Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure

To investigate the possible role of peripheral-type benzodiazepine receptors (PBR) in hepatic encephalopathy, we examined expression of PBR in mouse brain following thioacetamide (TAA)-induced acute liver failure. Treatment of mice with TAA resulted in an increase in the number of binding sites of the PBR ligand [3H] Ro5-4864 to brain homogenates, with no significant change in affinity of the ligand. The order of potency of different ligands to compete against [3H] Ro5-4864 binding in the brain of TAA-treated mice was Ro5-4864 > PK11195 > diazepam > protoporphyrin IX, findings similar to those in the control. Northern blot analysis revealed an increase in PBR/isoquinoline binding protein (PBR/IBP) mRNA in mouse brain following TAA treatment, in a time- and dose-dependent manner. These results indicate that the increased number of PBR in the brains of TAA-treated mice relates to the induction of PBR/IBP expression and suggest that the induction of PBR in brain may contribute to pathogenesis of hepatic encephalopathy.     

Induction of acute phase gene expression by brain irradiation

PURPOSE: To investigate the in vivo acute phase molecular response of the brain to ionizing radiation. METHODS AND MATERIALS: C3Hf/Sed/Kam mice were given midbrain or whole-body irradiation. Cerebral expression of interleukins (IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6), interferon (IFN-gamma), tumor necrosis factors (TNF-alpha and TNF-beta), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), alpha 1-antichymotrypsin (EB22/5.3), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay. The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined. RESULTS: Levels of TNF-alpha, IL-1 beta, ICAM-1, EB22/5.3 and to a lesser extent IL-1 alpha and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain. Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy. Most responses were rapid, peaking within 4-8 h, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsided. Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely. Dexamethasone was more inhibitory than pentoxifylline at the doses chosen. CONCLUSIONS: The initial response of the brain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy. This mechanism explains the beneficial effects of the clinical use of steroids in such circumstances.     

Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.     

Induction and inhibition of cytochrome P450 isoforms by imazalil, a food contaminant, in mouse small intestine and liver

1. The effects of imazalil, a food contaminant used as a fungicide, were investigated on the expression and activity of cytochrome P450 in the small intestinal mucosa and liver of mice. Imazalil was orally administered to mice daily at 1 or 10 mg/kg for 3 days. 2. Imazalil enhanced cytochrome P450-catalysed ethoxyresorufin O-deethylase and pentoxyresorufin O-depentylase (PROD) activities in both tissue microsomes at the 10 mg/kg/day dose level, indicating the induction of cytochrome P450 subfamilies CYP1A and CYP2B. In addition, immunochemical analyses also demonstrated an enhanced expression of CYP2B, CYP2C and CYP3A subfamilies in both tissues. 3. Imazalil was a potent inhibitor of cytochrome P450-dependent monooxygenase activities (PROD, aminopyrine N-demethylase and erythromycin demethylase) in in vitro assays using both small intestinal and liver microsomes. 4. From these findings, imazalil has been demonstrated to have not only a potent inhibitory activity but also a significant inducing ability of P450 isoforms in the small intestine. Prolonged ingestion of such a food contaminant may modulate the xenobiotic-metabolizing enzyme system at the site of a primary portal of xenobiotic entry to the systemic circulation.     

Combating hyperthermia in acute stroke: a significant clinical concern

OBJECTIVE: There is only limited evidence from adequately controlled clinical trials to support high-dose methylprednisolone therapy for attacks of multiple sclerosis (MS) and none supporting oral administration. We assessed the effect of oral high-dose methylprednisolone therapy in attacks of MS. METHODS: Twenty-five patients with an attack of MS lasting less than 4 weeks were randomized to placebo treatment. Twenty-six patients received oral methylprednisolone (500 mg once a day for 5 days with a 10-day tapering period). The patients received scores on the Scripps Neurological Rating Scale (NRS) and Kurtzke Expanded Disability Status Scale. The symptoms were scored on a visual analog scale (VAS) before treatment and after 1, 3, and 8 weeks of treatment. Primary efficacy measures were NRS and VAS scores in the first 3 weeks and changes in NRS score and answers to an efficacy questionnaire administered after 8 weeks of treatment. RESULTS: Changes in NRS scores among methylprednisolone- and placebo-treated patients differed significantly in the first 3 weeks and after 8 weeks (p = 0.005 and p = 0.0007). VAS scores the first 3 weeks and treatment efficacy after 8 weeks also favored a beneficial effect of methylprednisolone treatment (p = 0.02 and p = 0.05). After 1, 3, and 8 weeks, 4%, 24%, and 32% in the placebo group and 31%, 54%, and 65% in the methylprednisolone group had improved one point on the Expanded Disability Status Scale score (all p < 0.05). No serious adverse events were seen. CONCLUSION: Oral high-dose methylprednisolone is recommended for managing attacks of MS.     

Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol 12,13-myristate-type tumor promoters in immortalized mouse liver cells

Increased expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, has been associated with growth regulation and carcinogenesis in several systems. COX-2 is known to be induced by cytokines and the skin tumor promoter 12-tetradecanoylphorbol-13-myristate (TPA). In the present study, we investigated the effects of several non-TPA-type tumor promoters on COX-2 expression in immortalized mouse liver cells. Specifically, we tested peroxisome proliferators (PPs), which are rodent liver tumor promoters that cause gross alterations in cellular lipid metabolism, the rodent liver tumor promoter phenobarbital, and the skin tumor promoters okadaic acid and thapsigargin. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, ciprofibrate ethyl ester, and eicosatetraynoic acid each caused large increases in COX-2 mRNA and protein, with maximal expression seen approximately 10 h after treatment of quiescent cells. COX-2 expression was also induced by thapsigargin, okadaic acid, and calcium ionophore A23187, but not by phenobarbital or the steroid PP dehydroepiandrosterone sulfate. Induction of COX-2 expression generally resulted in increased synthesis of prostaglandin E2 (PGE2). However, the PPs caused little or no increase in PGE2 levels, and they inhibited serum-induced PGE2 synthesis. Unlike non-steroidal anti-inflammatory drugs, the PPs do not directly inhibit cyclooxygenase enzyme activity in vitro. Thus, PPs regulate prostaglandin metabolism via both positive (COX-2 induction) and inhibitory mechanisms. In summary, the strong induction of COX-2 expression by PPs, thapsigargin, and okadaic acid suggests a possible role for COX-2 in the growth regulatory activity of these non-TPA-type tumor promoters.     

Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased in normal mice but virtually not affected in the transgenic animals. This suggests that ectopically expressed parvalbumin is involved in the regulation of Ca2+ signals in the sinusoidal endothelial cells. This animal model could be of interest to those working on the physiology of liver circulation.     

Induction of apoptosis in liver tumors by the monoterpene perillyl alcohol

The monoterpenes d-limonene and perillyl alcohol (POH) inhibit the growth of mammary tumors. In this investigation we tested whether POH is also effective in reducing liver tumor growth. Diethylnitrosamine was used to induce liver tumors in male Fischer 344 rats. Two weeks after diethylnitrosamine exposure was discontinued, the animals were divided into POH-treated and untreated groups. The mean liver tumor weight for the POH-treated rats after 19 weeks of POH treatment was 10-fold less than that for the untreated animals. POH did not influence tumor cell proliferation but increased the apoptotic index approximately 10-fold. The mRNA levels for the mannose 6-phosphate/insulin-like growth factor II receptor and the transforming growth factor beta type I, II, and III receptors were also significantly increased in the liver tumors from the POH-treated animals when compared to the corresponding receptor mRNA levels in the normal tissue surrounding the tumors and in the tumors of untreated animals. These results demonstrate that POH does not promote the formation of liver tumors, but rather inhibits their growth by enhancing tumor cell loss through apoptosis.     

Induction of differentiation in mouse neuroblastoma cells

In the presence of 1--2% dimethyl sulfoxide (DMSO), mouse neuroblastoma cells are induced to differentiate morphologically as well as electrically. In addition, treatment of neurolbastoma cells with 2% DMSO results in a marked increase in the veratridine-activated K+ or Rb+ efflux. At 4% DMSO, neurite outgrowth is completely repressed and electrical activity is poorly developed. However, at this concentration, the cells have a relatively high resting potential which suggested that membrane components determining passive and active permeability properties are not necessarily under coordinated control. Induction of differentiation by 2% DMSO is also accompanied by an increase in a heavier molecular form of acetylcholinesterase sedimenting at 10.5S. The effect of other agents on the growth and differentiation of neuroblastoma cells is also presented.     

Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse

We describe a patient, RM, who suddenly became amnesic for premorbid autobiographic events in the absence of any known precipitating event. Learning abilities as well as semantic knowledge were normal. Knowledge of famous facts and persons was good, although not perfect. Whether RM suffered from organic or psychogenic isolated retrograde amnesia (IRA) could not be established on the basis of available clinical and neuropsychological elements. Regardless of its aetiology, RM's case respects the boundaries between semantic and episodic memory and so gives further support to the distinction between these two memory systems.     

Inactivation of protein kinase C in rat liver during late hypoglycemic phase of sepsis

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat liver during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. Hepatic PKC was extracted and partially purified by ammonium sulfate fractionation and DEAE-cellulose chromatography. PKC activity was assayed based on the rate of incorporation of 32p from [gamma-32P]ATP into histone. The results show that during early sepsis, both membrane-associated and cytosolic PKC activities remained relatively unaltered. During late sepsis, membrane-associated PKC was unaffected while cytosolic PKC activity was decreased by 19.5-34.4%. Kinetic analysis of the data on cytosolic PKC during late phase of sepsis reveals that the Vmax values for ATP, histone, Ca2+, phosphatidylserine, and diacylglycerol were decreased by 23.4, 22.1, 19.5, 25, and 34.4%, respectively, with no changes in their Km values. These data indicate that cytosolic PKC activity was inactivated in rat liver during late hypoglycemic phase of sepsis. Since PKC-mediated phosphorylation plays an important role in regulating hepatic glucose metabolism, an inactivation of cytosolic PKC may contribute to the development of hypoglycemia during late phase of sepsis.     

The acute phase reaction syndrome: the acute phase reactants (a review)

The acute phase reaction of the organism, which determines the response that follows the injury of tissues and organs and helps the survival of the individual is reviewed. The main participants, the induction of the reaction and the regulatory mechanisms are shortly discussed, with special respect to the prominent role of interleukin-6. The relationship between the production and synthesis of the acute phase reactants under physiological and pathophysiological conditions is analyzed. The particular functions of orosomucoid and alpha 2HS-glycoprotein are summarized. Finally, the common biological actions of IL-6 and the acute phase reactants are mentioned.     

Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope

Self proteins are handled in the same way as foreign proteins by antigen presenting cells, but because of T-cell tolerance the presentation of self peptides does not normally lead to T cell activation. By providing physically linked T-cell help it is possible to overcome the B cell non-responsiveness toward self antigens. We have shown previously that a very potent antibody response, cross-reactive with a self protein, can be rapidly induced by immunizing with a recombinant immunogen consisting of the self protein with a foreign immunodominant T helper epitope inserted into its sequence (Dalum, I., Jensen, M. R., Hindersson, P., Elsner, H. I. and Mouritsen, S. (1996) J. Immnunol. 157, 4796). In this study we compare this approach for inducing autoantibodies against a self protein with the traditional method of conjugating the self antigen to a foreign carrier protein. The highly conserved self protein ubiquitin with an inserted epitope from ovalbumin (UbiOVA) is used as a model protein and compared to two traditionally conjugated immunogens consisting of ubiquitin chemically conjugated to a peptidic T helper epitope or to ovalbumin. The traditionally conjugated immunogens induce much slower and low titered ubiquitin specific antibody responses than the recombinant construct which also is capable of inducing antibodies directed against a much broader range of potential ubiquitin B cell determinants than the chemically conjugated immunogens. All three constructs are processed by antigen presenting cells and ovalbumin derived T cell epitopes are presented to T helper cells. From these observations it seems likely that the presence of non-shielded autologous B cell determinants on the immunogen is critical for the ability to induce a strong autoantibody response with a diverse fine specificity. Furthermore, the ubiquitin specific antibodies induced by UbiOVA contain higher levels of IgG2a/b relative to IgG1 compared to the conjugates. We therefore speculate that the insertion of a T cell epitope directly into the self antigen could possibly induce an immune response with a different Th1/Th2 balance than a response induced with traditional conjugates.     

Induction of acute inflammatory lung injury by staphylococcal enterotoxin B

Superantigens stimulate T lymphocytes at high frequency by interacting with appropriate Vbeta segments of the TCR. Challenge of mice with bacterial superantigens such as staphylococcal enterotoxin B (SEB) induces the systemic release of cytokines resulting in septic shock and death of sensitized animals. Analysis of putative pathogenic mechanisms of T cell-dependent septic shock revealed that administration of SEB results in acute inflammatory lung injury characterized by a profound increase in vascular permeability. Injury was associated with marked leukocyte infiltration of the lung and induction of cell adhesion molecules including VCAM-1, ICAM-1, and P-selectin, but not E-selectin. Lung infiltrating leukocytes consisted of granulocytes, mononuclear phagocytes and NK cells with granulocytes representing the major fraction. Consistent with a role of neutrophils as cellular mediators of inflammatory organ injury, we demonstrate activation of circulating granulocytes in mice treated with SEB. When compared with granulocytes of control mice, peripheral blood granulocytes of SEB-treated mice were found to express increased levels of cell surface Mac-1, to down-regulate expression of L-selectin, and to respond with an increased production of toxic oxygen metabolites upon exposure to FMLP. Interestingly, TNF-alpha further enhanced FMLP-induced oxidant production by granulocytes from SEB-treated but not control mice, suggesting that the systemic response to SEB increases granulocyte sensitivity to TNF-alpha-mediated signals. Together, these results suggest that acute inflammatory lung injury may contribute to the pathogenesis of T cell-dependent lethal shock in mice challenged with bacterial superantigens and indicate common pathogenic mechanisms of lung injury induced by a large number of distinct inflammatory stimuli.