Direct correlation between diffusion of Loxosceles reclusa venom and extent of dermal inflammation.

OBJECTIVES: Envenomation by Loxosceles species (brown recluse) spiders results in large dermal inflammatory lesions. Venom-induced dermal inflammation occurs indirectly via soluble mediators of inflammation. This study aimed to explore whether the anatomic extent of dermonecrotic arachnidism is due to the cascade of soluble proinflammatory mediators elicited by venom deposited at the bite site, or due to diffusion of the venom per se. METHODS: Three New Zealand white rabbits received intradermal L. reclusa venom (3-microg) injections in the flank. At the time of maximum dermal inflammation (24 hr), paired 4-mm dermal biopsies were obtained in 2-cm intervals extending 0 to 12 cm from the inoculation site. Normal dermal tissue was obtained from the opposite flank to serve as a negative control. One biopsy sample from each interval was homogenized and assayed for myeloperoxidase (MPO) activity and for the presence of venom via an enzyme immunoassay (EIA). The other paired dermal biopsy was sectioned, and examined for the presence of polymorphonuclear neutrophils (PMNs) by microscopy. Lesional areas were measured using digital images imported into imaging software. RESULTS: Mean +/- SD lesional diameter 24 hours post inoculation measured 9.18 +/- 0.64 cm. Venom was detected in biopsies 0 to 10 cm from the injection site. As expected, the highest venom concentrations were measured at the inoculation site (4.28 +/- 3.9 ng/4 mm). In addition, PMNs and MPO were detected up to 8 and 10 cm from the inoculation site, respectively. Neither PMNs nor MPO was detected in tissue absent of venom (kappa = 0.88, p < 0.001). CONCLUSIONS: Loxosceles venom diffuses from the envenomation site. The extent of dermal inflammation mirrors the extent of Loxosceles venom diffusion. This observation implies that the venom itself defines the extent and magnitude of tissue injury following Loxosceles envenomation.

Antigenic cross-reactivity of venoms from medically important North American Loxosceles spider species.

We characterized the antigenic cross-reactivity of two medically important North American Loxoxceles species: L. reclusa (native to southeastern US) and L. deserta (native to southwestern US). Dermonecrosis resulting from bites from these two North American spider species are indistinguishable clinically. Polyclonal IgG antivenins directed against L. reclusa and L. deserta were raised in rabbits and used to develop specific enzyme immunoassays (EIAs). Antigenic differences in the two venoms were evaluated as follows: (1) Comparison of the sensitivities and correlation coefficient (R(2)) of anti-L. reclusa (alpha LoxR) and anti-L. deserta antibodies (alpha LoxD) in the detection of varying concentrations of the two venoms; (2) separation and western blot comparison of venom components; (3) protein sequence analysis of L. desertavenom and comparison to the L. reclusa protein sequence analysis present in a US national database; and (4) in vivo evaluation of alpha LoxR and alpha LoxD antivenins in attenuating dermal lesions (rabbit model). Correlation coefficients for alpha LoxR (R(2)=0.99) and alpha LoxD (R(2)=0.99) polyclonal antibodies in the measurements of standard concentrations of venoms were virtually identical. Western blot analysis revealed multiple common bands between the two venoms. Amino acid data (amino acids 1-35, N-terminal) of the active venom components of the two venoms revealed only three non-identical amino acids. alpha LoxR and alpha LoxD antivenins were similarly effective in blocking the development of rabbit skin lesions (ANOVA p<0.05). In summary, L. reclusa and L deserta spider venoms possess several common protein bands as identified by western blot, greater than 90% amino acid sequence identity, and marked antigenic cross-reactivity.

Human milk lipids bind Shiga toxin.

Hemolytic uremic syndrome, a serious complication of Shiga toxin-associated diarrhea, is rare before 6 months of age. Immunologic and nonimmunologic factors present in human milk may partially explain this observation. In prior studies, we have demonstrated that human milk contains Gb3, the receptor for the B subunit of Shiga toxin, and also contains secretory IgA (sIgA) against the toxin. We therefore sought to determine the relative importance of milk glycolipid and toxin-specific sIgA in toxin binding. We studied two populations that differed in their frequency of exposure to Shiga toxin. Human milk samples obtained from healthy donors from Boston and Buenos Aires were separated by centrifugation into aqueous (antibody enriched) and cream (glycosphingolipid enriched) fractions. An emulsion of equal volumes of aqueous phase or cream layer of each sample and purified Shiga toxin was incubated, and the amount of free toxin present in each was determined by enzyme immunoassay. The cream layers bound 85%+/-2 (mean +/- SE) (Argentina milk samples) and 86%+/-1 (Boston milk samples) of Shiga toxin. In contrast, the soluble fraction in samples from Buenos Aires, a population expected to frequently have antibodies to Shiga toxin, bound more toxin (48%+/-2) than did this fraction in samples from Boston, an area where toxin exposure is infrequent (30%+/-3) (P < 0.0001). Toxin-binding lipids present in human milk are biologically active and may contribute to the putative protective effect of human milk. In a population frequently exposed to Shiga toxins (Argentina), protection may be due to both immune (sIgA), and nonimmune (lipid) factors present in human milk. In a population infrequently exposed to Shiga toxins, cream fraction-associated glycolipids represent the major toxin binding activity in human milk.

Protective role of human lactoferrin against invasion of Shigella flexneri M90T.

Lactoferrin is an iron-binding protein found in human mucosal secretions such as milk. A variety of functions have been ascribed to this protein, it appears to contribute to antimicrobial host defense. Still its overall physiological role remains to be defined. We sought to study the role of recombinant human lactoferrin (rhLf) in Shigella infection. Invasion of epithelial cells is essential to the development of bacillary dysentery. Shigella flexneri 5 M90T, a virulent strain, was evaluated in the classic HeLa cell invasion model, in immunoblots, and by transmission electron microscopy, immunofluorescence, and deconvolved microscopy Bacteria not exposed to rhLf were used as controls. We found that rhLf decreased significantly the invasiveness of S. flexneri 5 M90T in a HeLa cell model. The immunoblot data showed that invasion plasmid antigen B (IpaB) was released from the bacteria during incubation with rhLf. Lactoferrin treatment did not directly dissociate the complex of IpaB and IpaC (IpaBC) once the complex had been formed. Furthermore, ferric iron had no effect on release of IpaB. Electron microscopy of rhLf-treated bacteria suggested a reduction in vacuolization of the HeLa cell cytoplasm and decreased number of bacteria within HeLa cells. At 40,000 x magnification the few rhLf-treated Shigella that invaded exhibited a dense ring completely surrounding them. Immunofluorescence and deconvolved microscopy suggested that rhLf-treated bacteria were completely surrounded by a thick layer of actin. The fact that two cell surface functions (invasion and actin-mediated movement) were deranged suggests that rhLf disrupts the integrity of the bacterial outer membrane in which virulence proteins are anchored. The mechanism by which rhLf impairs Shigella invasiveness may be relevant to other enteropathogens that share similar virulence strategies.

Detection of Loxosceles venom in lesional hair shafts and skin: application of a specific immunoassay to identify dermonecrotic arachnidism.

Loxosceles spiders, of which the brown recluse is the best known, are indigenous to southcentral and southwestern regions of the United States. Loxosceles spider envenomation frequently results in painful, centrally necrotic, erythematous skin lesions that evolve over 24 to 48 hours and may take several weeks to completely heal. The diagnosis of loxoscelism is typically is based on the presence of the characteristic dermal lesion, because no definitive clinical diagnostic assay exists, and the spider is generally not available for identification. We used a rapid Loxosceles-specific enzyme immunoassay to detect spider venom in a dermal biopsy and hairs plucked from a suspicious skin lesion on the lower extremity of a 52-year-old man. This report indicates that in using a novel Loxosceles-specific immunoassay, venom can be detected in dermonecrotic skin and hair specimens for up to 4 days after envenomation.

Severe gamma-hydroxybutyrate withdrawal: a case report and literature review.

We report a case of gamma-hydroxybutyrate (GHB) withdrawal resulting in severe agitation, mental status changes, elevated blood pressure, and tachycardia hours after stopping chronic use of GHB. The patient admitted to substantial GHB abuse on a daily basis for 2.5 years. Previous attempts at cessation reportedly resulted in diaphoresis, tremors, and agitation. The patient's symptoms, negative polypharmacy history, and negative urine and blood toxicological analysis for alcohol, benzodiazepines, sedative-hypnotics, or other substances suggested the diagnosis of GHB withdrawal. Later analysis of a patient drug sample confirmed the presence of GHB. The patient required 507 mg of lorazepam and 120 mg of diazepam over 90 h to control agitation. This is one of the few reported cases of GHB withdrawal and one of the most severe. Given the increasing use of GHB, more cases of severe GHB withdrawal should be anticipated.

Intradermal anti-loxosceles Fab fragments attenuate dermonecrotic arachnidism.

OBJECTIVE: Bites from the brown recluse spider and other arachnids from the genus Loxosceles frequently induce necrotic skin lesions that can be recalcitrant to treatment and disfiguring. The authors used a rabbit model of dermonecrotic arachnidism to address the therapeutic efficacy of intradermal (id) polyclonal anti-Loxosceles Fab fragments (alphaLoxd Fab) raised against Loxosceles deserta spider venom. METHODS: Fab fragments were prepared by papain digestion and affinity chromatography from the IgG fraction of L. deserta antivenom raised in rabbits. Eighteen inbred New Zealand white rabbits were assigned to six groups of three. The rabbits received L. deserta venom (3 microg, id) injections into each flank. Cohorts of rabbits received single id injections (at one venom site/rabbit) of 30 microg alphaLoxd Fab at different times (T = 0, 1, 2, 4, 8, and 12 hours) after venom injection. In each rabbit the opposite flank was left untreated. As an additional control, one group of rabbits (T = 0) received nonspecific Fab (30 microg, id) in the opposite flank. Dermal lesions were quantified as a function of time through the use of a series of digital photographs and imaging software. In addition, myeloperoxidase (MPO) activity, a measure ofneutrophil accumulation, was determined in lesion biopsies. Lesion areas and MPO activities were analyzed by repeated-measures analysis of variance (ANOVA). RESULTS: Lesion areas and MPO activity were markedly reduced when alphaLoxd Fab was administered very early after venom injections. As the interval between venom inoculation and antivenom treatment increased, the therapeutic benefit of alphaLoxd Fab decreased. The final time tested that demonstrated therapeutic efficacy of alphaLoxd Fab was T = 4 hours. Lesion attenuation was no longer apparent when alphaLoxd Fab was given 8 hours post inoculation. CONCLUSIONS: Intradermal administration of alphaLoxd Fab attenuates Loxosceles-induced dermonecrotic lesion formation when given up to 4 hours after venom inoculation in this rabbit model.

Loxosceles deserta spider venom induces NF-kappaB-dependent chemokine production by endothelial cells.

BACKGROUND: Loxosceles spider evenomation in man frequently results in disfiguring necrotic skin lesions. Recent studies suggest that several proinflammatory mediators participate in lesion development. We have observed that Loxosceles deserta venom induces production of the chemokines interleukin-8, growth-related oncogene alpha, and monocyte chemoattractant protein-I by human umbilical vein endothelial cells. Members of the Rel/Nuclear factor (NF)-kappaB family of transcription factors are important regulators of many genes involved in immune and inflammatory responses. We hypothesized that Loxosceles-venom-induced chemokine expression in human umbilical vein endothelial cells is mediated by NF-kappaB. METHODS: Human umbilical vein endothelial cell monolayers were exposed to activating concentrations of Loxosceles deserta venom. Nuclear extracts of these monolayers were analyzed by electrophoretic mobility shift assay. A direct cause and effect linkage between NF-kappaB activation and chemokine expression by Loxosceles venom was established through examination of the effect of SN50 on interleukin-8 and monocyte chemoattractant protein-1 production using a whole-cell enzyme immunoassay. SN50 is a cell-permeable peptide that specifically blocks cytosolic to nuclear translocation of NF-kappaB. Furthermore, the venom-induced synthesis of chemokine mRNAs was investigated by RNase protection assays. RESULTS: Loxosceles deserta venom induces the activation of NF-kappaB in human umbilical vein endothelial cells. Antibodies to p50 and p65, but not to p52, c-Rel, or Rel B, induce supershifts of the DNA-protein complexes formed by oligonucleotide probes and nuclear extracts from venom-activated human umbilical vein endothelial cells. SN50 peptide inhibits NF-kappaB translocation and interleukin-8 and monocyte chemoattractant protein-1 production in activated human umbilical vein endothelial cells. CONCLUSIONS: Loxosceles deserta venom induces synthesis of interleukin8 and monocyte chemoattractant protein-1 mRNAs in human umbilical vein endothelial cells. The expression of chemokines occurs via an NF-kappaB-dependent pathway.

Loxosceles spider venom induces the production of alpha and beta chemokines: implications for the pathogenesis of dermonecrotic arachnidism.

Bites from the brown recluse spider and other Loxosceles arachnids result in dermonecrotic skin lesions. Neutrophils (PMN) are essential to the development of Loxosceles-induced skin lesions, but paradoxically, in vitro PMN activation is inhibited by direct exposure to Loxosceles venom. Neutrophil activation occurs in response to a myriad of soluble mediators that include members of both the alpha and beta chemokine families. Because arachnid envenomation results in the exposure of several different cell types to venom, we investigated venom-induced expression of alpha and beta chemokines in both endothelial cells (human umbilical vein; HUVEC) and epithelial cells (A549 pneumocytes). Chemokine-specific capture enzyme immunoassays (EIA) were used to measure Loxosceles deserta venom-induced alpha chemokines: interleukin-8 (IL-8), growth-related oncogene-alpha (GRO-alpha), and beta chemokines: monocyte chemoattractant protein-1(MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in cell-free conditioned media from HUVEC and A549 cell monolayers. Exposure of HUVECs (8 h) to Laxosceles venom resulted in the production of IL-8 (5.2+/-1.30 ng/ml), MCP-1 (1.44+/-0.11 ng/ml) and GRO-alpha (1.97+/-0.15 ng/ml) in a dose and time-dependent manner. Exposure of A549 cell monolayers to venom resulted in IL-8 (7.74+/-0.30 ng/ml), and MCP-1 (2.61+/-0.31 ng/ml), but neither GRO-alpha nor RANTES accumulated during an 8-hour incubation period. Chemokines accumulated in a venom dose and time-dependent manner. Neither cell type secreted RANTES in response to Loxosceles venom. These data indicate that Loxosceles spider venom is a potent inducer of alpha and beta chemokines in both endothelial and epithelial cell types. Based on the established roles of IL-8, MCP-1, and GRO-alpha, in inflammation, these observations have relevance to the pathophysiology of Loxosceles-induced dermonecrosis.

Mediators of microvascular injury in dermal burn wounds.

In previous studies we have demonstrated that second-degree thermal injury of skin in rats leads to secondary effects, such as systemic complement activation, C5a-mediated activation of blood neutrophils, their adhesion-molecule-guided accumulation in lung capillaries and the development of acute pulmonary injury, largely caused by neutrophil-derived toxic oxygen metabolites. In the dermal burn wound, however, pathophysiologic events are less well understood. The injury is fully developed at four hours post-burn. To further elucidate the pathogenesis of the "late phase" dermal vascular damage, rats were depleted of neutrophils or complement by pretreatment with rabbit antibody against rat neutrophils or with cobra venom factor, respectively. In other experiments, rats were treated with blocking antibodies to IL-6, IL-1, and TNF alpha immediately following thermal burning or were pretreated with hydroxyl radical scavengers (dimethyl sulfoxide, dimethyl thiourea). Extravasation of 125I-labeled bovine serum albumin into the burned skin was studied, as well as, skin myeloperoxidase levels. The studies revealed that, like in secondary lung injury, neutrophils and toxic oxygen metabolites, are required for full development of microvascular injury. In contrast, however, development of dermal vascular damage in thermally injured rats was not affected by complement depletion. Our data suggest that the development of microvascular injury in the dermal burn wound is complement-independent, involves the pro-inflammatory cytokines IL-1, TNF alpha and IL-6, and may result from reactive oxygen metabolites generated by neutrophils accumulating in the burn wound.

Prevention of gastrointestinal iron absorption by chelation from an orally administered premixed deferoxamine/charcoal slurry.

STUDY OBJECTIVE: To investigate the effect of an orally administered premixed slurry of deferoxamine mesylate (DFO) and activated charcoal (AC) on the gastrointestinal (GI) absorption of ferrous sulfate under physiologic conditions. METHODS: This was a prospective, crossover, controlled human volunteer study. Participants were healthy adult subjects aged 25 to 38 years. Volunteers ingested either 5 mg/kg ferrous sulfate alone, 5 mg/kg ferrous sulfate added to 25 g of 20% (weight/ volume) AC, or 5 mg/kg ferrous sulfate added to a premixed slurry consisting of 8 g of DFO and 25 g of 20% (weight/volume) AC. The same group of volunteers was used in each limb of the study. Serum iron concentrations were measured at baseline and at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 hours after ingestion for all subjects. Urinary iron was determined over the first 12 hours after ingestion for each limb. The maximum iron concentration (Cmax), the time to maximum iron concentration (Tmax), and the area under the curve (AUC) were compared for all three limbs. RESULTS: The AUC (P = .042) and Cmax (P = .017) were significantly lower in all subjects in the DFO/AC limb compared with the two control limbs. There was no significant difference in the Tmax iron concentration (P = .77). In the ferrous sulfate control limb, female volunteers had a significantly higher mean Cmax (P = .008) and AUC (P = .014) than males. Iron was undetectable in all baseline and 12-hour urine collections. CONCLUSION: In this model, a premixed 1:3 (weight/weight) DFO/ AC slurry reduced the GI absorption of ferrous sulfate in adult volunteers under physiologic conditions.

Clinical ergotism with lingual ischemia induced by clarithromycin-ergotamine interaction.

The macrolide class of antibiotics, including erythromycin and troleandomycin, is associated with clinically significant adverse drug interactions. This results from macrolide inhibition of cytochrome P-450 metabolism of numerous xenobiotics, resulting in elevated serum drug levels and clinical intoxication. Animal studies, however, suggest that clarithromycin, the newest approved macrolide antibiotic, has has less potential for adverse drug reactions. We describe a patient who, on her fifth day of clarithromycin therapy, developed clinical ergotism (i.e., hypertension, lingual ischemia, and peripheral cyanosis) several hours after administration of her usual 2-mg dose of ergotamine tartrate. To our knowledge, this is the first report of clinical ergotism precipitated by clarithromycin-ergotamine interaction and suggests that, like other macrolide antibiotics, ergot preparations should be avoided in patients who are taking clarithromycin.

Postmortem acetaminophen pharmacokinetics: an experimental study of site and time-dependent concentration changes.

Postmortem blood drug concentrations are obtained routinely for assessment of the cause of mortality. However, the relationship of postmortem drug concentration to blood concentrations at the time of death remains poorly characterized. Using Ketamine sedation, 10 New Zealand white rabbits were sacrificed 20 minutes after oral gavage with liquid acetaminophen 160 mg/kg as a model drug. Blood samples were obtained from peripheral (femoral vein) and central sites (heart & inferior cava) over time and compared with heart blood concentrations obtained at the time of sacrifice. The mean +/- SE antemortem acetaminophen concentration was 63.1 +/- 14.6 mcg/ml. Postmortem central blood concentrations were as follows: T = 3 h: 200.8 +/- 129.2 micrograms/mL, T = 6 h: 100.8 +/- 39.6 micrograms/mL and T = 12 h: 480.8 +/- 128.8 micrograms/mL. Postmortem peripheral site results were: T = 3 h: 50.2 +/- 21.4 micrograms/mL, T = 6 h: 100.8 +/- 18.1 and T = 12 h: 117.7 +/- 37.2 micrograms/mL. Overall, blood acetaminophen concentrations increased significantly over time for central sampling sites. Drug concentration increases seen in the central sampling sites were several times higher than that seen in peripheral blood. Blood samples taken from peripheral sites did not alter significantly. The results of this controlled study were consistent with previous autopsy case series and case reports suggesting that postmortem drug concentrations do not reflect premortem values. Variables affecting postmortem drug concentrations include both postmortem sampling time and anatomic blood collection site.

Incomplete hemolytic-uremic syndrome in Argentinean children with bloody diarrhea.

Argentina has an exceptionally high frequency of hemolytic-uremic syndrome (HUS). We sought to define prospectively the role of verocytotoxins (Shiga-like toxins [SLTs]) in 254 Argentinean children with grossly bloody diarrhea during spring and summer. Free fecal SLTs (I/II) and/or DNA probe-positive isolates were found in 99 (39%) of the children. During the follow-up period, HUS developed in 6 patients (4 with evidence of recent SLT infection based on stool studies); another 14 patients had some, but not all, of the abnormalities seen in typical HUS. The development of HUS or incomplete HUS in these children was significantly associated with recent SLT-Escherichia coli infection (p = 0.024). The high incidence of SLT-associated bloody diarrhea in Argentina explains, at least partially, the unusually high frequency of HUS. Our data indicate that incomplete forms of HUS may be common in patients with SLT-associated bloody diarrhea.

Tumor necrosis factor concentrations in hemolytic uremic syndrome patients and children with bloody diarrhea in Argentina.

Hemolytic uremic syndrome (HUS) is thought to be a vascular endothelial injury disease. The mechanism of injury is unknown although verocytotoxins (Shiga-like toxins (SLTs)) are known to be associated with it. Recent evidence suggests that in vitro treatment of some endothelial cells with tumor necrosis factor alpha (TNF-alpha) dramatically increases their susceptibility to SLTs. We studied 25 children with HUS, 63 children with SLT-positive bloody diarrhea, 62 children with bloody diarrhea not associated with SLTs and 39 children admitted for elective surgery, included as an age- and season-matched control group. The TNF-alpha concentrations were found to be significantly elevated in children with HUS (range, 1 to 95 pg/ml; geometric mean, 32.2 pg/ml) compared with the healthy controls (range, 0 to 53 pg/ml; mean, 12.5 pg/ml; P < 0.001). Because it is hypothesized that TNF-alpha elevation might precede development of HUS, we also studied children with blood diarrhea. The TNF-alpha serum concentrations were significantly higher during the first 10 days after onset of bloody diarrhea than after the first 10 days (P < 0.02). Such elevation could be associated with vascular endothelial glycolipid receptor up-regulation and increased susceptibility to the effects of SLTs.

Adsorption of botulinum toxin to activated charcoal with a mouse bioassay.

STUDY OBJECTIVE: We evaluated the effectiveness of activated charcoal (AC) in adsorbing Clostridium botulinum type A toxin using a mouse bioassay. DESIGN: Prospective, blinded, randomized, controlled animal study. SETTING: Animal care facility. PARTICIPANTS: One hundred forty Swiss/Webster ND-4 strain mice. INTERVENTION: Food contaminated with type A botulinum toxin was homogenized in a phosphate/gel buffer (pH 6.2). The concentrate was diluted by factors of 1:10, 1:50, and 1:100. AC was added to aliquots of the dilutions to a 20% final concentration. The samples were centrifuged, supernatant was removed, and separate groups of mice were injected intraperitoneally with .5 mL of each dilution (those treated with AC and controls untreated with AC). The animals were then observed over 5 days for signs of botulism. RESULTS: None of the 60 animals injected intraperitoneally with dilutions treated with AC was observed to have any signs of botulism. In contrast, deaths were observed in 10 of 20, 9 of 20 and 4 of 20 mice injected with untreated dilutions of 1:100, 1:50, and 1:10, respectively (P < .004). CONCLUSION: In this model, treatment of botulinum toxin with AC before administration resulted in greatly reduced morbidity and mortality.

Elevation of breath ethanol measurements by metered-dose inhalers.

STUDY OBJECTIVE: Metered-dose inhalers (MDIs) may contain as much as 38% ethanol. We evaluated the effects of ethanol-containing MDIs on breath alcohol testing. DESIGN: Prospective, single-blind, crossover, controlled study. PARTICIPANTS: Three healthy male volunteers 29 to 36 years old. INTERVENTION: We studied three brands: Tornalate, (38% ethanol), Bronkometer, (30% ethanol), and Alupent, (0% ethanol). The effects of each MDI on breath and blood ethanol measurements were evaluated separately. Two puffs of each brand of MDI were administered. Breath ethanol measurements were obtained at baseline and .25, .5, 1, 2, 3, 5, and 10 minutes after MDI use. Blood ethanol measurements were obtained at baseline and 1 and 10 minutes after MDI use. RESULTS: Overall, Tornalate had the highest breath ethanol readings, with a mean ethanol level of 189 mg/dL recorded just after MDI use. Breath ethanol levels subsequently decreased rapidly over time. Mean breath ethanol concentrations were lower after the use of Bronkometer and undetectable after the use of Alupent. Blood ethanol levels were undetectable at all times tested. CONCLUSION: MDIs may cause elevations of breath alcohol above the legal criteria for intoxication. These effects are transient and may be prevented by a 10-minute interval between the use of an MDI and breath alcohol testing.

Vitreous humor cocaine and metabolite concentrations: do postmortem specimens reflect blood levels at the time of death?

The interpretation of postmortem cocaine concentrations is made in an attempt to estimate drug concentrations present at the time of death and thus infer not only drug presence but drug toxicity. Previous data suggest that changes in postmortem blood cocaine concentrations over time are not predictable and interpretation of cocaine levels should be done with caution. However, these data come from autopsy case series where vital information, such as blood cocaine concentration at the time of death, dose and time since last use, and postmortem interval is often not known. The purpose of this study was to characterize postmortem changes in cocaine and metabolite concentrations relative to premortem concentrations over time at two anatomic sites: peripheral blood and vitreous humor, in a controlled, large animal model. Juvenile swine were given cocaine HCl 10 mg/kg as an IV bolus which resulted in seizures and wide complex tachycardia. Five minutes after cocaine administration, animals were euthanized. At time of death and eight hours postmortem, femoral venous blood and vitreous humor (VH) samples were obtained for quantitation of cocaine, benzoyl ecgonine (BE), and ecgonine methyl ester (EME) by GC/MS. There were no significant increases over time in mean femoral vein concentrations of cocaine or BE. However, a large interanimal variability in direction and magnitude of concentration changes was seen. Mean EME concentrations at the femoral site increased significantly over 8 hours (P < 0.03). Mean VH cocaine concentrations at time of death were significantly lower than corresponding blood concentrations (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal immune response of volunteers ingesting a strain of enteroadherent (HEp-2 cell-adherent) Escherichia coli.

Enteroadherent Escherichia coli (EAEC) strains identified by adherence to HEp-2 tissue culture cells have been incriminated epidemiologically as important etiologic agents of diarrheal disease in both adult travelers and children in developing countries. One strain, JM 221, with no recognized E. coli virulence characteristics other than adherence to HEp-2 cells, caused diarrhea in 5 of 16 volunteers ingesting it. We studied the secretory immunoglobulin A (sIgA) responses to EAEC JM 221 of five volunteers with diarrhea and five volunteers who remained healthy after challenge. sIgA was extracted from stools obtained prechallenge and 7 days postchallenge. Total sIgA was standardized for all specimens. Specific sIgA titers were determined by dot blotting with the following JM 221 antigens: water-extractable surface antigens, whole cells, lipopolysaccharides, and outer membrane proteins. All five subjects who became ill had fourfold or greater rises in titers against each of the four antigens. The five subjects who remained healthy following challenge did not exhibit significant rises in titers to any JM 221 antigens, but their mean titers were significantly higher than the mean prechallenge titers of the volunteers with diarrhea, suggesting that high intestinal sIgA titers may be protective. The significant increases in intestinal antibody against JM 221 in the subjects who became ill is further evidence of the enteropathogenicity of EAEC strains.