Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6–8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H. shock; bone marrow cells     

Nature of the change in cytostatic and cytotoxic splenocyte functions in mice after immobilization stress

It has been shown that at late periods after immobilization stress there is an essential reduction in the cytostatic activity of CBA mice splenocytes detected by inhibition of mastocytoma P 815 cell proliferation in the test with 3H-uridine incorporation. The cytostatic activity returns to normal 5 to 14 days after stress. The changes in the cytolytic and cytostatic activity of mouse splenocytes following stress are correlated and a possible biological value of changes in the immunoassays for the host are discussed.     

Effect of interleukin-15 on depressed splenic dendritic cell functions following trauma-hemorrhage

Although trauma-hemorrhage (T-H) induces suppressed splenic dendritic cell (DC) maturation and antigen presentation capacity, it remains unclear whether IL-15 modulates splenic DC functions. The aim of this study therefore was to investigate the effect of IL-15 on splenic DC functions after T-H. Male C3H/HeN mice (6-8 wk old) were randomly assigned to T-H or sham operation. T-H was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x the shed blood volume in the form of Ringer lactate). Two hours later, mice were killed, splenic DCs were isolated, and the effects of exogenous IL-15 on their costimulatory factors, major histocompatibility class II expression, ability to produce cytokines, and antigen presentation were measured. The results indicate that IL-15 production capacity of splenic DCs was reduced following T-H. Ex vivo exposure to IL-15 attenuated the suppressed production of TNF-alpha, IL-6, and IFN-gamma from splenic DCs following T-H. In addition, expression of surface antigen studies demonstrate that exogenous IL-15 attenuated T-H-induced downregulation of the activation of DC. The suppressed splenic DC antigen presentation function following T-H was also attenuated by IL-15 treatment. Moreover, IL-15 enhanced IL-12-induced IFN-gamma production and antigen presentation by splenic DCs. These data suggest that ex vivo treatment with IL-15 following T-H provides beneficial effects on splenic DCs. The depression in IL-15 production by splenic DCs could contribute to the host's enhanced susceptibility to infections following T-H.     

Metoclopramide: a novel adjunct for improving cardiac and hepatocellular functions after trauma-hemorrhage

Although metoclopramide (MCP) administration after trauma-hemorrhage restores the depressed immune functions, it remains unknown whether this agent has any salutary effects on the depressed cardiovascular and hepatocellular functions under those conditions. Adult male Sprague-Dawley rats underwent a midline laparotomy (i.e., induction of soft-tissue trauma) and were then bled to and maintained at a mean arterial pressure (MAP) of 40 mmHg until 40% of the maximal shed blood volume was returned in the form of Ringer lactate (RL). The rats were then resuscitated with four times the shed blood volume in the form of RL over 60 min. MCP (2 mg/kg body wt) or vehicle was administered subcutaneously at the end of resuscitation. At 24 h after resuscitation, cardiac index and hepatocellular function (i.e., the maximum velocity and the overall efficiency of indocyanine green clearance) were determined. Plasma levels of interleukin (IL)-6 and prolactin were also assayed. The results indicate that treatment with MCP after trauma-hemorrhage and resuscitation significantly improved the depressed cardiac output and hepatocellular function. Furthermore, MCP administration significantly increased circulating levels of prolactin and decreased the plasma levels of the proinflammatory cytokine IL-6. Thus, administration of MCP, which increased prolactin secretion, appears to be a useful adjunct for restoring the depressed cardiac and hepatocellular functions and downregulating inflammatory cytokine release after trauma and hemorrhagic shock.     

Transgenic mice: beyond the knockout

Transgenic mice have had a tremendous impact on biomedical research. Most researchers are familiar with transgenic mice that carry Cre recombinase (Cre) and how they are used to create conditional knockouts. However, some researchers are less familiar with many of the other types of transgenic mice and their applications. For example, transgenic mice can be used to study biochemical and molecular pathways in primary cultures and cell suspensions derived from transgenic mice, cell-cell interactions using multiple fluorescent proteins in the same mouse, and the cell cycle in real time and in the whole animal, and they can be used to perform deep tissue imaging in the whole animal, follow cell lineage during development and disease, and isolate large quantities of a pure cell type directly from organs. These novel transgenic mice and their applications provide the means for studying of molecular and biochemical events in the whole animal that was previously limited to cell cultures. In conclusion, transgenic mice are not just for generating knockouts.     

Immunomodulatory effects of dehydroepiandrosterone in proestrus female mice after trauma-hemorrhage.

Studies indicate that administration of the adrenal steroid dehydroepiandrosterone (DHEA) after trauma-hemorrhage in male mice improved cellular immune functions and reduced mortality rates from subsequent sepsis. There is evidence, however, that DHEA is converted to estrogens in males and that estrogens are immunoprotective after trauma-hemorrhage (TH). In contrast, DHEA in females can be converted to testosterone that has deleterious effects on immune functions. The aim of our study, therefore, was to determine whether administration of DHEA in proestrus females after TH would deteriorate immune responses. Proestrus female C3H/HeN mice (age 7-8 wk) were subjected to laparotomy (i.e., soft tissue trauma induced) and hemorrhagic shock (35 +/- 5 mmHg for 90 min) or sham operation. The mice then received DHEA (100 micro/25 g body wt) or vehicle subcutaneously followed by fluid resuscitation (4x the shed blood volume). Plasma IL-6, splenocyte proliferation, splenocyte IL-2, IL-3, IFN-gamma, IL-10 release, and splenic Mphi IL-1beta, IL-6, IL-10, and IL-12 release were determined 24 h after TH. Plasma IL-6 levels were significantly increased in vehicle-treated females, and DHEA administration markedly attenuated this response. In vehicle-treated females, splenocyte proliferation, IL-2, IL-3, and IFN-gamma release, and splenic Mphi IL-1 beta, IL-6, and IL-12 release were maintained or slightly enhanced after TH. In DHEA-treated females, however, these immune functional parameters were either unaltered compared with vehicle-treated animals or even further enhanced, but surprisingly were not depressed. Moreover, DHEA reduced splenocyte and splenic M phi anti-inflammatory cytokine (i.e., IL-10) production after TH compared with vehicle-treated females. Because DHEA further enhances the immune responsiveness in proestrus females after TH, this hormone might be a useful adjunct even in females for further enhancing immune responses and decreasing the mortality rate after trauma and severe blood loss.     

N-acetyl-beta-endorphin1-31 antagonizes the suppressive effect of beta-endorphin1-31 on murine splenocyte proliferation via a naloxone-resistant receptor.

High affinity binding sites for beta-endorphin1-31 (beta-EP) have been observed on transformed mononuclear cells such as the human U937 monocyte-like cell line and the murine EL4-thymoma line, and on normal murine splenocytes. Binding of beta-EP at these sites is resistant to competition by naloxone and other opiate receptor ligands but sensitive to N-acetyl-beta-endorphin1-31 (N-Ac), cations and GTP-gamma-sulfate. Thus, the following studies were done to determine the functional significance of binding beta-EP and N-Ac. beta-EP suppressed phytohemagglutinin (PHA)-stimulated [3H]thymidine uptake in a dose-dependent, naloxone-insensitive fashion. beta-Endorphin1-27, (des)-tyrosine beta-endorphin2-31, or N-Ac failed to duplicate the suppressive effect of beta-EP. However, N-Ac, which is equipotent to beta-EP at displacing 125I-beta-EP bound to murine splenocytes or U937 cells, antagonized the suppressive effect of beta-EP. Taken together with previous binding studies, the present observations suggest that beta-EP effects receptor-mediated responses on normal immunocytes that do not depend on the activation of neuronal-like opiate receptors which are naloxone-sensitive. N-Ac, which shows minimal binding to such brain opiate receptors, is a potent functional antagonist of the naloxone-insensitive immunocyte receptor for beta-EP.     

1,25-Dihydroxyvitamin D3 induces splenocyte apoptosis and enhances BALB/c mice sensitivity to toxoplasmosis

The hormonal form of Vitamin D, 1,25-dihydroxyvitamin D3, is well known for its immunosuppressive, anti-proliferative and pro-apoptotic activities. In the present work, we studied the effect of 1,25-dihydroxyvitamin D3 on Toxoplasma gondii-infected mice. We observed that 1,25-dihydroxyvitamin D3 reduces the survival rate of infected mice by up to 37% at day 10 post-infection compared to untreated infected mice (P < 0.0001). IFN-gamma and IL-12p40 levels were significantly reduced by 1,25-dihydroxyvitamin D3 in infected mice sera indicating an inhibition of Th-1-type cytokines. CD4+ T lymphocyte and splenocyte counts were also reduced following 1,25-dihydroxyvitamin D3 treatment and a marked induction of apoptosis, accompanied with down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L), was observed. The above results indicate that 1,25-dihydroxyvitamin D3 induces splenocyte apoptosis and enhances host susceptibility to toxoplasmosis.     

Estrus cycle: influence on cardiac function following trauma-hemorrhage

Since cardiac function is depressed in males but not in proestrus (PE) females following trauma-hemorrhage (T-H), we examined whether different estrus cycles influence cardiac function in female rats under those conditions. We hypothesized that females in the PE cycle only will have normal cardiac function following T-H and resuscitation. Sham operation or T-H was performed in five groups of rats (250-275 g) including PE, estrus (E), metestrus (ME), diestrus (DE), and ovariectomized (OVX) females (n = 6-7 per group). Cardiac function was determined 2 h after T-H, following which cardiomyocytes were isolated and nuclei extracted. Cardiomyocyte IL-6 and NF-kappaB expressions were measured using Western blotting. Moreover, plasma IL-6, estradiol, and progesterone levels were measured using ELISA or EIA kits. Results (1-way ANOVA) indicated that following T-H, 1) cardiac function was depressed in DE, E, ME, and OVX groups but maintained in the PE group; 2) the PE group had the highest plasma estrogen level; 3) plasma IL-6 levels increased significantly in DE, E, ME, and OVX groups, but the increase was attenuated in the PE group; 4) cardiomyocyte IL-6 protein level increased significantly in DE, E, ME and OVX groups after TH, but the increase was attenuated in the PE group; and 5) cardiomyocyte NF-kappaB expression increased significantly but was attenuated in the PE group. These data collectively suggest that the estrus cycle plays an important role in cardiac function following TH. The salutary effect seen in PE following TH is likely due to a decrease in NF-kappaB-dependent cardiac IL-6 pathway.     

Endothelial lipase modulates HDL but has no effect on atherosclerosis development in apoE-/- and LDLR-/- mice

Endothelial lipase (EL) is a determinant of high density lipoprotein-cholesterol (HDL-C) level, which is negatively correlated with atherosclerosis susceptibility. We found no difference in aortic atherosclerotic lesion areas between 26-week-old EL+/+ apolipoprotein E-deficient (apoE-/-) and EL-/- apoE-/- mice. To more firmly establish the role of EL in atherosclerosis, we extended our study to EL-/- and EL+/+ low density lipoprotein receptor-deficient (LDLR-/-) mice that were fed a Western diet. Morphometric analysis again revealed no difference in atherosclerosis lesion area between the two groups. Compared with EL+/+ mice, we found increased HDL-C in EL-/- mice with apoE-/- or LDLR-/- background but no difference in macrophage content between lesions of EL-/- and EL+/+ mice in apoE-/- or LDLR-/- background. EL inactivation had no effect on hepatic mRNAs of proteins involved in reverse cholesterol transport. A survey of lipid homeostasis in EL+/+ and EL-/- macrophages revealed that oxidized LDL-induced ABCA1 was attenuated in EL-/- macrophages. This potentially proatherogenic change may have nullified any minor protective increase of HDL in EL-/- mice. Thus, although EL modulated lipoprotein profile in mice, there was no effect of EL inactivation on atherosclerosis development in two hyperlipidemic atherosclerosis-prone mouse models.     

In vitro studies of splenocyte cytotoxicity against syngeneic mammary tumors of mice

Summary Spleen cells from BALB/c mice immunized with D7T4S (MTV-negative) or D14 (MTV-positive) mammary tumors exhibited marked cytotoxic activity for the corresponding tumor cells in a terminal ⁵¹ -Cr-labeling cytotoxicity assay. A pronounced, seemingly nonspecific cytotoxic effect was displayed by splenocytes derived from normal BALB/c and BALB/cfC3H mice subjected to various surgical procedures 10–14 days before testing. Possible mechanisms underlying this phenomenon are discussed.     

Synergistic effect of phorbol myristate acetate on bacterial lipopolysaccharide induced rat splenocyte proliferation

1. Bacterial lipopolysaccharide (LPS) stimulated [3H]TdR incorporation of rat splenocytes in a concentration dependent manner. 2. Phorbol 12-myristate 13-acetate (PMA) alone has little effect on rat splenocyte proliferation but it exerted a marked synergistic effect on LPS-induced [3H]TdR incorporation when added at the first few hours of incubation with LPS. Minimal synergistic effect of PMA was observed if it was added later than 4 hr after LPS application. 3. Both LPS-stimulated and PMA synergized incorporation of [3H]TdR in rat splenocytes were inhibited by H-7, a protein kinase C inhibitor. 4. The results support the notion that the activation of protein kinase C is a necessary but insufficient cellular signal in the initiation of proliferative response of rat splenocytes by LPS.     

Protective effect of Acorus calamus on kidney and liver functions in healthy mice

Acorus calamus (AC), is an herbal medicine commonly used as traditional practice in pharmacological applications. Present study initiated was evident to proof the hepatoprotective and nephroprotective activity with supporting histopathological status of kidneys and liver. Investigation done with the 5% (w/v) of AC dissolved in tap water (50 g/l) given for 15 days compared with control tap water to 5-7 week old C57Bl/6 mice both sexes. Renal function, liver function, biochemical and complete blood count was evaluated. AC significantly reduced food intake, body weight, also plasma concentration of electrolytes such as Na⁺, K⁺, Ca²⁺, were reduced as the excretion of electrolytes were increased in urine, significantly increased Fluid Intake, with Urinary urea, Urinary creatinine, Glomerular Filtration Rate, creatinine clearance, High-density lipoproteins, Mean Corpuscular Volume. The biochemical findings showed the hepatoprotective and histopathological changes showed the nephroprotective nature of AC by normal structure with no necrosis.     

Comparison of longitudinal leukocyte gene expression after burn injury or trauma-hemorrhage in mice.

A primary objective of the large collaborative project entitled "Inflammation and the Host Response to Injury" was to identify leukocyte genes that are differentially expressed after two different types of injury in mouse models and to test the hypothesis that both forms of injury would induce similar changes in gene expression. We report here the genes that are expressed in white blood cells (WBCs) and in splenocytes at 2 h, 1 day, 3 days, and 7 days after burn and sham injury or trauma-hemorrhage (T-H) and sham T-H. Affymetrix Mouse Genome 430 2.0 GeneChips were used to profile gene expression, and the results were analyzed by dCHIP, BRB Array Tools, and Ingenuity Pathway Analysis (IPA) software. We found that the highest number of genes differentially expressed following burn injury were at day 1 for both WBCs (4,989) and for splenocytes (4,715) and at day 1 for WBCs (1,167) and at day 3 for splenocytes (1,117) following T-H. The maximum overlap of genes that were expressed after both forms of injury were at day 1 in WBCs (136 genes) and at day 7 in splenocytes (433 genes). IPA revealed that the cell-to-cell signaling, cell death, immune response, antiapoptosis, and cell cycle control pathways were affected most significantly. In summary, this report provides a database of genes that are modulated in WBCs and splenocytes at sequential time points after burn or T-H in mice and reveals that relatively few leukocyte genes are expressed in common after these two forms of injury.     

Transgenic mice expressing dominant-negative activin receptor IB in forebrain neurons reveal novel functions of activin at glutamatergic synapses

The transforming growth factor beta family member activin is an important regulator of development and tissue repair. It is strongly up-regulated after acute injury to the adult brain, and application of exogenous activin protects neurons in several lesion models. To explore the role of endogenous activin in the normal and acutely damaged brain, we generated transgenic mice expressing a dominant-negative activin receptor IB (dnActRIB) mutant in forebrain neurons. The functionality of the transgene was verified in vivo. Hippocampal neurons from dnActRIB mice were significantly more vulnerable to intracerebroventricular injection of the excitotoxin kainic acid than those from control littermates, indicating a crucial role of endogenous activin in the rescue of neurons from excitotoxic insult. Because dnActRIB is only expressed in neurons, but not in glial cells, activin affords protection at least in part through a direct action on endangered neurons. Unexpectedly, the transgenic mice also revealed a prominent novel role of activin in glutamatergic neurotransmission in the intact adult brain. Electrophysiologic examination of excitatory synapses onto CA1 pyramidal cells in hippocampal slices of dnActRIB mice showed a reduced NMDA current response, which was associated with impaired long term potentiation. This is the first demonstration that activin receptor signaling is essential to optimize the performance of neuronal circuits in the mature brain under physiological conditions.     

Transgenic mice express human MPO -463G/A alleles at atherosclerotic lesions, developing hyperlipidemia and obesity in -463G males

Myeloperoxidase (MPO) is an oxidant-generating enzyme present in macrophages at atherosclerotic lesions and implicated in coronary artery disease (CAD). Although mouse models are important for investigating the role of MPO in atherosclerosis, neither mouse MPO nor its oxidation products are detected in lesions in murine models. To circumvent this problem, we generated transgenic mice expressing two functionally different human MPO alleles, with either G or A at position -463, and crossed these to the LDL receptor-deficient (LDLR(-/-)) mouse. The -463G allele is linked to higher MPO expression and increased CAD incidence in humans. Both MPO alleles were expressed in a subset of lesions in high-fat-fed LDLR(-/-) mice, notably at necrotic lesions with cholesterol clefts. MPOG-expressing LDLR(-/-) males (but not females) developed significantly higher serum cholesterol, triglycerides, and glucose, all correlating with increased weight gain/obesity, implicating MPO in lipid homeostasis. The MPOG- and MPOA-expressing LDLR(-/-) males also exhibited significantly larger aortic lesions than control LDLR(-/-) males. The human MPO transgenic model will facilitate studies of MPO involvement in atherosclerosis and lipid homeostasis.     

Differential effect of culture epimastigotes and blood-form trypomastigotes on normal mouse splenocyte responsiveness to mitogens

Blood form trypomastigotes of the Y strain of T. cruzi, produced a strong inhibition of the blastogenic response to T and B cell mitogens, of the C3H/He, C57BL/6 and BALB/cJ strains of mice, while culture epimastigotes of the Y strain kept in a medium that allows parasite growth at 26 degrees, 30 degrees, 34 degrees and 37 degrees C produced a strong stimulatory effect that was even higher than the effect of the mitogens alone. Both the inhibitory or the stimulatory effects were dose-dependent. The stimulatory effect of epimastigotes was also temperature-dependent producing increased stimulation indexes as the temperature of parasite cultures was raised. Metabolically active, living parasites seemed to be necessary for an improved lymphocyte stimulation suggesting a potential role of secreted metabolites as polyclonal activators of mouse lymphocytes.     

转基因小鼠在现代生命科学中的应用

随着分子生物学的发展,转基因技术取得了巨大的进步,并且在现代生命科学中得到了广泛的应用.针对转基因技术在生物医学研究领域中的重要作用,结合近年来转基因技术及转基因动物的研究成果,着重介绍转基因小鼠的发展,转基因的方法及在医学中的应用,并对转基因小鼠在现代生命科学中的应用前景进行了展望.     

Plant KT/KUP/HAK potassium transporters: single family - multiple functions

BACKGROUND AND AIMS: Potassium transporters belonging to the KT/KUP/HAK family are important for various aspects of plant life including mineral nutrition and the regulation of development. Genes encoding these transporters are present in the genomes of all plants, but have not been found in the genomes of Protista or Animalia. The aim of this Botanical Briefing is to analyse the function of KT/KUP/HAK transporters from evolutionary, molecular and physiological perspectives. SCOPE: This Briefing covers the phylogeny and evolution of KT/KUP/HAK transporters, the role of transporters in plant mineral nutrition and potassium homeostasis, and the role of KT/KUP/HAK transporters in plant development.