Severe poisoning after self-reported use of 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine, a novel substituted amphetamine: a case series.

Significant toxicity from amphetamine and cathinone derivatives is being increasingly reported. We describe a series of self-reported exposures to 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25-I-NBOME or 25-I), a novel amphetamine derivative. Ten patients with an average age of 17 years presented to local emergency departments (EDs) in our community after ingestion and/or insufflation of a drug referred to as "25-I." Of 10 patients, 6 reported taking 25-I alone; other substances included ethanol; 2,5-dimethoxy-4-ethylphenethylamine; marijuana; and ketamine. Most common effects included tachycardia (90%), hypertension (70%), agitation (60%), and hallucinations (50%). The average heart rate was 123 beats per minute. Two patients were found in status epilepticus, and another was found unresponsive. One patient who had a seizure had multiple, discrete intraparenchymal hemorrhages and acute kidney injury. Six patients were admitted to the intensive care unit, two were treated in the ED and released, and 1 each was admitted to psychiatry or managed in a clinical decision unit and subsequently discharged. Three patients required emergent intubation, and all admitted patients (7/10) were given intravenous benzodiazepines for sedation. Urine and blood specimens were obtained from 1 patient, which showed analytic confirmation of 25-I. In addition to sympathomimetic effects, methoxy and other substituent groups impart serotonergic effects, resulting in hallucinogenic properties. 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine appears to be extremely potent with a reported "dose" of 500 µg resulting in increased potential for inadvertent overdose. This case series describes significant morbidity in a local cluster of young patients after self-reported use of 25-I, a newly identified drug of abuse.

A novel method for direct measurement of complement convertases activity in human serum.

Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.

Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior.

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Recommended guidelines for submission, trimming, margin evaluation, and reporting of tumor biopsy specimens in veterinary surgical pathology.

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Correlation of serum cardiac troponin I and myocardial damage in cattle with monensin toxicosis.

BACKGROUND: Cardiac troponin I (cTnI) is used as a biomarker of myocardial injury in people and small animals. Little is known about the diagnostic use of cTnI in cattle. HYPOTHESIS: Serum cTnI correlates to myocardial function and histopathologic lesions in cattle with monensin-induced myocardial injury. ANIMALS: Ten healthy cows. METHODS: Experimental study. Animals received 1 dose of monensin PO; 30 mg/kg (n = 1) or 40 mg/kg (n = 1) (Group A) or 50 mg/kg monensin (n = 8) (Group B) of body weight. Repeated measurements were performed of serum cTnI, biochemistry, and ECG and echocardiography until study termination at 80 (Group A) and 144 hours (Group B) after dosing. Semiquantitative histopathologic examinations of the heart were performed in each cow. A scoring system with regard to the magnitude of myocardial injury was established and a total heart score was compared with maximum cTnI concentration measured after monensin administration. Five hearts from healthy cows served as controls. RESULTS: Increased cTnI (>0.07 ng/mL) was found in 9/10 cows. cTnI was significantly associated with left ventricular shortening fraction (r(2)= 0.51; P= .02) and myocardial histopathologic lesion score (r(2)= 0.49; P= .021). All cows (n = 7) with evidence of myocardial necrosis had a cTnI concentration > or = 1.04 ng/mL. CONCLUSION AND CLINICAL IMPORTANCE: cTnI is related to myocardial necrosis and severity of myocardial damage in cattle with monensin toxicosis. cTnI could become a useful diagnostic tool in the noninvasive assessment of myocardial injury in cattle with naturally occurring cardiac disease.

Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine.

Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death.

Alcohol dehydrogenase 2 is a major hepatic enzyme for human retinol metabolism.

The metabolism of all-trans- and 9-cis-retinol/ retinaldehyde has been investigated with focus on the activities of human, mouse and rat alcohol dehydrogenase 2 (ADH2), an intriguing enzyme with apparently different functions in human and rodents. Kinetic constants were determined with an HPLC method and a structural approach was implemented by in silico substrate dockings. For human ADH2, the determined K(m) values ranged from 0.05 to 0.3 microM and k(cat) values from 2.3 to 17.6 min(-1), while the catalytic efficiency for 9-cis-retinol showed the highest value for any substrate. In contrast, poor activities were detected for the rodent enzymes. A mouse ADH2 mutant (ADH2Pro47His) was studied that resembles the human ADH2 setup. This mutation increased the retinoid activity up to 100-fold. The K(m) values of human ADH2 are the lowest among all known human retinol dehydrogenases, which clearly support a role in hepatic retinol oxidation at physiological concentrations.

Polychlorinated biphenyl concentrations, congener profiles, and ratios in the fat tissue, eggs, and plasma of snapping turtles (Chelydra s. serpentina) from the Ohio Basin of Lake Erie, USA.

Concentrations and profiles of polychlorinated biphenyls (PCBs) were determined in three tissues of adult snapping turtles (Chelydra serpentina serpentina) from six locations in the Ohio Basin of Lake Erie to characterize tissue variation and geographic trends. The locations included the Ohio Areas of Concern, i.e., the Ashtabula, Black, and Maumee Rivers; the Ottawa River near Toledo; and two reference sites. Mean total PCBs were greatest in turtles from the Ottawa River followed by the Maumee, Ashtabula, and Black Rivers. All three types of samples-fat tissue (FT), eggs, and plasma-showed the same geographic trend in PCB levels. On a wet-weight basis, mean concentrations ranged from 2,148 to 18,669 ng/g in FT, from 183 to 3,683 ng/g in eggs, and from 18 to 201 ng/g in plasma. Across all sites, total PCB concentrations between the tissues were significantly correlated (0.001 < p < 0.005; Pearson correlation coefficient (r ( P )) was between 0.720 and 0.954). Two distinctly different profiles with respect to relative congener and homologue concentrations were found among the sites. One that included four of the six sites examined was characterized by hexa-chlorobiphenyl (hexa-CB) dominance followed by hepta-CBs, with PCBs no. 138 + 163, 153 + 132 + 105, and 180 being the most abundant congeners. The second profile, specific for turtles from the Ottawa River, was different from the first in that tetra-CBs were the most abundant congeners followed by hexa-CBs. Analysis of variance (ANOVA) indicated significant intertissue differences in the PCB homologue profiles, i.e., FT had a higher percentage of hepta-, octa-, and nona-CBs compared with eggs and plasma, whereas eggs showed a higher percentage of hexa-CBs. At any listed location, FT, eggs, and plasma had the same congener profile. An intertissue distribution of lipid-normalized individual congener concentrations examined by regression analyses revealed significant egg-FT, egg-plasma, and FT-plasma relations for >40 congeners (0.001 < p < 0.05). The distribution ratios determined for these congeners from the slope of the regression lines averaged 1.235 +/- 0.279, 0.430 +/- 0.170, and 0.387 +/- 0.115, respectively. The plasma wet weight-FT lipid-normalized concentration ratios for these congeners averaged 0.012 +/- 0.006. Both egg-FT and plasma wet weight-FT lipid-normalized ratios regressed against log K(ow) showed significant decreases, with increasing log K(ow), indicating greater accumulation of highly chlorinated congeners in FT than in other compartments. The estimated 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents ranged from 0.007 ng/g at reference sites to 0.060 ng/g at contaminated sites and from 0.099 to 1.992 ng/g in plasma and eggs, respectively. In both plasma and eggs, coplanar-CBs were the major contributors to total toxic equivalents (TEQs). Eggs from all contaminated sites had TEQs that exceeded the lowest observed effect level TEQs proposed for bald eagle chicks, in addition to high SigmaPCB levels at some of these sites, especially the Ottawa and Maumee River sites, indicate potentially increased risk to turtles and possibly other wildlife species inhabiting these ecosystems. Significant correlations of total PCBs and individual congeners between FT, eggs, and plasma indicate that blood sampling can provide a good nonlethal measure of PCB exposure and can be used to monitor environmental contamination.

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2.

The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.

A mixture of cottonseed meal, soybean meal and animal byproduct mixture as a fish meal substitute: growth and tissue gossypol enantiomer in juvenile rainbow trout (Oncorhynchus mykiss).

Diets incorporating three different sources of extracted cottonseed meal (CM), soybean meal and an animal protein mixture were evaluated for juvenile rainbow trout. Fish averaging 0.96 g were divided into groups of 30; 3 groups per treatment, and each group was fed one of four diets for a 16-week period. Fish meal (FM) was replaced on a 25% protein basis by each of three different sources of CM from California (CA), Tennessee (TN), and Arkansas (AR), U.S.A. In the three CM-containing diets another 25% soybean meal protein and 50% animal protein mixture were also incorporated to completely replace FM protein. The results of growth rate and feed utilization showed that FM could be entirely replaced by a mixture of plant proteins (CM and soybean meal) and animal by-product proteins. Hematocrit levels were significantly lower in the group fed CM-containing diets than in the control. The findings suggest that CM can be used as a good protein source by the incorporation of at least 15% in diets (25% of fish meal protein replacement), and that the nutritive values of CM in juvenile trout can be different due to their different origin. Significantly higher concentrations of total gossypol were found in faeces of CM-TN (5.8 +/- 0.4 micromol/g) and CM-AR (5.6 +/- 0.6) groups than in that of CM-CA (3.7 +/- 0.4) group. It was documented that gossypol enantiomers, present in an equal proportion in diets, selectively accumulated in liver and bile, whereas equal proportions of (+)- and (-)-enantiomers were found in whole-body and faeces. Depending on CM source, fish can absorb approximately 35-50% of dietary gossypol, and the majority of the absorbed gossypol seemed to be excreted.

Functional polymorphism in the alcohol dehydrogenase 3 (ADH3) promoter.

The ADH3 gene encodes alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase, the ancestral and most conserved form of alcohol dehydrogenase. ADH3 is expressed in all tissues examined and the enzyme is essential for formaldehyde scavenging. We have screened the promoter region including exon 1 and exons 5, 6 and 7 of the ADH3 gene for allelic variants. Using 80 samples of genomic DNA from Swedes as template, the various parts of the gene were PCR amplified and subsequently analyzed on single strand conformation polymorphism (SSCP) gels. No abnormal migration patterns could be detected by SSCP analysis of exons 5, 6 and 7 while for the promoter region, a large number of the samples displayed differences in SSCP gel migration patterns. Cloning and sequence analysis revealed four possible base pair exchanges in the promoter region. Two transitions were found at position -197 and -196, GG --> AA, one at position -79, G --> A and finally, close to the transcription start site, a fourth transition was found at position +9, C --> T. An allele specific PCR method was developed and allele frequencies were determined in three populations: Chinese, Spanish and Swedish. GG-197,-196 and AA-197,-196 alleles were common in all three populations, G-79 and A-79 were common in Swedes and Spaniards but only A-79 was found among Chinese. T+9 was the most rare allele with an allele frequency of 1.5% in Swedes. Finally, promoter activity assessments and electrophoretic mobility shift assays demonstrated that the C+9 --> T+9 exchange resulted in a significant transcriptional decrease in HeLa cells and a decreased binding of nuclear proteins. These base pair exchanges may have an effect on the expression of the enzyme and thereby influence the capacity of certain individuals to metabolize formaldehyde.

Cutaneous neosporosis in two adult dogs on chronic immunosuppressive therapy.

Antemortem diagnosis of generalized ulcerative and pyogranulomatous dermatitis with numerous intralesional tachyzoites was made from skin biopsy specimens from 2 adult dogs on chronic immunosuppressive therapy. A 9-year-old Italian Greyhound was on long-term corticosteroid therapy for the treatment of a lupus-like systemic autoimmune disorder, and a 7-year-old Labrador Retriever had received several months of chemotherapy for lymphosarcoma. The tachyzoites were identified as Neospora caninum by immunoperoxidase immunohistochemistry. Both dogs were treated with clindamycin. Lesions in the Greyhound resolved; however, the Labrador Retriever was euthanized because of evidence of neuromuscular disease, despite improvement of the skin lesions. These 2 cases indicate that cutaneous neosporosis can occur in adult dogs on chronic immunosuppressive therapy. The disease may result from reactivation of a congenital infection and/or a recently acquired primary infection.

Class II alcohol dehydrogenase (ADH2)--adding the structure.

Class II alcohol dehydrogenase (ADH2) represents a highly divergent class of alcohol dehydrogenases predominantly found in liver. Several species variants of ADH2 have been described, and the rodent enzymes form a functionally distinct subgroup with interesting catalytic properties. First, as compared with other ADHs, the catalytic efficiency is low for this subgroup. Second, the substrate repertoire is unique, e.g. rodent ADH2s are not saturated with ethanol as substrate, and while omega-hydroxy fatty acids are common substrates for the human ADH1-ADH4 isoenzymes, including ADH2, these compounds function as inhibitors rather than substrates. The recently determined structure of mouse ADH2 reveals a novel substrate-pocket topography that accounts for the observed substrate specificity and may, therefore, be important for the exploration of orphan substrates of ADH2. It is possible to improve the catalytic efficiency of mouse ADH2 by an array of mutations at position 47. Residue Pro47 of the wild type ADH2 enzyme seems to strain the binding of coenzyme, which prevents a close approach between the coenzyme and substrate for efficient hydrogen transfer. Based on crystallographic and mechanistic investigations, the effects of residue replacements at position 47 are multiple, affecting the distance for hydride transfer, the pK(a) of the bound alcohol substrate as well as the affinity for coenzyme.

Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6.

Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5'-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma.

Mammalian alcohol dehydrogenase - functional and structural implications.

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1-ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system.

Human class V alcohol dehydrogenase (ADH5): A complex transcription unit generates C-terminal multiplicity.

The human ADH5 gene was reported to lack the last exon compared to other mammalian ADHs and consequently should be expressed as a truncated protein. Here we show with PCR amplification of 3'-cDNA ends that the ADH5 gene harbors the "missing" exon. Besides a cDNA identical to the published sequence, we found full-length transcripts that contained additional codons for eight amino acid residues. Northern blot analysis established the full-length variant as the major transcript with the strongest signal from adult liver. Sequence analysis of genomic DNA confirmed that the ADH5 gene displays composite internal/terminal exons, which can be differentially processed; i.e., 3'-end generation is a result of competition between polyadenylation and splicing.

Transfer of the feline erythropoietin gene to cats using a recombinant adeno-associated virus vector.

Chronic renal failure and the associated erythropoietin-responsive anemia afflicts over 2 million domestic cats in the United States, resulting in morbidity that can affect the owner-pet relationship. Although treatment of cats with recombinant human erythropoietin (Epo) protein can be effective, response to the drug often dissipates over time, probably due to the development of antibodies reactive with the human protein. As an alternate approach to the treatment of this disease, we have developed a recombinant adeno-associated virus vector containing the feline erythropoietin gene (rAAV/feEpo). This vector, when administered intramuscularly to normal healthy cats, caused a dose-related increase in hematocrit over a 7-week period after injection. Thus, the rAAV/feEpo vector holds promise as a simple, safe and effective therapy for the anemia of chronic renal failure in domestic cats.