 and Rh3/Rh4 could act in wavelength modulation of short wavelength sensitive pigments and substantiate the proposed external two-point charge model which accounts for the spectral sensitivity of visual pigments [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A. and Motto, M. (1979). Journal of the American Chemical Society, 101, 7084-7086].

Radiosensitization and radioprotection of E. coli by alcohols.

The survival of E. coli K12 strain AB1157 and the isogenic repair-deficient mutant E. coli AB2480 (recA13, uvrA6) was measured after gamma-irradiation in the presence of various alcohols as well as after incubation and subsequent removal of the alcohols before irradiation. Irradiation in the presence of alcohols leads to the already known protection effect, which has been attributed to OH radical scavenging. However, it was not possible to explain the protection solely in terms of the reactivity of the OH radical with the various alcohols, because addition of some of the alcohols did not yield the expected survival values. It was found that incubation with and subsequent removal of various alcohols before irradiation led to radiosensitization. The degree of radiosensitization increases with the hydrophobicity of the alcohol. In the case of glycerol no radiosensitization was observed. We can conclude that alcohols protect predominantly by OH radical scavenging. The comparatively small protection of cell survival by the more hydrophobic alcohols can be attributed to the sensitizing effect of these alcohols.

Double-strand breaks in plasmid DNA and the induction of deletions.

Double-strand breaks (dsbs) have been produced in plasmid DNA by various restriction endonucleases and the survival and the deletion mutation incidence have been measured in E. coli. The deletion formation is known to depend upon the occurrence of short direct repeats within the DNA molecule. In order to study the role of these repeats we constructed plasmid molecules with repeats of various lengths or with a 10-base pair repeat at different distances from each other. Furthermore the influence of the location and the structure of the dsb was studied. Repair and deletion frequencies of the linearized plasmids were measured after transformation of E. coli. The yield of the specific deletion mutation (the one which occurs between the introduced repeats) increases nearly linearly with the square of the length of the repeat, while the yield of the correctly repaired DNA and the yield of all other deletion mutants remained constant. The slope of the linear increase of the yield of the specific deletion depends on the location and the structure of the dsb. The yield of the specific deletion mutation decreases with increasing distance between the repeats. A proposal for the rate-determining step of the deletion formation is made.

Chromophore-protein interaction controls the complexity of the phytochrome photocycle.

A new protocol for the preparation of recombinant phytochromes results in significantly higher yields which, for the first time, have made kinetic studies possible. Flash photolysis with nanosecond laser excitation reveals that, in recombinant and native phytochromes, the decay kinetics of the primary photoproducts I700i and the kinetics of the formation of the Pfr form are similar. Phycocyanobilin-containing recombinant phytochrome, however, shows only a monoexponential decay of the I700 intermediate with a time constant of approximately 90 microseconds, and a biexponential formation of the Pfr form, albeit with time constants (approximately 13 and 100 ms) somewhat shorter than those from native phytochrome. Thus the seemingly small structural modification of the chromophore (substitution of the native vinyl for an ethyl group) has a profound influence on the availability of protein conformational rearrangement pathways. The result is therefore of general interest in chromoprotein dynamics.

Deaths among drug addicts in Denmark in 1987-1991.

In the period 1987-1991 a total of 739 fatalities among drug addicts was investigated at the three University Institutes of Forensic Medicine in Denmark. The annual number rose from 130-140 in the first 4 years to 192 in 1991, and 80% were males. The mean and median age for both males and females increased by 1 year in the period. The main drug of abuse was heroin, in most cases supplemented by various other drugs, and in almost all cases taken intravenously. In about one-third of the cases each year there was information of abuse of alcohol in addition. In the poisoning cases, the main drug of poisoning was morphine/heroin, constituting 35-55% of the cases each year. As regards methadone-poisoning cases, the number increased significantly in 1991 compared to the first 4 years. Furthermore, the number and proportion of addicts dying while in methadone treatment increased during the 5-year period. In about half of the methadone poisoning cases, there was information of methadone treatment at the time of death. The other half obviously obtained the methadone completely illegally. Ketobemidone was the third most frequent drug of poisoning, while propoxyphene and barbituric acid only were found in a very few cases each. The results are compared to those from an earlier investigation concerning drug deaths in Denmark in 1968-1986. The importance of registering drug deaths is emphasized.

Development of the KVL segregating inbred strain of the Syrian hamster (Mesocricetus auratus).

A new colony of a single segregating inbred strain of the Syrian hamster (Mesocricetus auratus), designated KVL, has recently been developed at the Royal Veterinary and Agricultural University, Copenhagen, Denmark. The segregation occurs with respect to coat colour (pied gray/pied brown) as a result of inbreeding with forced heterozygosity. After 20 generations average litter size was 6.7 at birth and 5.57 at weaning. Mean generation interval was 89.4 days. An initially significant decrease in productivity (inbreeding depression) levelled out with further generations and has now stabilized. The colony is being maintained.

Pregnancy-associated murine protein-1 plasma levels during oestrus cycle, pseudopregnancy and pregnancy in the mouse.

A cyclic variation in plasma levels of pregnancy-associated murine protein-1 (PAMP-1) during the oestrus cycle in outbred Pan: Thei mice was recorded. PAMP-1 plasma levels were significantly elevated in dioestrus as compared with the three other stages of the murine oestrus cycle. Until day 7 of gestation the PAMP-1 plasma levels remained low, and no significant differences could be observed between pregnant and pseudopregnant female mice. The PAMP-1 levels increased markedly in the circulation on day 8 of pregnancy, and continued to increase until peak values were reached at day 11 of pregnancy. In the latter half of pregnancy the PAMP-1 levels declined until day 17 of pregnancy, at which stage the normal non-pregnant values were recorded.

Induction of pregnancy-associated murine protein-1 in dwarf mice by human growth hormone.

Pregnancy-associated murine protein-1 (PAMP-1) could not be detected in peripheral blood of female dwarf mice (genotype dw/dw of the DW strain). By contrast the normal size females of the DW strain (genotypes +/+ and +/dw) had PAMP-1 serum levels of 18.9 AU +/- 15.7 AU/ml. Following administration of biosynthetic human growth hormone (hGH) every 2 h for 52 h PAMP-1 was detected in all dwarf females at concentrations of 16.0 AU +/- 3.3 AU/ml. The albumin levels in the circulation of DW females of normal size were significantly higher (P less than 0.05) than those of DW dwarfs, and the hGH administration did not change the serum albumin levels. The present experiment adds weight to the suggestion that the PAMP-1 serum level is regulated by GH.

Prehospital treatment of patients with i.v. heroin overdose: what are we treating?

OBJECTIVE: To measure blood levels of morphine and additional drugs in patients suspected of intravenous (i.v.) heroin abuse and to evaluate the effects of antidote treatment. DESIGN: Prehospital blood sampling in 52 patients. RESULTS: Forty-five patients were blood-positive for heroin, eight of whom were hospitalized. Forty-one patients also had abused additional drugs: minor tranquilizers, ethanol, amphetamine, cocaine, and/or carbamazepine. Seven patients had taken either only methadone or ketobemidione: one was admitted. Treatment with increasing doses of naloxone indicated a necessity for hospitalization. Six of 14 patients treated with naloxone (1.8 mg were hospitalized. Seven patients had an extremely high blood level of morphine (0.2 mg/kg), that could be reverted with naloxone in moderate doses. CONCLUSION: This study indicates that under prehospital conditions, it is difficult to identify a patient intoxicated only with intravenous heroin. Nearly all patients treated were cases of multiple drug/alcohol overdoses. Even the symptoms associated with extremely high blood levels of morphine could be reversed with naloxone in moderate doses.