Expression profile of multiple secretory phospholipase A2 isoforms in the rat CNS: Enriched expression of sPLA2-IIA in brainstem and spinal cord

Phospholipases A₂ (PLA₂) are enzymes which cleave the sn-2 ester bond in membrane phospholipids to release free fatty acids and lysophospholipids. The present study aimed to elucidate the expression profile of multiple secretory phospholipase A₂ (sPLA₂) isoforms in the normal rat CNS with focus on sPLA₂-IIA in the brainstem and spinal cord. Quantitative RT-PCR analysis showed that sPLA₂-IB expression was low throughout the CNS, sPLA₂-IIA expression was high in the brainstem and spinal cord, sPLA₂-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/hypothalamus, sPLA₂-V expression was high in the olfactory bulb and cerebellum, and sPLA₂-X was expressed at very low levels in the normal CNS. Of the isoforms, sPLA₂-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway. Western blot analysis showed high level of sPLA₂-IIA expression in the brainstem and cervical, thoracic and lumbar spinal segments but low level of expression in other parts of the brain. sPLA₂-IIA was localized by immunohistochemistry to the spinal trigeminal and facial motor nuclei and dorsal- and ventral-horns of the spinal cord. The enzyme was found on the endoplasmic reticulum of neuronal cell bodies and small diameter dendrites or dendritic spines at electron microscopy. The expression of sPLA₂-IIA in the dorsal horn and spinal trigeminal nucleus is consistent with previous results which showed an important role of CNS sPLA₂ in nociceptive transmission.     

Necrosectomy and postoperative local lavage in patients with necrotizing pancreatitis: Results of a prospective clinical trial

Seventy-four patients with necrotizing pancreatitis were included in a prospective clinical trial of a surgical management protocol comprising necrosectomy and postoperative local lavage of the lesser sac and of the necrosis cavity. Fifty-eight patients showed preoperative organ failures such as pulmonary dysfunctions (57%), renal dysfunctions (37%), shock (12%), and sepsis (26%) in spite of intensive care treatment. The median value of the early prognostic signs was 4.5 points. Intraoperatively, 62% of the patients revealed extensive intrapancreatic parenchymal necrosis, 69% had extrapancreatic necrosis, and 39% showed bacterial contamination of the necrotic material. Following the necrosectomy, postoperative local lavage was performed for an average period of 25 days with 7 liters (median) of lavage fluid per 24 hours. In each of 18 studied patients, a considerable release of immunoreactive trypsin was demonstrated and, in each of 20 studied patients, a high concentration of immunoreactive phospholipase A₂ was demonstrated in the lavage fluid up to the 12th/14th postoperative day. The intensive care period averaged 6 1/2 days, the hospital stay averaged 54 days. The hospital mortality rate was 8.1%. It is concluded that restricted necrosectomy and postoperative local lavage treatment correspond in particular to the pathomorphologic conditions and to the local release of biologically active compounds such as bacteria, endotoxin, trypsin, and phospholipase A₂ in patients with necrotizing pancreatitis.

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Resumen Setenta y cuatro pacientes con pancreatitis necrotizante fueron incluídos en un ensayo clínico prospectivo aplicando un protocolo de manejo quirÚrgico que comprende necrosectomía y lavado peritoneal postoperatorio de la transcavidad de los epiplones y de la cavidad necrótica. Cincuenta y ocho pacientes exhibierion fallas orgánicas postoperatorias tales como disfunción pulmonar (57%), disfunción renal (37%), shock (12%), y sepsis (26%) a pesar de cuidado intensivo. El valor promedio de los signos précoces pronóstico (Ranson), con exclusión de la retención de líquido fue de 4.5 puntos. Los hallazgos intraoperatorios revelaron necrosis pancreática extensa en 62% de los pacientes, necrosis extrapancreática en 69%, y contaminación bacteriana del material necrótico en 39%. Realizada la necrosectomía se instauró lavado peritoneal postoperatorio por un período promedio de 25 días con 7 litros (promedio) de líquido por cada 24 horas. En cada uno de los 18 pacientes estudiados se demostró liberación considerable de tripsina inmunorreactiva, así como una elevada concentración de fosfolipasa A₂ inmunorreactiva, en el líquido de lavado hasta el 12/14 días postoperatorios. El período de cuidado intensivo fue de 6 1/2 días, y la hospitalización de 54 días en promedio. La mortalidad hospitalaria fue de 8.1%. En conclusión, se plantea que el tratamiento mediante la necrosectomía restringida y el lavado peritoneal local postoperatorio está indicado en pacientes con las condiciones patomorfológicas de pancreatitis necrotizante que resultan en la liberación local de compuestos biológicamente activos tales como bacterias, endotoxina, tripsina, y fosfolipasa A₂. Serán necesarios ulteriores estudios clínicos controlados para confirmar los resultados favorables que hemos obtenido con la necrosectomía y el lavado peritoneal postoperatorio en pacientes con pancreatitis necrotizante y extensa e infectada necrosis pancreática.

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Résumé Un essai prospectif d'une méthode de traitement chirurgical consistant en nécrosectomie associée au lavage de l'arrière cavité des épiploons et de la cavité nécrotique a concerné 74 malades présentant une pancréatite nécrotique. Malgrè le traitement intensif 58 d'entre eux ont accusé des complications telles que troubles pulmonaires (57%), rénaux (37%), choc (12%), et infection (26%). La valeur moyenne des signes de pronostic précoce fut de 4.5 points. A l'intervention 62% des opérés présentaient une nécrose pancréatique étendue, 69% des opérés une nécrose extra-pancréatique, 39% une surinfection du tissu pancréatique. Après l'exèrése de la nécrose le lavage fut pratiqué quotidiennement avec en moyenne 7 litres de liquide pendant une période de 25 jours. Chez 18 malades fut constaté une libération importante de trypsine immunoactive et chez 20 malades un taux élevé de phospholipase cA dans le liquide de lavage pendant 12/14 jours après l'intervention. La durée des soins intensifs fut en moyenne de 6.5 jours et celle de l'hospitalisation de 54 jours. Le taux de mortalité opératoire fut de 8.1%. On peut conclure de ces faits que la nécrosectomie limitée, associée au lavage local constitue un traitement adapté aux lésions et à la libération locale d'éléments biologiques pathologiques: bactérie, endotoxine, trypsine, et phospholipase A au cours de la pancréatite nécrotique.

     

Serum α- and γ-tocopherol concentrations and risk of advanced beta cell autoimmunity in children with HLA-conferred susceptibility to type 1 diabetes mellitus

Aims/hypothesis The aim of our study was to assess the associations of serum α- and γ-tocopherol concentrations with the risk of advanced beta cell autoimmunity in children with HLA-conferred genetic susceptibility to type 1 diabetes mellitus. Methods A case–control study with 108 cases with advanced beta cell autoimmunity and 216 matched control participants nested within the birth cohort of the Type 1 Diabetes Prediction and Prevention Project. A serum sample for vitamin E analyses was collected from all the children in the cohort at the age of 1 year and thereafter at 12 month intervals. For each case–control group, all the repeated serum samples up to the age of seroconversion to autoantibody positivity in the case were analysed. A conditional logistic regression model was used to determine potential associations between seroconversion and serum tocopherol concentrations. Results Serum α- or γ-tocopherol concentrations were not significantly associated with the risk of advanced beta cell autoimmunity. The odds ratio (95% CI) for μmol/l increase in serum concentration of the first-year sample was 0.97 (0.92–1.03) for α-tocopherol and 1.10 (0.70–1.74) for γ-tocopherol. However, there was an interaction between high values of γ-tocopherol at the age of 1 year and the time of seroconversion (p = 0.024). Conclusions/interpretation It seems unlikely that high concentrations of α- or γ-tocopherol protect against advanced beta cell autoimmunity in young children.     

Urine albumin/creatinine ratio and echocardiographic left ventricular structure and function in hypertensive patients with electrocardiographic left ventricular hypertrophy: the LIFE study. Losartan Intervention for Endpoint Reduction

Background Albuminuria, reflecting systemic microvascular damage, and left ventricular (LV) geometric abnormalities have both been shown to predict increased cardiovascular morbidity and mortality. However, the relationship between these markers of cardiovascular damage has not been evaluated in a large hypertensive population. Methods The urine albumin/creatinine ratio (UACR) and echocardiographic measures of LV structure and function were obtained in 833 patients with stage I to III hypertension and LV hypertrophy determined by electrocardiogram (ECG) (Cornell voltage-duration or Sokolow-Lyon voltage criteria) after 14 days of placebo treatment. Results Patients' mean ages were 66 years, 42% were women, 23% had microalbuminuria, and 5% had macroalbuminuria. Patients with eccentric or concentric LV hypertrophy had higher prevalences of microalbuminuria (average 26%-30% vs 9%, P < .001) and macroalbuminuria (6%-7% vs <1%, P < .001). Furthermore, patients with microalbuminuria and macroalbuminuria had a significantly higher LV mass and lower endocardial and midwall fractional shortening. Patients with abnormal diastolic LV filling parameters had a significantly increased prevalence of microalbuminuria. In univariate analyses, UACR correlated positively to LV mass, systolic blood pressure, age (all P < .001) and pulse pressure/stroke volume and negatively to relative wall thickness (both P < .01) and endocardial (P < .05) and midwall shortening (P < .001) but not to diastolic filling parameters. In multiple regression analysis higher UACR was associated with higher LV mass (β = .169, P < .001) independently of older age (β = .095, P < .01), higher systolic pressure (β = .163), black race (β = .186), and diabetes (β = .241, all P < .001). Conclusions In hypertensive patients with ECG LV hypertrophy, abnormal LV geometry and high LV mass are associated with high UACR independent of age, systolic blood pressure, diabetes, and race, suggesting parallel cardiac and microvascular damage. (Am Heart J 2002;143:319-26.)     

Alcohol and the preventive paradox: serious harms and drinking patterns

AIMS: The preventive paradox prevails if the majority of alcohol problems accrue to the lesser-drinking majority of population, not to heavy drinkers. Evidence for the paradox has been criticized for being based on self-report. The aim was to examine whether the paradox also applies to deaths and hospital admissions. DESIGN: Data from four surveys representing the Finnish population aged 15-69 years in 1969, 1976, 1984 and 1992 were pooled; those from 1969, 1976 and 1984 (n = 6726) to study alcohol-related hospital admissions and alcohol-related deaths, and those from 1984 and 1992 (n = 5558) to study self-reported problems. The former data were linked with register data on hospital admission and death up to the end of 2002. METHODS: Comparisons were made separately for men and women (1) between the 10% of population with the highest average alcohol consumption and the remaining 90% of drinkers and (2) between those who reported and those who did not report drinking to intoxication. RESULTS: A total of 3025 men and 2693 women were available for the study of self-reported problems and 2945 men and 2615 women for deaths and hospital admissions. Seventy per cent of all self-reported problems, 70% of alcohol-related hospitalizations, 64% of alcohol-related deaths and 64% of the premature life-years lost before the age of 65 occurred among the 90% of men consuming less. The respective figures for women were 64%, 60%, 93% and 98%. Drinking five or more drinks per occasion was related to more harm than not drinking that much. CONCLUSIONS: In men, the "prevention paradox" appears to apply to a broadly similar degree to hospitalizations and deaths as self-report alcohol-related problems; in women the phenomenon was apparent to a greater degree for deaths than for other markers of harm.     

Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.

Group II PLA2 has been implicated in inflammatory processes in both man and other animals and has been shown to be involved in inflammatory conditions, such as arthritis and sepsis. Transgenic mice expressing the human group II PLA2 gene have been generated using a 6.2-kb genomic fragment. These mice express the group II PLA2 gene abundantly in liver, lung, kidney, and skin, and have serum PLA2 activity levels approximately eightfold higher than nontransgenic littermates. The group II PLA2 transgenic mice reported here exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia. The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. However, unlike what is seen with these dermatopathies, no significant inflammatory-cell influx was observed in the skin of these animals, or in any other tissue examined. These mice provide an important tool for examining group II PLA2 expression, and for determining the role of group II PLA2 in normal and disease physiology. They serve as an in vivo model for identifying inhibitors of group II PLA2 activity and gene expression.     

The Effect of Dividing Walls, a Tunnel, and Restricted Feeding on Cardiovascular Responses to Cage Change and Gavage in Rats (Rattus norvegicus)

Cage change and gavage are routine procedures in animal facilities, yet little is known about whether housing modifications change responses to these procedures. Telemetric activity and cardiovascular parameters were assessed in this experiment. BN and F344 male rats were housed in open or individually ventilated cages, each containing 3 rats, 1 of which had a transponder. A crossover design was used, in which 2 groups were given dividers made of 2 intersecting boards (1 form contained holes loaded with food pellets; the other did not) and 1 group was given a rectangular tunnel. On day 8 of each 2-wk period, the cages were changed; on day 11, rats were gavaged. The parameters were evaluated for the first hour and for the following 17 h. Baseline values for each rat were subtracted from the corresponding response values. The presence of objects did not affect the responses of F344 rats to cage changing or gavage. In BN rats with IVCs, the presence of the plain divider modified the response to both procedures. Responses to procedures appeared to be dependent on both the strain and the cage object, thus complicating the establishment of valid general recommendations.Abbreviations: bpm, beats per minute; HR, heart rate; IVC, individually ventilated cage; MAP, mean arterial pressureThe new European regulations on laboratory rodents^12,15 mandate the provision of sufficient nest material to build a complete, covered nest or, if doing so is not possible, providing a nest box. Because rats are poor nest-builders,¹⁹ they must be provided with objects for this purpose. Moreover, objects that provide cover or divide the cage area may allow the rats to initiate or avoid contact with cagemates.³⁵All these regulations are rather specific, but generally the same for all laboratory rodents, that is, rats, mice, gerbils, hamsters, and guinea pigs. However, all rodent species and even strains and stocks within a species may have different needs. These differences raise the question of whether general guidelines, which may be valid for 1 species, may have a negative effect on welfare in other species and strains.Cage change is a frequent routine procedure in animal facilities that induces temporary, but significant, cardiovascular and behavioral changes in rats.^{9,10,13,27,29,31-33} Similarly the frequency^9,10 and time²⁹ of changing, type of the bedding material,⁹ light intensity, and length of the dark period³ all modify the intensity of the response to cage changing. The effects on physiologic parameters, such as blood pressure and heart rate, after the cage change seem to be a consequence of the transfer procedure itself and of the novel environment.Two features of rats suggest potential advantages of placing objects in the cage. First, rats are known to have a good sense of smell—1493 specific olfactory receptor genes have been identified on the cilia of the olfactory neurons—and smell is their primary sense for monitoring their environment.²⁴ Second, rats have dominance hierarchies in which fighting is essentially territorial, rather than for any specific object.⁴ The term ‘skirmishing’ has been used to describe a pattern of behaviors often assumed to be aggressive in rats; in 1 study,¹⁰ the frequency of skirmishing was increased during the first 15 min after a cage change. Consequently, cage objects may retain a familiar odor cue during cage change; the presence of the old item in the new cage reduces the aggressive behavior of rats that is triggered by regrouping.¹⁰Gavage is a method widely used to administer test compounds into the stomach of laboratory rats. Rats display increased blood pressure and heart rate (HR) immediately after gavage, and these increases may persist for 30 to 60 min after the procedure.^7,40 Furthermore, elevations in plasma corticosterone levels have been measured in rats after gavage.⁸ The selection of the correct administration volume^2,7,8,40 and a suitable probe material⁴⁰ are important to performing this procedure properly, but whether housing can be advantageous is unknown.Housing refinements have not been assessed in regard to their effect on refining the performance of procedures in rats, although housing modifications can alter their physiology and behavior. Rats have lower blood pressure and HR when housed on bedding compared with a grid or plastic floor.²² Rats also prefer a cage with shelter to a barren environment,³⁶ perhaps because they prefer to spend most of the light phase inside the shelter.¹⁴ Rats with a furnished environment are more active than those which lack such objects,³⁸ and the presence of a shelter in the cage decreases fearfulness.³⁶ Finally, the availability of cage objects may allow the rats to exhibit species-specific behaviors.¹¹In many previous studies of the effects of cage changing or gavage,^{7,8,13,27,29,31-33} the presence of objects in the cages was not described. However, 1 study of both Sprague–Dawley and spontaneously hypertensive male rats showed that providing a multifaceted enrichment program over a week did not affect HR or systolic blood pressure responses to placement in a standard rodent restrainer for 60 min.³⁴ However, after removal from the restrainer, the rats showed a secondary increase in HR and systolic blood pressure that was significantly attenuated in enriched compared with nonenriched rats of both strains. Moreover, enriched rats of both strains had lower HR and systolic blood pressure responses to a variety of procedures, including removal of a cagemate, tail-vein injection, and exposure to the odor of urine and feces of stressed male or female rats.³⁴We hypothesized that cage objects would alter the effect of cage change and gavage on telemetrically recorded cardiovascular parameters and locomotor activity. This study was designed to evaluate the effect of an aspen wall divider with or without restricted feeding and of the presence of an aspen tunnel in the cage on these measures after routine cage changing and gavage of laboratory rats in both open-top cages and IVCs.     

Bactericidal/permeability-increasing protein in colonic mucosa in ulcerative colitis.

BACKGROUND/AIMS: Increased mucosal concentration of bactericidal/permeability-increasing protein (BPI) has been shown in inflammatory bowel diseases. The purpose of the present study was to investigate the relationship between the mucosal concentration of BPI and the grade of mucosal inflammation in ulcerative colitis. METHODOLOGY: Samples of colonic mucosa from 12 patients with ulcerative colitis and from 8 control patients were studied. The concentration of BPI in tissue extracts was measured by a time-resolved fluoroimmunoassay. The concentration of BPI was compared between samples with histological inflammatory changes of different severity. BPI was localized in tissue sections by immunohistochemistry. RESULTS: The concentration of BPI was higher (p < 0.001) in samples of colonic mucosa from patients with ulcerative colitis (median: 3.2 micrograms/g, range: 0.3-22.6 micrograms/g) than in control samples (0.4 microgram/g, 0.1-0.6 microgram/g,). Moreover, the concentration of BPI was higher (p = 0.015) in samples with severe inflammation (2.5 mu/g, 0.3-22.6 micrograms/g) than in those with mild inflammation (0.5 mu/g, 0.3-2.5 micrograms/g). The concentration of BPI in mucosal samples correlated well with the degree of histological inflammation (Spearman R = 0.70, p = 0.01). BPI was localized in polymorphonuclear leukocytes in the mucosa and stroma of the colonic wall. CONCLUSIONS: The concentration of BPI is increased in the colonic mucosa of patients with ulcerative colitis. The increase in the concentration of BPI in colonic mucosa seems to be closely associated with the inflammatory activity of ulcerative colitis.