High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats

 NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly but not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAb 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3^bright cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3^bright cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation. Received: 11 July 1995 / Accepted: 16 November 1995     

Host-mediated antiviral activity ofLactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killedLactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated orLactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD₅₀) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bg^J/bg^J) mice, when compared with that in their littermate (bg^J/+) mice. Poor protection of bg^J/bg^J mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Long-term influence of adjuvant therapy on natural killer cell activity in breast cancer

Summary Unstimulated IFN- and IL2-stimulated (NK) cell activities were investigated in patients with breast cancer who had received either local radiotherapy alone or adjuvant chemotherapeutic treatment with CMF combined with radiotherapy 12 to 18 months previously. When tested against the primarily NK-sensitive K562 cell line, patients who had received adjuvant chemotherapeutic treatment with CMF were shown to have a significantly decreased unstimulated and IFN-stimulated NK cell activity, as compared to both patients after radiotherapy only (P<0.002) and P<0.005, respectively) and healthy control persons (P<0.05). The former group of patients also had a significantly decreased IFN-stimulated NK cell activity, when tested against the primarily NK-insensitive Chang hepatoma cell line, as compared to patients after radiotherapy only (P<0.005) and healthy controls (P<0.05). Moreover, patients after radiotherapy only proved to have a significantly increased unstimulated (P<0.01) and IFN-stimulated NK cell activity (K562: P<0.05; Chang hepatoma cell line: P<0.05), as compared to healthy control individuals. In contrast, no difference in IL2-stimulated NK cell activity was detected. The investigation for the expression of CD3 and/or Leu 19 antigens as phenotypic markers of cells with non-MHC restricted cytotoxicity showed a significantly lower percentage of cells with the CD3+ phenotype in patients with breast cancer, irrespective of the chosen post-operative treatment, as compared to healthy controls (P<0.01). Finally, patients with breast cancer who had received radiotherapy only had a significant trend towards an increased percentage of CD³+/Leu 19+ PMNC, as compared to both patients after CMF treatment (P<0.05) and healthy controls (P<0.025). We conclude that patients with breast cancer vary on a long-term basis in their NK activity and in the phenotype of their PMNC depending on their post-operative adjuvant management.Abbreviations NK natural killer cells - IFN interferon - IL 2 interleukin 2 - CMF cyclophosphamide, methotrexate, fluorouracil - ER oestrogen receptor - PMNC peripheral blood mononuclear cells - MHC major histocompatibility complex - CTL cytotoxic T lymphocytes     

Dietary selenium levels determine epidermal langerhans cell numbers in mice

Selenium (Se) is a dietary trace element that is essential for effective immunity and protection from oxidative damage induced by ultraviolet radiation (UVR). Langerhans cells (LC) represent the major antigen-presenting cells resident in the epidermis; a proportion migrate from the skin to the draining lymph nodes in response to UVR. Because it is known that Se deficiency impairs immune function, we determined what effect this has on LC numbers. CH3/HeN mice were weaned at 3 wk and placed on diets containing <0.005 ppm of Se (Se deficient) or 0.1 ppm of Se (Se adequate, control mice). After 5 wk on the diet, the epidermal LC numbers in the Se-adequate group were 966±51 cells/mm² and LC counts in the epidermis of the Se-deficient mice were 49% lower (p<0.05). Glutathione peroxidase-I (GPx) activity was measured in the epidermis, lymph nodes, and liver. In the epidermis, the activity of GPx in the Se-deficient mice was only 39% (p<0.01) of that seen in epidermis from Se-adequate mice (1.732 U/mg protein). The mice were then irradiated with one dose of 1440 J/m² of broadband UVB or mock irradiated. After 24 h, the decrease in LC number after UVB was greater in the Se-adequate mice, (40% decrease) compared to the Se-deficient group (10%). Thus, Se deficiency reduces epidermal LC numbers, an effect that might compromise cutaneous immunity.     

Low‐carbohydrate High‐fat Diets: Regulation of Energy Balance and Body Weight Regain in Rats

The aim of the current investigations was to examine the effects of a low‐carbohydrate high‐fat diet (LC‐HFD) on body weight, body composition, growth hormone (GH), IGF‐I, and body weight regain after stopping the dietary intervention and returning the diet back to standard laboratory chow (CH). In study one, both adolescent and mature male Wistar rats were maintained on either an isocaloric LC‐HFD or CH for 16 days before having their diet switched. In study two, mature rats were maintained on either LC‐HFD or CH for 16 days to determine the effects of the LC‐HFD on fat pad weight. LC‐HFD leads to body weight loss in mature rats (P < 0.01) and lack of body weight gain in adolescent rats (P < 0.01). Despite less body weight, increased body fat was observed in rats maintained on LC‐HFD (P < 0.05). Leptin concentrations were higher (P < 0.05), and IGF‐I (P < 0.01) concentrations were reduced in the LC‐HFD rats. When the diet was returned to CH following LC‐HFD, body weight regain was above and beyond that which was lost (P < 0.01). The LC‐HFD resulted in increased body fat and had a negative effect upon both GH and IGF‐I concentrations, which might have implications for the accretion and maintenance of lean body mass (LBM), normal growth rate and overall metabolic health. Moreover, when the LC‐HFD ceases and a high‐carbohydrate diet follows, more body weight is regained as compared to when the LC‐HFD is consumed, in the absence of increased energy intake.     

Enhanced dopamine D<Subscript>2</Subscript> receptor function in hypothalamus and corpus striatum: their role in liver,plasma and in vitro hepatocyte ALDH regulation in ethahol treated rats

Dopamine D₂ receptors are involved in ethanol self- administration behavior and also suggested to mediate the onset and offset of ethanol drinking. In the present study, we investigated dopamine (DA) content and Dopamine D₂ (DA D₂) receptors in the hypothalamus and corpus striatum of ethanol treated rats and aldehyde dehydrogenase (ALDH) activity in the liver and plasma of ethanol treated rats and in vitro hepatocyte cultures. Hypothalamic and corpus striatal DA content decreased significantly (P < 0.05, P < 0.001 respectively) and homovanillic acid/dopamine (HVA/DA) ratio increased significantly (P < 0.001) in ethanol treated rats when compared to control. Scatchard analysis of [³H] YM-09151-2 binding to DA D₂ receptors in hypothalamus showed a significant increase (P < 0.001) in B_max without any change in K_d in ethanol treated rats compared to control. The K_d of DA D₂ receptors significantly decreased (P < 0.05) in the corpus striatum of ethanol treated rats when compared to control. DA D₂ receptor affinity in the hypothalamus and corpus striatum of control and ethanol treated rats fitted to a single site model with unity as Hill slope value. The in vitro studies on hepatocyte cultures showed that 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in 10% ethanol treated cells to near control level. Sulpiride, an antagonist of DA D₂, reversed the effect of dopamine on 10% ethanol induced ALDH activity in hepatocytes. Our results showed a decreased dopamine concentration with enhanced DA D₂ receptors in the hypothalamus and corpus striatum of ethanol treated rats. Also, increased ALDH was observed in the plasma and liver of ethanol treated rats and in vitro hepatocyte cultures with 10% ethanol as a compensatory mechanism for increased aldehyde production due to increased dopamine metabolism. A decrease in dopamine concentration in major brain regions is coupled with an increase in ALDH activity in liver and plasma, which contributes to the tendency for alcoholism. Since the administration of 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in ethanol treated cells to near control level, this has therapeutic application to correct ethanol addicts from addiction due to allergic reaction observed in aldehyde accumulation.     

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross-priming of EGFR-specific CD8+ T cells

Background

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10–20 % of patients. An immunological mechanism of action such as natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells.

Objective

To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells.

Methods

Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8⁺ T cells were isolated and analyzed using ⁵¹Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR_853-861 tetramer staining.

Results

TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8⁺ T cells in the presence of cetuximab.

Discussion

VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.     

A comparative evaluation of the biological effects of environmental cadmium-contaminated control diet and laboratory-cadmium supplemented test diet

This study was designed to examine the effects of a diet contaminated by environmental cadmium on organ/body weight ratio and selected toxicological indices. It was also designed to permit a comparative analysis of the effects of the diet contaminated by environmental cadmium and that of cadmium supplemented diet based on the same parameters. Our results show that even though the cadmium content of the environmental cadmium contaminated diet was 300% less than that of the cadmium supplemented diet, the former caused statistically significant changes in Superoxide dismutase (SOD) activity (P < 0.025), Na⁺/K⁺ ATPase activity (P < 0.005) and Malondialdehyde (MDA) level (P < 0.025) when values at the end of 1 month exposure were compared to the values at the end of 3 months exposure. These parameters were altered in the same manner by the cadmium content of the supplemented diet in addition to significant reduction in liver/body weight ratio(P < 0.005) within the exposure periods examined. By virtue of the very close nature of the values of these parameters in rats exposed to the two different diets, it appears that the background cadmium (cadmium from the environment) content of the diets is largely responsible for the observed changes, except in the case of liver/body weight ratio.Published online: March 2005     

Trypanosoma evansi: Adenosine deaminase activity in the brain of infected rats

The study was undertaken to evaluate changes in the activity of adenosine deaminase (ADA) in brains of rats infected by Trypanosoma evansi. Each rat was intraperitoneally infected with 10⁶ trypomastigotes either suspended in fresh (group A; n = 13) and cryopreserved blood (group B; n = 13). Thirteen animals were used as control (group C). ADA activity was estimated in the cerebellum, cerebral cortex, striatum and hippocampus. No differences (P > 0.05) in ADA activity were observed in the cerebellum between infected and non-infected animals. Significant (P < 0.05) reductions in ADA activity occurred in cerebral cortex in acutely (day 4 post-infection; PI) and chronically (day 20 PI) infected rats. ADA activity was significantly (P < 0.05) decreased in the hippocampus in acutely infected rats, but significantly (P < 0.05) increased in the chronically infected rats. Significant (P < 0.05) reductions in ADA activity occurred in the striatum of chronically infected rats. Parasites could be found in peripheral blood and brain tissue through microscopic examination and PCR assay, respectively, in acutely and chronically infected rats. The reduction of ADA activity in the brain was associated with high levels of parasitemia and anemia in acute infections. Alterations in ADA activity of the brain in T. evansi-infected rats may have implications for pathogenesis of the disease.     

Evidence for the role of natural immunity in the control of metastatic spread of head and neck cancer

Summary Deficient natural killer (NK) cell activity may contribute to the development of distant metastases in the head and neck cancer patient. A total of 246 previously untreated patients expressed deficient NK activity against K562 target cells when compared to 110 age-matched healthy controls (70±48 lytic units (LU) versus 95±52 LU) (P<0.001). Some 164 consecutive patients have undergone definitive therapy subsequent to NK cell assessment and have been followed for a minimum of 12 months (median=16 months), and 23 have developed recurrent disease in distant sites. The risk of subsequently (1) developing distant metastases, (2) developing regional metastases, and (3) dying of progressive cancer was inversely related to pretreatment NK LU values (P<0.02, <0.02, <0.005, respectively, by the Cox proportional hazards model). NK cell function within the peripheral blood of the patient with head and neck cancer could be related to the percentage of Leu 11+ NK cell subsets (P<0.01 by linear regression analysis) as determined by both single-parameter and multiparameter flow cytometric assessment. Contrastingly, no relationship could be identified between NK function with the percentage of circulating Leu 7+ cell subsets. In vitro measured NK cell function identifies a population at increased risk for developing distant metastases, thus supporting the role of natural immunity as defense mechanism against blood-borne disease.Funded in part by a grant from the Kimberly-Clark Corporation and by grant CA16672 awarded by The National Cancer Institute, Department of Health and Human Services     

Effect of hypoxic stress on the immune response and the resistance to vibriosis of the shrimpPenaeus stylirostris

The immune response of shrimp exposed to severe hypoxia, 1 mg O₂ml⁻¹for 24 h, was measured in terms total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity (PO) and respiratory burst by NBT reduction. Results showed that hypoxia induced a significant decrease (P<5%) of the THC due to a significant decrease of semi-granular cells (SGC) (P<5%) and hyaline cells (HC) (P<5%). There was also a decrease in the NBT reduction. On the other hand, PO activity increased significantly (P<1%) and seemed to be related to a lower amount of plasma inhibitors regulating the prophenoloxidase system. Experimental infection with a virulentVibrio alginolyticuswas carried out by intramuscular injection of 2×10⁴colony forming units (cfu) per shrimp. The mortality of stressed shrimp was 48% while the mortality of control shrimp was 32%. This difference was significant (P<1%). The variations of immunological parameters, except for the PO activity, seem to be consistent with thein vivoresponse against the pathogenicVibrio. Coyright 1998 Academic Press     

运动干预对肥胖大鼠睾丸自噬和凋亡的影响

目的 探讨肥胖对大鼠生精小管结构及自噬和凋亡相关蛋白质的影响,并探讨运动对睾丸自噬和凋亡的影响及其调控机制。方法 将50只6周龄雄性SD大鼠随机分为标准饲养组（SD组,n=20）和高脂饲养组（HFD组,n=30）。HFD组喂养8周建立肥胖大鼠模型,并随机筛选出20只肥胖大鼠进行运动干预。SD组和HFD组分别随机分为标准对照组（CC组）、标准运动组（CE组）、肥胖对照组（OC组）、肥胖运动组（OE组）,每组10只。其中CE组和OE组进行8周中等强度跑台运动干预,60 min/d,5 d/周,其他两组维持原饲养条件。在最后一次运动结束48 h后,将大鼠腹腔麻醉,称重,取大鼠左右两侧睾丸、称量睾丸重量并计算睾丸指数。制作睾丸石蜡切片,利用HE染色法观察睾丸组织结构。采用蛋白质印迹法（Western blot）检测睾丸组织中p62、LC3II、LC3I、BCL-2、Bax和AMPK蛋白表达量并计算LC3II/LC3I比值,采用免疫荧光检测睾丸中LC3和BCL-2蛋白表达位置。结果 与CC组相比,OC组大鼠睾丸指数降低,生精小管直径显著降低（P<0.01）,精子细胞减少,睾丸组织中有脂滴沉...     

Enhanced [<Superscript>3</Superscript>H] Glutamate Binding in the Cerebellum of Insulin-Induced Hypoglycaemic and Streptozotocin-Induced Diabetic Rats

Aim Energy deprivation causes neuronal death affecting the cognitive and memory ability of an individual. The kinetic parameters of glutamate dehydrogenase (GDH), the enzyme involved in the production of glutamate, was studied in the cerebellum and liver and the binding parameters of glutamate receptors in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats were studied to reveal the role of glutamate excitotoxicity. Methods A single intrafemoral dose of streptozotocin was administered to induce diabetes. Hypoglycaemia was induced by appropriate doses of insulin subcutaneously in control and diabetic rats. The kinetic parameters V _max and K _m of GDH were studied spectrophotometrically at different substrate concentrations of α-ketoglutarate. Glutamate receptor binding assay was done with different concentrations of [³H] Glutamate. Results The GDH enzyme assay showed a significant increase (P < 0.001) in the V _max of the enzyme in the cerebellum of hypoglycaemic and diabetic rat groups when compared to control. The V _max of hypoglycaemic groups was significantly increased (P < 0.001) when compared to diabetic group. In the liver, the V _max of GDH was significantly increased (P < 0.001) in the diabetic and diabetic hypoglycaemia group when compared to control. The V _max of GDH increased significantly (P < 0.001) in the diabetic hypoglycaemic rats compared to diabetic group, whereas the control hypoglycaemic rats showed a significant decrease in V _max (P < 0.001) when compared to diabetic and diabetic hypoglycaemic rats. The K _m showed no significant change amongst the groups in cerebellum and liver. Scatchard analysis showed a significant increase (P < 0.001) in B _max in the cerebellum of hypoglycaemic and diabetic rats when compared to control. The B _max of hypoglycaemic rats significantly increased (P < 0.001) when compared to diabetic group. In hypoglycaemic groups, B _max of the control hypoglycaemic rats showed a significant increase (P < 0.001) compared to diabetic hypoglycaemic rats. The K _d of the diabetic group decreased significantly (P < 0.01) when compared to control and control hypoglycaemic rats. There was a significant decrease (P < 0.05) in the K _d of diabetic hypoglycaemic group when compared to the control hypoglycaemic rats. Conclusion Our studies demonstrated the increased enzyme activity in the hypoglycaemic rats with increased production of extracellular glutamate. The present study also revealed increased binding parameters of glutamate receptors reflecting an increased receptor number with increase in the affinity. This increased number of receptors and the increased glutamate production will lead to glutamate excitotoxicity and neuronal degeneration which has an impact on the cognitive and memory ability. This has immense clinical significance in the management of diabetes and insulin therapy.     

Comparative effect of water and food-chain mediated cadmium exposure in rats

This study sets out to compare the absorption and toxicity of Cadmium (Cd) administered via the food-chain and inorganic Cd administered in drinking water after 1 and 3 months exposure using rats as animal model. The food-chain was mimicked by exposing rats to diet containing Cd pre-exposed fish. The uptake of Cd by the rats after both mode of exposure was calculated by summing up the Cd burden in the liver and kidneys and was expressed in terms of % intake. The toxicity of Cd was assessed by monitoring biochemical indices of liver function in the plasma and liver. Regardless of the mode of exposure of the rats, the Cd load in the liver and kidney was significantly (P < 0.05) higher than the respective controls with the kidney having a significantly higher load than the liver after both periods of exposure. However irrespective of the mode of exposure, more Cd was accumulated in the liver and kidney of the 3 months exposed rats relative to those exposed for 1 month. The uptake of Cd by rats exposed to Cd via the food-chain for 1 and 3 months was significantly (P < 0.05) lower when compared to the corresponding water mediated Cd exposed rats, except for the liver after 3 months of exposure. The liver l-ALT activity of rats administered inorganic Cd in drinking water for 1 and 3 months was significantly (P < 0.05) lower as compared to controls. Parallel analysis of the plasma showed no significant (P > 0.05) difference in l-ALT activity between both groups after the same periods of exposure. The l-AST activity in the plasma of rats similarly exposed to Cd for 1 and 3 months was significantly (P < 0.05) higher as compared to controls with a corresponding reduction in the liver. Conversely no significant (P > 0.05) change was observed in plasma and liver l-ALT and l-AST activities after food-chain mediated exposure to Cd for 1 and 3 months in relation to their respective controls. These findings indicate that Cd incorporated in fish is more easily bioavailable, but less toxic relative to inorganic Cd salts at the end of 3 months of exposure in rats.     

Aldolase A as a prognostic factor and mediator of progression via inducing epithelial–mesenchymal transition in gastric cancer

Glycolysis is regarded as the hallmark of cancer development and progression, which involves a multistep enzymatic reaction. This study aimed to explore the clinicopathological significance and potential role of glycolytic enzyme aldolase A (ALDOA) in the carcinogenesis and progression of gastric cancer (GC). ALDOA was screened from three paired liver metastasis tissues and primary GC tissues and further explored with clinical samples and in vitro studies. The ALDOA protein level significantly correlated with a larger tumor diameter (P = .004), advanced T stage (P < .001), N stage (P < .001) and lymphovascular invasion (P = .001). Moreover, the expression of ALDOA was an independent prognostic factor for the 5‐year overall survival and disease‐free survival of patients with GC in both univariate and multivariate survival analyses (P < .05). Silencing the expression of ALDOA in GC cell lines significantly impaired cell growth, proliferation and invasion ability (P < .05). Knockdown of the expression of ALDOA reversed the epithelial–mesenchymal transition process. Mechanically, ALDOA could affect the hypoxia‐inducible factor (HIF)‐1α activity as demonstrated by the HIF‐1α response element–luciferase activity in GC cells. Collectively, this study revealed that ALDOA was a potential biomarker of GC prognosis and was important in the carcinogenesis and progression of human GC.     

T‐Cell Recruitment and Th1 Polarization in Adipose Tissue During Diet‐Induced Obesity in C57BL/6 Mice

The role of adaptive immunity in obesity‐associated adipose tissue (AT) inflammation and insulin resistance (IR) is controversial. We employed flow cytometry and quantitative PCR to assess T‐cell recruitment and activation in epididymal AT (eAT) of C57BL/6 mice during 4–22 weeks of a high‐fat diet (HFD (60% energy)). By week 6, eAT mass and stromal vascular cell (SVC) number increased threefold in mice fed HFD, coincident with onset of IR. We observed no increase in the proportion of CD3⁺ SVCs or in gene expression of CD3, interferon‐γ (IFN‐γ), or regulated upon activation, normal T‐cell expressed and secreted (RANTES) during the first 16 weeks of HFD. In contrast, CD11c⁺ macrophages (MΦ) were enriched sixfold by week 8 (P < 0.01). SVC enrichment for T cells (predominantly CD4⁺ and CD8⁺) and elevated IFN‐γ and RANTES gene expression were detected by 20–22 weeks of HFD (P < 0.01), coincident with the resolution of eAT remodeling. HFD‐induced T‐cell priming earlier in the obesity time course is suggested by (i) elevated (fivefold) interleukin‐12 (IL‐12)p40 gene expression in eAT by week 12 (P ≤ 0.01) and (ii) greater IFN‐γ secretion from phorbol myristate acetate (PMA)/ionophore‐stimulated eAT explants at week 6 (onefold, P = 0.08) and week 12 (fivefold, P < 0.001). In conclusion, T‐cell enrichment and IFN‐γ gene induction occur subsequent to AT macrophage (ATMΦ) recruitment, onset of IR and resolution of eAT remodeling. However, enhanced priming for IFN‐γ production suggests the contribution of CD4⁺ and/or CD8⁺ effectors to cell‐mediated immune responses promoting HFD‐induced AT inflammation and IR.     

Decreased dopamine D2 receptor function in cerebral cortex and brain stem: their role in hepatic ALDH regulation in ethanol treated rats

Ethanol exerts numerous pharmacological effects through its interaction with various neurotransmitters. The dopaminergic pathway is associated with cognitive, endocrine, and motor functions, and reinforcement of addictive substances or behaviours. Aldehyde dehydrogenase (ALDH) is a vital enzyme involved with alcohol metabolism and detoxification. In the present study, we investigated the role of cerebral cortex and brain stem dopamine D₂ receptors in the functional regulation on ALDH enzyme activity, in ethanol administrated rats. Two groups of rats were selected viz. control and alcoholic. Cerebral cortex, brain stem and the liver dopamine content was decreased significantly (P < 0.05, 0.05, 0.001, respectively) and homovanillic acid/dopamine (HVA/DA) ratio has significantly increased (P < 0.05, 0.001 and 0.001), respectively in ethanol treated rats when compared to control. Scatchard analysis of [³H]YM-09151-2 binding to synaptic membrane preparations of cerebral cortex and brain stem showed a significant decrease (P < 0.001, 0.05, respectively) in B _max in ethanol treated rats compared to control and the K _d also decreased significantly (P < 0.05). The ALDH analysis showed a significant increase (P < 0.05) in V _max in cerebral cortex, plasma and liver of experimental rats when compared with control without having significant change in brain stem but with decreased K _m (P < 0.001). Our results suggest that decreased function of dopamine mediated through DA D₂ receptor in the cerebral cortex and brain stem enhanced the brain, plasma and liver ALDH activity in ethanol treated rats. This ALDH regulation has significance to correct alcoholics from addiction due to allergic reaction observed in aldehyde accumulation.     

Differential modulation of natural and adaptive immunity in fischer rats exposed for 6 weeks to 60 Hz linear sinusoidal continuous-wave magnetic fields

Two separate, independent experiments were conducted to evaluate the effect of 60 Hz linearly polarized, sinusoidal, continuous-wave magnetic fields (MFs) on immune system performances in rats born and raised under these fields. Each experiment lasted for 6 weeks. A total of 96 animals, divided into groups of eight animals each, was exposed for 20 h/day to MFs of different intensities, i.e., sham (<0.02 μT) and 2, 20, 200, and 2000 μT. Another group of animals, which was housed in a separate room, served as cage controls (CC). These animals were exposed to ambient MFs of <0.02 μT. The following immune responses were evaluated in both experiments: total T and B cells; CD4⁺ and CD8⁺ subpopulation and natural killer (NK) cell activity in splenic lymphocytes; hydrogen peroxide (H₂O₂), nitrous oxide (NO), and tumor necrosis factor (TNF) production by peritoneal macrophages. Our results show that a 6 week exposure to MFs induced a significant decrease in the number of CD5⁺, CD4⁺, and CD8⁺ populations. These changes were even more significant in rats that were exposed to fields of 2000 μT. A lower, although significant, decrease in the CD5⁺ population was also observed in animals that were exposed to fields of 200 μT. Linear regression analysis demonstrated a dose effect with MF intensity. B lymphocyte (Ig⁺ cell) populations also showed a 12% decrease (P < .05) in the groups that were exposed to fields of 20 and 200 μT. However, these results were not significant, and no relation with MF intensities could be demonstrated. In contrast, evaluation of splenic NK cell activity revealed a 50% increase (P < .05) in animals that were exposed to fields of 2000 μT. No significant results were obtained from the evaluation of TNF activity and NO secretion in peritoneal macrophages. Phorbol 12-myristate 13-acetate (PMA)-stimulated and net H₂O₂ productions for a minor subpopulation of peritoneal cells showed positive dose-response correlations by linear regression analysis. Taken together, our results suggest that an in vivo exposure of rats for 6 weeks to 60 Hz MFs can induce significant immunological perturbations on effector cells of both natural and adaptive immunity in a dose-dependent fashion. © 1996 Wiley-Liss, Inc.     

ATP Accelerates Respiration of Mitochondria From Rat Lung and Suppresses Their Release of Hydrogen Peroxide

Lung mitochondria were isolated by differential centrifugation from pentobarbital-anesthetized male rats. One to three millimolar Mg²⁺-ATP increased the consumption of oxygen of lung mitochondria oxidizing 10 mM succinate > fourfold (P < 0.01) whereas ATP increased the respiration of liver mitochondria by < 35%. ATP also hyperpolarized partially uncoupled lung mitochondria in the presence of the mitochondria-specific antagonist, oligomycin. However, only 20% of the ATPase activity in the lung mitochondria was blocked by oligomycin compared to a blockade of 91% for liver mitochondria. We investigated the effect of reducing the non-mitochondrial ATPase activity in the lung preparation. A purer suspension of lung mitochondria from a Percoll gradient was inhibited 95% by oligomycin. The volume fraction identified as mitochondria by electron microscopy in this suspension (73.6± 3.5%) did not differ from that for liver mitochondria (69.1± 4.9%). ATP reduced the mean area of the mitochondrial profiles in this Percoll fraction by 15% (P <0.01) and increased its state 3 respiration with succinate as substrate by 1.5-fold (P < 0.01) with no change in the state 4 respiration measured after carboxyatractyloside. Hence, ATP increased the respiratory control ratio (state 3/state 4, P <0.01). In contrast, state 3 respiration with the complex 1-selective substrates, glutamate and malate, did not change with addition of ATP. The acceleration of respiration by ATP was accompanied by decreased production of H₂O₂. Thus ATP-dependent processes that increase respiration appear to improve lung mitochondrial function while minimizing the release of reactive oxygen species.     

Increased metabolism of retinoic acid after chronic ethanol consumption in rat liver microsomes

In rat liver microsomes, all-trans-[11,12-³H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[³H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.