Aurelio Pasi

Fulminant Liver Failure in Association with the Emetic Toxin of Bacillus cereus

BACKGROUND A 17-year-old boy and his father had acute gastroenteritis after eating spaghetti and pesto that had been prepared four days earlier. Within two days, fulminant liver failure and rhabdomyolysis developed in the boy and he died. The father had hyperbilirubinemia and rhabdomyolysis but recovered. We investigated the cause of these illnesses. METHODS Bacteria were isolated and characterized by conventional methods, and bacterial toxins were quantified by immunoassays and cell-culture techniques. The effect of the isolated toxin on the rates of oxidation of various substrates was analyzed in rat-liver mitochondria. RESULTS Autopsy of the boys liver revealed diffuse microvesicular steatosis and midzonal necrosis that suggested impaired beta-oxidation of liver mitochondria due to a mitochondrial toxin. There was no evidence of ingestion of heavy metals, halogenated compounds, hepatotoxic drugs, or staphylococcal enterotoxin. However, high concentrations of Bacillus cereus emetic toxin were found in both the residue from the pan used to reheat the food and the boys liver and bile. B. cereus was cultured from the intestinal contents and the pan residue. The emetic toxin isolated from the B. cereus cultures was found to be a mitochondrial toxin. CONCLUSIONS Fulminant liver failure developed after the ingestion of food contaminated with the B. cereus emetic toxin. The toxin inhibits hepatic mitochondrial fatty-acid oxidation, indicating that it caused liver failure in this patient.

Parallel distribution of immunoreactive α-neo-endorphin and dynorphin in rat and human tissue

Abstract Using antibodies directed against α-neo-endorphin and dynorphin-(1–13) a striking parallelism was found between the regional distribution of the two opioid peptides in various rat and human tissues. In the rat, the highest levels were found in the neurointermediate pituitary, followed by those in the adenohypophysis, the hypothalamus and the striatum. High levels were also found in the spinal cord, whereas the cerebellum contained only small amounts of the immunoreactive peptides. In peripheral tissues, high concentrations of both were detected in the gut and surprisingly, the lungs, whereas other tissues such as the adrenal glands, the kidney and the liver did not contain measurable amounts. The distribution pattern in the human brain was similar to that in the rat. A particularly high concentration of both of the peptides was found in the substantia nigra. In contrast to the rat pituitary, the human pituitary contained only small amounts of the immunoreactive opioid peptides. In all tissues of both species, the levels of immunoreactive (ir-) α-neo-endorphin were somewhat higher than those of ir-dynorphin with the ir-α-neo-endorphin/ir-dynorphin ratio varying between 1 and 3. In response to dehydration, ir-α-neo-endorphin, ir-dynorphin, vasopressin and leu-enkephalin were depleted from the neurohypophysis of the rat indicating a similar regulation of the pools of these peptides in the neurohypophysis. A common biosynthetic origin of ir-α-neo-endorphin and vasopressin, is unlikely, since Brattleboro rats which do not synthesize vasopressin possess levels of ir-α-neo-endorphin in the neurohypophysis comparable to those of control rats. Gelfiltration and HPLC analysis of the immunoreactive components in the rat pituitary lobes revealed that the vast majority of the immunoreactivity in the neurointermediate pituitary coelutes with synthetic mα-neo-endorphin, whereas a substantial part of that in the adenohypophysis appears to comprise a material with a molecular size of about 8000 daltons. There was no evidence for the existence of substantial amounts of β-neo-endorphin in the rat pituitary. The possibility is discussed that the close parallelism in the distribution and secretion of ir-α-neo-endorphin and ir-dynorphin might indicate a common biosynthetic origin for these peptides.

Characterization and localization of immunoreactive dynorphin, α-neo-endorphin, met-enkephalin and substance P in human spinal cord

Abstract By use of specific antisera, the distributions of immunoreactive dynorphin (ir-DYN), α-neo-endorphin (ir-α-NEO), Met-enkephalin (ir-MET) and substance P (ir-SP) were evaluated in discrete regions of human spinal cord and spinal ganglia. The relative concentrations of immunoreactive peptides in particular regions were as follows: sacral > lumbar > cervical > thoracic. Concentrations of ir-DYN, ir-α-NEO and ir-SP were 2–10-fold, but of ir-MET 1–2-fold, higher in the dorsal as compared to the ventral parts of cervical, lumbar and sacral cord. The concentrations of all peptides (when examined in discrete areas of thoracic cord) were found to be highest in the substantia gelatinosa. All peptides were present in the gray matter but only ir-MET was found in white matter. Gel-permeation chromatography of dorsal sacral spinal cord extracts revealed two major ir-DYN peaks. The smaller molecular weight peak, eluted at the position of synthetic dynorphin 1–17 . ir-α-NEO and ir-SP comigrated exactly with their respective synthetic marker peptides. Substantial amounts of ir-SP and also, as confirmed by high pressure liquid chromatography, ir-MET, were found in the dorsal and ventral roots and spinal ganglia, and very low concentrations of ir-DYN or ir-α-NEO were also detected in these tissue. These results suggest that dynorphin and α-neo-endorphin, in addition to enkephalins, may be involved in transmission of somatosensory information in the human spinal cord.

A subclassification of κ-sites in human brain by use of dynorphin 1–17

Abstract The interaction of dyn 1–17 with κ-receptor sites in human amygdala membranes was studied by means of competitive binding techniques. κ-sites were labeled with [ 3 H]diprenorphine in the presence of saturating concentrations of δ- and μ-opiate receptor ligands. Dynorphin 1–17 was an extremely potent ligand, and β-endorphin a moderately potent ligand at one half of the κ-binding component but had 2000-fold lower affinity to the remaining κ-fraction, suggesting a subclassification of the κ-opiate binding site. In contrast, various benzomorphane κ-agonists, the sigma agonist SKF 10,047 and naloxone completely displaced [ 3 H]diprenorphine at low concentrations.

Angiogenesis : modulation with opioids

1. The effect of beta-endorphin (beta-EP) and morphine sulfate (MS), in presence and absence of naloxone (NX), on chicken chorioallantoic membrane was studied as a function of blood vessel proliferation. 2. A 50% reduction in blood vessel proliferation occurred by 10 micrograms of beta-EP or by 5 micrograms of MS per egg compared to controls. 3. An individual dose, i.e. 5 micrograms of beta-EP, did not significantly inhibit blood vessel counts after initial 24 hr period of the drug application when given alone compared to inhibition occurring with combined use of NX. 4. NX (1 microgram) did not significantly reverse the angiostatic effects of MS (10 micrograms) or of beta-EP (5 micrograms). 5. The observed modulation of angiogenesis by opioids suggests involvement of beta-EP and MS in the proliferation of vascular endothelial cells. 6. This may be due to an effect of beta-EP and MS on cell-mediated immunity factors such as interferons, interleukins and prostaglandin E2.

Social conflict-induced changes in nociception and β-endorphin-like immunoreactivity in pituitary and discrete brain areas of C57BL/6 and DBA/2 mice

The present study characterizes the time course of social conflict analgesia and its reversibility by opioid antagonist drugs in the C57BL/6 and DBA/2 inbred strains of mice and examines the relationship between alterations in brain and pituitary levels of beta-endorphin-like immunoreactivity (beta-ELIR) and the antinociception elicited by social stress. Data revealed statistically significant strain differences in regard to beta-ELIR in control animals. The pituitary content of beta-ELIR was higher in DBA/2, while the values in the periaqueductal grey (PAG) and in the amygdala were higher in C57BL/6 mice. No interstrain differences were found in the hypothalamus. Exposure to 50 attack bites resulted in a 6-fold higher analgesia in DBA/2 mice and in a strain-independent fall of beta-ELIR in pituitary (approximately 27%) and PAG (23%). PAG but not pituitary beta-ELIR levels in C57BL/6 mice correlated positively with the increase in tail-flick latency after attack. Mere confrontation with a non-aggressive opponent failed to induce analgesia and was associated in C57BL/6 mice with a significant reduction in the beta-ELIR content of both the pituitary and the PAG. The data are discussed in terms of genotype-dependent sensitivity of the beta-endorphin system to stress and its relation to analgesia.

Effects of repeated as compared to single aggressive confrontation on nociception and defense behavior in C57BL/6 and DBA/2 mice

Behavioral reactions (submissive postures, escape, immobility, activity, locomotion) in C57BL/6 and DBA/2 test mice were recorded during single (50 bites) or three repeated (3 X 50 bites, separated by 24 hr) aggressive confrontations, as well as during a nonaggressive confrontation 24 hr after the last aggressive confrontation with opponents of the opposite strain. Nociception (hot plate response latency) was measured 1 min after aggressive or nonaggressive confrontations. During repeated aggressive confrontation, DBA mice reacted with a stable pattern of escape and analgesia, whereas C57 mice failed to develop an analgesic response and changed their behavioral defense strategy during repeated aggressive confrontations (decrease of escape, increase of defensive upright). The conditioned display of submission and of escape behavior during nonaggressive confrontation did not change as a function of earlier repeated aggressive confrontations in DBA mice, while C57 mice showed a significant increase of defensive upright postures and immobility. Conditioned analgesia was not observed after nonaggressive confrontations. The results point toward a dissociation between attack-elicited behavior and antinociception and suggest that encounter-induced analgesia may influence the processing of aversive experience.

Proenkephalin-a-derived peptides are present in human brain

The opioid peptides [Met] enkephalin-Arg6-Phe7, [Met]-enkephalin-Arg6-Gly7-Leu8, and BAM-12P were identified in various ares of human brain by use of a combination of radioimmunoassay (RIA), gel filtration, and high performance liquid chromatography (HPLC). The highest levels of each peptide were found in striatum, followed by substantia nigra, hypothalamus, and medulla. In all areas, the relative concentrations were [Met] enkephalin-Arg6-Phe7 less than [Met] enkephalin-Arg6-Gly7-Leu8 less than BAM-12P. The apparent existence of BAM-12P suggests that the enzymatic processing of the proenkephalin A precursor in human brain is not restricted to cleavage at sites with two basic amino acids. Our results provide evidence that a precursor similar or identical to that present in the adrenal medulla exists in human brain. It appears that the processing mechanisms are different, as virtually none of the immunoreactivity found in the brain was comprised of the higher molecular weight forms of the peptides seen in the adrenal.

β-endorphin-like immunoreactivity levels in the hypothalamus, the periaqueductal grey and the pituitary of the DBA mouse: Determination by ELISA and relationship to nociception

The present paper describes the development and application of an enzyme-linked immunosorbent assay (ELISA) for the assessment of beta-endorphin-like immunoreactivity (beta-ELIR) level in the hypothalamus, the periaqueductal grey (PAG) and the pituitary of DBA/2 mice that were subjected to mild social stress (aggressive confrontation). After confrontation these subjects showed elevated tail-flick latencies (TFL) when compared to controls, a finding that indicates stress-induced analgesia (SIA). A positive correlation was found between individual TFLs and beta-ELIR levels in the PAG but not in the hypothalamus and the pituitary. These results suggest that individual baseline PAG beta-ELIR levels may be taken as a predictor of high degrees of stress-induced analgesia.

Characterization of proenkepahlin B-derived opioid peptides in the human hypothalamo-neurohypophyseal axis

Proenkephalin B-derived opioid peptides, such as dynorphin1-17, dynorphin1-8, dynorphin B, alpha-neo-endorphin and beta-neo-endorphin in the human hypothalamo-neurohypophyseal tract were quantitated and characterized by the combined use of various radioimmunoassays, gel filtration, high performance liquid chromatography and enzymatic cleavage. Chromatographic analysis of immuno-reactive peptide levels determined that, in each case, these were comprised almost exclusively of the authentic peptides both in the neurohypophysis and hypothalamus. Concentrations of authentic proenkephalin B-peptides were 100-5000-fold lower in the human as compared to the rat neurohypophysis. However, in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and certain other nuclei of the human hypothalamus concentrations of authentic peptides were found to be in the same range as those in the rat hypothalamus. The ratio of proenkephalin B-peptides in PVN and SON to those of the neurohypophysis in the rat was ca. 1:50. Conversely, in man these ratios were shown to be 80:1 for dynorphin B, 6:1 for alpha-neo-endorphin and 1:1 for all other peptides evaluated. Examination of postmortem degradation of peptides indicated that these lower levels in the neurohypophysis are not due to a higher rate of postmortem breakdown. Since levels of both vasopressin and beta-endorphin were very high, these deficits in proenkephalin B-peptides were selective and do not represent a generalized property of the human pituitary. Experiments involving enzymatic cleavage demonstrated the occurrence of higher molecular weight forms containing the Leu-enkephalin sequence which were not recognized by the antisera employed.(ABSTRACT TRUNCATED AT 250 WORDS)