Outbreak of abdominal distension and obstipation in a C57BL/6J experimental autoimmune encephalomyelitis study.

Seventy-four 9-week old female C57BL/6J mice housed in a conventional facility were manipulated to induce experimental autoimmune encephalomyelitis, among which 26 developed clinical signs including lethargy, absence of defecation, and abdominal distension. By gross necropsy examination, there was distension of the cecum and colon with fecal impaction. By histologic examination, there was severe ulcerative and proliferative typhlocolitis. Fecal ELISA confirmed the presence of toxins A and B of Clostridium difficile. Alteration in immune status of the immunocompetent mice, due to stress caused by experimental manipulation or autoimmune disease, may have led to intestinal dysbiosis, followed by opportunistic infections resulting in C. difficile-associated disease. This report brings to light the occurrence of the disease in immunocompetent laboratory mice during experimental manipulations associated with alteration in immune status, and it discusses potential hazards associated with conventional housing within a hospital-associated research institute.

Detailed analysis of food-reinforced operant lever pressing distinguishes effects of a cannabinoid CB1 inverse agonist and dopamine D1 and D2 antagonists.

Overt similarities exist between the effects of systemic cannabinoid CB1 inverse agonists and dopamine (DA) antagonists on appetitive behavior. The present set of studies was undertaken to apply a fine-grained analysis of food-reinforced operant lever pressing in rats in order to compare the pattern of effects produced by administration of the CB1 inverse agonist AM 251 and those induced by the DA D1 antagonist SKF 83566, and the D2 antagonist raclopride. Three groups of rats were trained on a fixed-ratio 5 (FR5) schedule and administered these compounds over a range of doses expected to suppress responding. All three drugs produced a dose-related suppression of total lever pressing. In addition to main effects of dose, regression analyses were performed to determine which of several response timing- and rate-related variables correlated most strongly with overall responding in each group. It was found that total session time spent pausing from responding was significantly better at predicting responding in the AM 251 group, while both DA antagonists produced significantly stronger regression coefficients (versus AM 251) from fast responding measures. These results suggest that, while several similarities exist, CB1, D1, and D2 antagonists are not identical in their pattern of suppression of food-maintained lever pressing.

Estimating the minimum number of judges required for test-centred standard setting on written assessments. do discussion and iteration have an influence?

Absolute standard setting procedures are recommended for assessment in medical education. Absolute, test-centred standard setting procedures were introduced for written assessments in the Liverpool MBChB in 2001. The modified Angoff and Ebel methods have been used for short answer question-based and extended matching question-based papers, respectively. Data collected has been analysed to investigate whether reliable standards can be achieved for small-scale, medical school-based assessments, to establish the minimum number of judges required and the effect of a discussion phase on reliability. The root mean squared error (RMSE) has been used as a measure of reliability and used to compute 95% confidence intervals for comparison to the examination statistics. The RMSE has been used to calculate the minimum number of judges required to obtain a predetermined minimum level of reliability, and the effect of the number of judges and number of items have been examined. Values of the RMSE obtained vary from 0.9 to 2.2%. Using average variances across each paper type, the minimum number of judges to obtain a RMSE of less than 2% is 10 or more judges before discussion or 6 or more judges after discussion. The results indicate that including a discussion phase improves the reliability and reduces the minimum number of judges required. Decision studies indicate that increasing the number of questions included in the assessments would not significantly improve the reliability of the standard setting.

The novel cannabinoid agonist AM 411 produces a biphasic effect on accuracy in a visual target detection task in rats.

Cannabinoid agonists have been shown to produce dose-related impairments in several measures of cognitive performance. However, it is unclear if low doses of cannabinoid CB1 agonists, or CB1 antagonists, can facilitate aspects of stimulus detection. The present study employed an operant procedure involving visual stimulus detection in rats. The task was found to be sensitive to the muscarinic acetylcholine antagonist scopolamine. The CB1 antagonist AM 251 did not affect stimulus detection processes across a broad range of doses. However, the novel CB1 agonist AM 411 produced a biphasic effect, with the two lowest doses (0.25 and 0.5 mg/kg) enhancing accuracy. AM 411 changed patterns of responding toward runs of consecutive errors on only one of the two levers. It produced a biphasic effect on consecutive errors on the lever associated with a higher level of errors, with decreases in errors following the lower doses (0.25 and 0.5 mg/kg) and increases following the highest dose (2.0 mg/kg). These effects were not accompanied by changes in measures of bias commonly used to uncover such patterns in rodent operant models of cognitive performance. In contrast to the cognitive impairment seen after administration of moderate to high doses of CB1 agonists, it appears that low doses of some CB1 agonists may be capable of enhancing stimulus detection processes.

The cannabinoid CB1 antagonist AM 251 produces food avoidance and behaviors associated with nausea but does not impair feeding efficiency in rats.

RATIONALE: A growing body of evidence suggests that cannabinoid CB1 receptor antagonists have potential therapeutic utility as appetite suppressants. However, the specific mechanisms underlying the reduction in food intake produced by these drugs are not well understood. OBJECTIVE: Considering the known antiemetic and motor-suppressive effects of CB1 agonists, the present studies were conducted to determine if the reductions in food intake induced by the CB1 antagonist AM 251 could result from nausea or impairments in intake-related motor control, rather than solely from appetite suppression. METHODS: Three experiments were conducted to examine the effects of AM 251 (2.0, 4.0, or 8.0 mg/kg or vehicle) on detailed parameters of food intake, on the development of conditioned taste avoidance, and on taste reactivity. RESULTS: In the first experiment, acute administration of AM 251 dose-dependently decreased food intake; nevertheless, feeding rate (grams consumed per time spent eating) and food handling were unaffected, which suggests that food intake was not reduced because of severe motor impairments. In the second experiment, AM 251 dose-dependently reduced intake of a flavor with which it had previously been associated, indicating that conditioned taste avoidance had developed. Lastly, AM 251 was found to induce conditioned rejection reactions in a dose-dependent manner. CONCLUSIONS: The CB1 antagonist AM 251 may reduce food intake in part by inducing nausea or malaise, but not because of incoordination or motor slowing related to feeding.

The cannabinoid CB1 antagonists SR 141716A and AM 251 suppress food intake and food-reinforced behavior in a variety of tasks in rats.

Cannabinoid CB1 receptor agonists, including delta-9-tetrahydrocannabinol (Delta 9-THC) (the main psychoactive ingredient in marijuana) have been shown to increase feeding in rats and humans. Conversely, it has been reported that acute administration of the CB1 receptor antagonist SR 141716A reduces food intake in rats. Based upon this observation, it has been suggested that CB1 antagonists could be useful as appetite suppressant drugs. The present studies were designed to provide a detailed examination of the effects of CB1 antagonists on food intake across a range of paradigms. Two CB1 antagonists (SR 141716A and AM 251) were administered to rats trained on fixed-ratio schedules with two different ratio requirements (fixed-ratio 1 and fixed-ratio 5). Both drugs produced a dose-dependent decrease in lever pressing, and had a relatively long duration of action (T1/2: SR 141716A, 15.1 h; AM 251, 22.0 h). Furthermore, intake of three diets with differing macronutrient composition (lab chow, high fat, high carbohydrate) was studied. Both drugs significantly suppressed intake of all three foods, and there were no significant interactions between drug dose and diet type. These findings support the hypothesis that CB1 receptor antagonists could be useful pharmacological tools for the suppression of appetite.

Dopamine receptor expression on human T- and B-lymphocytes, monocytes, neutrophils, eosinophils and NK cells: a flow cytometric study.

This study documents expression of dopamine (DA) receptors on leukocyte subpopulations using flow cytometric techniques to identify dopamine receptors with subtype-specific antibodies. Of the D1-like receptor family (D(1) and D(5)), only D(5) was detected, and of the D2-like receptor family (D(2), D(3) and D(4)), all dopamine receptors were detected. T-lymphocytes and monocytes had low expression of dopamine receptors, whereas neutrophils and eosinophils had moderate expression. B cells and NK cells had higher and more consistent expression. Dopamine receptors D(3) and D(5) were found in most individuals whereas D(2) and D(4) had more variable expression. D(1) was never found.

A study of tissue interface membranes from revision accord knee arthroplasty: the role of T lymphocytes.

Despite four decades of advances in the design of orthopaedic devices aseptic loosening remains a major cause for the revision of total joint arthroplasty. This study used the techniques of immunohistochemistry and reverse transcription polymerase chain reaction to identify the inflammatory cell types, cytokines and chemokines within the interface tissue surrounding failed Accord Knee prostheses. Many T cells were identified within the tissue: however, the classical marker of activation, CD25 was expressed on very few cells. Molecular analysis failed to detect the synthesis of either Th1 or Th2 cytokines. These results suggest that the T cells are being actively recruited to the site of inflammation along the chemokine gradients but are not participating in a classical immune response.

Transplacental transfer of the opioid growth factor, [Met(5)]-enkephalin, in rats.

Placental transfer of the pentapeptide [Met5]-enkephalin, known to function as a growth regulating factor and neuromodulatory agent, was studied in pregnant Sprague-Dawley rats. Using separation by reversed phase high-performance liquid chromatography, and analysis by derivative spectroscopy, [Met5]-enkephalin was detected in 20-day-old fetal tissue including brain, heart, lung, and kidney. Fetal tissues from pregnant rats given an injection of 40 mg/kg [Met5]-enkephalin on gestation day 20 had markedly elevated levels of peptide within 1 h, indicating the transplacental transfer of this opioid. [Met5]-enkephalin levels were increased from control samples at 1, 2, 4, and 14 h post-injection of peptide, but not at 24 h. Evaluation of breakdown products of [Met5]-enkephalin, along with the related peptide [Leu5]-enkephalin, revealed that elution times differed substantially from [Met5]-enkephalin. These data indicate that [Met5]-enkephalin is present in fetal organs, crosses the placenta, does not appear to be restrictive in organ specificity, and is sustained in fetal tissues at detectable levels for at least 14 h. Given that [Met5]-enkephalin tonically inhibits DNA synthesis in the fetus, these results raise the question of whether an elevated level of this peptide (either maternally or from the fetus) may be detrimental to cellular ontogeny in the fetus, and perhaps have long-term implications for postnatal development.

Epithelial adhesion complexes and organ culture of the human cornea.

The effects of extended organ culture of human cornea on the structural integrity, particularly adhesion complexes, of the epithelium were determined. Human corneas were placed in organ culture using an immersion method. The structure of the cornea prior to culture (0 h) and at 7, 14, and 18 days in culture was evaluated by staining with hematoxylin/eosin, and by ultrastructural analysis that included a morphometric study of the type and number of adhesion complexes. Human corneas prepared immediately (0 h) and those in culture after 7 days showed similar structural organization and anatomical features. In contrast to 0 h specimens, the corneal epithelium at 14 days in culture exhibited signs of deterioration, with increases in cellular contraction, extracellular space, electron density of the cytoplasm, nuclear invaginations, and nucleoplasmic opacity, as well as aggregations of junctional complexes between cells. At 18 days in culture, the ocular surface epithelium was markedly reduced in thickness and consisted of no more than 2-3 cell layers; a distinct basal layer was not detected, and the morphology of the suprabasal and basal layers were similar. The basement membrane was disorganized, and anchoring complexes composed of hemidesmosomes were often absent. The number and type of the anchoring complexes associated with the basal epithelium and Bowman's membrane were comparable until 14 days of age, although the total number of hemidesmosomes per microm of epithelial plasmalemma was subnormal. After 2 weeks in culture, there were 38-72% fewer anchoring complexes and a decrease of 44% in the number of hemidesmosomes/microm of membrane from samples prepared immediately and after 7 days in culture. These results indicate that the structural integrity of human corneal epithelium in organ culture is compromised after 14 days in vitro using an immersion system of tissue culture. Thus, long-term use of cultures to define homeostasis and wound healing of the ocular surface epithelium, which necessitates normal architecture including anchoring complexes between epithelium and Bowman's membrane, may not be appropriate and requires careful monitoring both qualitatively and quantitatively at the electron microscopic level of resolution.

Gonadectomy impairs T-maze acquisition in adult male rats.

Recent studies have shown that chronic gonadectomy increases the density of dopaminergic axons in prefrontal but not sensorimotor cortices in adult male rats. Since supranormal prefrontal cortical dopamine stimulation is known to impair rats' performance in T-maze delayed alternation paradigms, we tested whether long-term gonadectomy might also impair T-maze performance. For comparison, sensorimotor functions were also assessed. Adult male rats were gonadectomized and placebo-, estradiol-, or testosterone propionate-treated or were sham operated and placebo-treated. Four weeks after surgery, the subjects were tested using a rotorod apparatus and in the acquisition of a T-maze delayed alternation paradigm. Gonadectomized placebo-treated and estradiol-treated rats took significantly longer to acquire the T-maze rule than controls, and gonadectomized, testosterone-treated rats acquired the task within the same time frame as controls. No group differences were detected in rotorod performance. Thus, chronic gonadectomy induced testosterone-sensitive, estradiol-insensitive acquisition deficits in a spatial learning task but had no demonstrable effects on the sensorimotor functions tested.

Molecular characterization and distribution of the opioid growth factor receptor (OGFr) in mouse.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. The receptor for OGF, OGFr, has been cloned and sequenced in humans and rats. Using primers based on the rat OGFr cDNA, and a mouse embryo expressed sequence tag, the full-length 2.1 kb mouse OGFr cDNA was sequenced. The open reading frame was found to encode a protein of 634 amino acids, and 14 imperfect repeats of 9 amino acids each were a prominent feature. The molecular weight of OGFr was calculated as 70679, and the isoelectric point was 4.5. Northern blot analysis revealed a 2.1 kb OGFr mRNA transcript in adult mouse brain, heart, lung, liver, kidney, and triceps surae muscle. The amino acids for mouse and rat OGFr were 93% similar and 91% identical, but the mouse and human shared only a 70% similarity and a 58% identity. These results emphasize the molecular validity of OGFr, and explain the interaction of OGF with respect to normal and abnormal growth in mouse cells and tissues.

Cellular dynamics of corneal wound re-epithelialization in the rat. III. Mitotic activity.

The number and distribution of mitotic epithelial cells in the ocular surface during homeostasis and in response to abrasion of the mammalian cornea were determined. Normal rats and those receiving a 3 mm diameter centrally located epithelial defect, received an intraperitoneal injection of colchicine 6 h prior to sacrifice. Mitosis in the basal epithelium during homeostasis was comparable in magnitude across the ocular surface epithelium, with the exception of a few mitotic figures in the midline. Thirty percent of the mitotic figures were in the basal layer (layer 1), and 70% were in layer 2; the cells in layer 2 were often noted to retain connection to the basal lamina by cytoplasmic stalks. Mitosis was rarely noted in the regenerating epithelium. However, summation of M phase cells in both the basal and suprabasal epithelium adjacent to the wound showed increases of 3- and 5-fold at 30 and 36 h after abrasion, respectively, from levels at homeostasis and the time of injury. In striking contrast to homeostatic epithelium, 80% of the mitotic cells were located in layer 1 of the corneal epithelium, with normal distribution observed by 72 h. Mitosis in the limbus and conjunctiva was increased 3-fold at 30 h and 24 h, respectively, from values at homeostasis and the time of debridement. These results, using rigorous statistical analysis and precise topographic assessment, showed that mitosis is not impeded - but rather often accelerated - following denuding of the corneal epithelium and that the spatial distribution of mitotic cells is correlated with wounding. The data revealed that re-epithelialization of the corneal epithelium is not dependent on mitosis in the regenerating epithelium, but rather in the adjacent unwounded epithelium of the cornea, with most cells being located in the basal layer until re-epithelialization is completed. Mitotic cells in the limbus and conjunctiva may be related to replenishment of ocular surface epithelial cells used in the repair process rather than directly supplying the abraded surface.

Opioid growth factor regulates the cell cycle of human neoplasias.

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

Elevated levels of opioid growth factor in the plasma of patients with pancreatic cancer.

Opioid growth factor (OGF, [Met5]-enkephalin) is an endogenous peptide that regulates the growth of human pancreatic cancer. To evaluate whether human subjects with pancreatic cancer have alterations in plasma levels of OGF, fasting blood samples were obtained from 15 patients with histologically confirmed pancreatic adenocarcinoma. Forty-five subjects with other malignancies, 20 patients with acute pancreatitis, and 30 aged-matched patients without cancer served as control populations. Individuals with pancreatic cancer had OGF values, as determined by radioimmunoassay, that were up to 7.3-fold greater than control subjects. No differences were found between OGF values obtained from patients with other malignancies, acute pancreatitis, or subjects without cancer. The sensitivity and specificity of OGF for pancreatic cancer were greater than either CA 19-9 or CEA. These data indicate that pancreatic cancer is associated with a marked increase in plasma OGF levels and suggest that this peptide may serve as a useful diagnostic tool in the screening for this disease.

Fine motor control in rats is disrupted by delta-9-tetrahydrocannabinol.

Evidence has suggested that cannabinoids such as THC, the active ingredient in marijuana, cause deficits in motor control and the production of movement. However, the specific components of motor control that are affected by cannabinoids have yet to be identified. The present study used an operant beam-press paradigm with a force criterion to determine the effects of THC on different parts of the force-time trajectory. Seven rats were trained to press a beam with at least 50 g of force to receive a sugar solution. THC was injected, as was apomorphine (APO), a selective dopamine D(2)/D(1) receptor agonist that acts as an antagonist at low doses. Low doses of APO, which have been found to cause deficits in motor execution, were used as a control for the effects of THC. Average peak force of a given press, as well as rate of rise of force, were significantly lowered by THC, as well as by apomorphine. Past research suggests that deficits in the rate of rise of force that can be attributed to depletions of dopamine in the nigrostriatal pathway, as in the case of low doses of APO, reflect failures of motor unit recruitment rather than of motor memory. Similarities in the motor effects of THC and APO suggest that THC plays a role in recruitment and synchronization of motor neurons appropriate for a given task.

Opioid growth factor modulates angiogenesis.

OBJECTIVE: Induced angiogenesis has recently been attempted as a therapeutic modality in patients with occlusive arterial atherosclerotic disease. We investigated the possible role of endogenous opioids in the modulation of angiogenesis. METHODS: Chick chorioallantoic membrane was used as an in vivo model to study angiogenesis. Fertilized chick eggs were incubated for 3 days, explanted, and incubated for an additional 2 days. Three-millimeter methylcellulose disks were placed on the surface of the chorioallantoic membrane; each disk contained opioid growth factor ([Met(5)]-enkephalin; 5 microgram), the short-acting opioid receptor antagonist naloxone (5 microgram), opioid growth factor and naloxone together (5 microgram of each), the long-acting opioid antagonist naltrexone (5 microgram), or distilled water (control). A second series of experiments was performed with distilled water, the angiogenic inhibitor retinoic acid (1 microgram), and vascular endothelial growth factor (1 microgram) to further evaluate our model. The developing vasculature was imaged 2 days later with a digital camera and exported to a computer for image analysis. Total number of blood vessels, total vessel length, and mean vessel length were measured within a 100-mm(2) region surrounding each applied disk. Immunocytochemical analysis was performed with antibodies directed against opioid growth factor and its receptor (OGFr). RESULTS: Opioid growth factor had a significant inhibitory effect on angiogenesis, both the number of blood vessels and the total vessel length being decreased (by 35% and 20%, respectively) in comparison with control levels (P <.005). The simultaneous addition of naloxone and opioid growth factor had no effect on blood vessel growth, nor did naloxone alone. Chorioallantoic membranes exposed to naltrexone displayed increases of 51% and 24% in blood vessel number and length, respectively, in comparison with control specimens (P <.005). These results indicate that the opioid growth factor effects are receptor mediated and tonically active. Immunocytochemistry demonstrated the presence of both opioid growth factor and OGFr within the endothelial cells and mesenchymal cells of the developing chorioallantoic membrane vessel wall. Retinoic acid significantly reduced the number and the total length of blood vessels, whereas vascular endothelial growth factor increased both the number and the length of blood vessels in comparison with the controls (P <.0001). The magnitude of opioid growth factor's effects were comparable to those seen with retinoic acid, whereas inhibition of opioid growth factor with naltrexone induced an increase in total vessel length comparable to that for vascular endothelial growth factor. CONCLUSIONS: These results demonstrate for the first time that endogenous opioids modulate in vivo angiogenesis. Opioid growth factor is a tonically active peptide that has a receptor-mediated action in regulating angiogenesis in developing endothelial and mesenchymal vascular cells.

The opioid growth factor, [Met5]-enkephalin, inhibits DNA synthesis during recornification of mouse tail skin.

Opioid peptides serve as tonically active negative growth regulators in renewing and regenerating epithelia. To examine the involvement of opioids in renewal of the stratum corneum after tape stripping of tail skin, C57BL/6 J mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX, 20 mg/kg i.p.) following injury. Blockade of opioid-receptor interaction by NTX for 4 h resulted in an elevation of 36-66% in basal cell DNA synthesis measured 24 h after injury. Injection of the endogenous opioid peptide, [Met5]-enkephalin (OGF, 10 mg/kg i.p.) 4 h before termination, suppressed radiolabelled thymidine incorporation in the basal cell layer by 37-46% at 24 h after wounding. The magnitude of the effects on DNA synthesis of OGF, but not NTX, depended on the timing of administration with respect to injury. OGF maximally depressed basal cell labelling (72%) when given 16 h after tape stripping. Concomitant administration of naloxone (10 mg/kg) with OGF blocked the inhibition of DNA synthesis; naloxone alone at the dosage utilized had no effect on cell labelling. Both OGF and its receptor, OGFr, were detected by immunocytochemistry in the basal and suprabasal cell layers, but not the cornified layer of tape stripped and uninjured tail skin. These results indicate: (a) a native opioid peptide and its receptor are expressed in epidermal cells of injured and uninjured mouse tail skin; (b) removal of the stratum corneum by tape stripping does not disrupt the function of the endogenous opioid growth system; (c) the proliferative response to wounding of the tail is tonically inhibited by the receptor-mediated action of an endogenous opioid peptide; and (d) DNA synthesis by basal cells can be elevated by disrupting opioid peptide receptor interactions.