Diet-induced obesity alters AMP kinase activity in hypothalamus and skeletal muscle

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10-12 weeks. Leptin acutely decreased food intake by approximately 30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased alpha2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited alpha2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters alpha2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.     

Increased tumor cell dissemination and cellular senescence in the absence of beta1-integrin function

Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization, as well as transduction of signals into cells, to promote various aspects of cellular behavior, such as proliferation or survival. Integrins participate in many aspects of tumor biology. Here, we have employed the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis to investigate the role of beta(1)-integrin in tumor progression. Specific ablation of beta(1)-integrin function in pancreatic beta cells resulted in a defect in sorting between insulin-expressing beta cells and glucagon-expressing alpha cells in islets of Langerhans. Ablation of beta(1)-integrin in beta tumor cells of Rip1Tag2 mice led to the dissemination of tumor cell emboli into lymphatic blood vessels in the absence of ongoing lymphangiogenesis. Yet, disseminating beta(1)-integrin-deficient beta tumor cells did not elicit metastasis. Rather, primary tumor growth was significantly impaired by reduced tumor cell proliferation and the acquisition of cellular senescence by beta(1)-integrin-deficient beta tumor cells. The results indicate a critical role of beta(1)-integrin function in mediating metastatic dissemination and preventing tumor cell senescence.     

An essential role for Akt1 in dendritic cell function and tumor immunotherapy

Current dendritic cell (DC) vaccine preparations involving ex vivo differentiation and maturation produce short-lived, transiently active DCs that may curtail T-cell responses in vivo. We demonstrate that Akt1, downregulation of which decreases DC lifespan, is critical for proinflammatory signal-mediated DC survival and maturation. Lipopolysaccharide or CD40 signaling stabilizes Akt1, promoting both activation and Bcl-2-dependent survival of DCs. Expression of a potent allele encoding a lipid raft-targeted Akt1, M(F)-DeltaAkt, is sufficient for maturation and survival of murine bone marrow-derived DCs in vivo. M(F)-DeltaAkt-transduced DCs enhanced T-cell proliferation, activation and long-term memory responses, enabling eradication of large pre-established lymphomas and aggressive B16 melanomas. Human myeloid DCs expressing constitutively active M(F)-DeltahAkt also survived significantly longer and promoted antigen-specific T-cell responses. Thus, Akt1 is a critical regulator of DC lifespan, and its manipulation in DCs can improve the clinical efficacy of DC-based tumor vaccines.     

Do-it-yourself tiling: dendritic growth in the absence of homotypic contacts

Many subtypes of neurons are precisely arranged with little overlap of their dendritic arbors, which has been suggested to arise due to homotypic dendritic repulsion. In this issue of Neuron, Lin et al. demonstrate that subtypes of retinal ganglion cells establish normal dendritic field sizes and morphologies in the absence of contact with cells of the same class.     

Memory B cell survival and function in the absence of secreted antibody and immune complexes on follicular dendritic cells

Ag, in the form of immune complexes retained on follicular dendritic cells, has been implicated in the development and maintenance of B cell memory. We addressed this question using a H chain transgenic (Tg) mouse model that lacks secreted Ig (mIg), and thus does not deposit Ag-containing immune complexes. We compared the ability of the mIg strain and a control Tg strain, which secretes IgM, to develop and maintain long-lived memory cells. After immunization, there was an increase of Ag-specific B cells in both strains that was maintained for at least 20 wk. We labeled the long-lived Ag-specific cells with BrdU and found that this population was similarly maintained. In addition, both Tgs were able to maintain a functional memory response as measured by secondary germinal center reactions. Our studies indicate that localization of Ag on follicular dendritic cells is not necessary for development and maintenance of B cell memory.     

Maternal obesity alters adiposity and monoamine function in genetically predisposed offspring

The impact of maternal obesity on brain monoamine function in adult offspring of dams selectively bred to express diet-induced obesity (DIO) or diet resistance (DR) was assessed by making dams obese or lean during gestation and lactation. After 12 wk on chow and 4 wk on a 31% fat diet, offspring hypothalamic nucleus size and [(3)H]nisoxetine binding to norepinephrine transporters (NET) and [(3)H]paroxetine binding to serotonin transporters (SET) were measured. Offspring of obese DIO dams became more obese than all other groups, but maternal obesity did not alter weight gain in DR offspring (25). Maternal obesity was associated with 10-17% enlargement of ventromedial nuclei (VMN) and dorsomedial nuclei in both DIO and DR offspring. Offspring of obese DIO dams had 25-88% lower NET binding in the paraventricular nuclei (PVN), arcuate nuclei, VMN, and the central amygdalar nuclei, while offspring of obese DR dams had 43-67% higher PVN and 90% lower VMN NET binding and a generalized increase in SET binding across all hypothalamic areas compared with other groups. Thus maternal obesity was associated with alterations in offspring brain monoamine metabolism, which varied as a function of genotype and the development of offspring obesity.     

Host absence of CCR5 potentiates dendritic cell vaccination

Previous work has shown that dendritic cells (DCs) express specific chemokine receptors that allow for coordinated movement in vivo. To test the in vivo relevance of this, we used a murine melanoma system and knockout mice to investigate the function of the chemokine receptor CCR5 and its ligands, CCR ligand (CCL)3 and CCL5. We found that the lack of CCR5 in the host mouse resulted in delayed tumor growth, but this effect was overcome at a higher tumor load. With the administration of tumor charged DCs, CCR5(-/-) mice that had previously been injected with tumor were completely protected from tumor. This effect was dependent on the dose of tumor cells and the expression of CCR5 on the DC and its absence in the host. In contrast, the loss of the CCR5 ligand, CCL3, led to an early delay in tumor growth that did not persist, while the absence of the CCR5 ligand, CCL5, had no effect. Blocking the activity of CCR5 in the host may represent a new strategy for enhancing the activity of a therapeutic melanoma DC vaccine.     

Diet-induced obesity suppresses ghrelin in rat gastrointestinal tract and serum

The aims of the present study were to examine ghrelin expression in serum and gastrointestinal tract (GIT) tissues, and to measure tissue ghrelin levels and obesity-related alterations in some serum biochemical variables in rats with diet-induced obesity (DIO). The study included 12 male rats, 60 days old. The rats were randomly allocated to two groups (n = 6). Rats in the DIO group were fed a cafeteria-style diet to induce obesity, while those in the control group were fed on standard rat pellets. After a 12 week diet program including an adaptation period all rats were decapitated, tissues were individually fixed, ghrelin expression was examined by immunohistochemistry , and tissue and serum ghrelin levels were measured by radioimmunoassay. Serum biochemical variables were measured using an autoanalyzer. When the baseline and week 12 body mass index and GIT ghrelin expression were compared between DIO and control rats, BMI had increased and ghrelin expression decreased due to obesity. The RIA results were consistent with these findings. Serum glucose, LDL cholesterol, and total cholesterol levels were elevated and HDL cholesterol significantly decreased in the DIO group. A comparison of GIT tissues between the control and obese groups demonstrated that ghrelin was decreased in all tissues of the latter. This decrease was brought about a decline in the circulating ghrelin pool. This suggests that rather than being associated with a change in a single tissue, obesity is a pathological condition in which ghrelin expression is changed in all tissues.     

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.     

Systemic tumor necrosis factor generated during lethal Plasmodium infections impairs dendritic cell function

Dendritic cells (DCs) initiate innate and adaptive immune responses including those against malaria. Although several studies have shown that DC function is normal during malaria, other studies have shown compromised function. To establish why these studies had different findings, we examined DCs from mice infected with two lethal species of parasite, Plasmodium berghei or P. vinckei, and compared them to DCs from nonlethal P. yoelii 17XNL or P. chabaudi infections. These studies found that DCs from only the lethal infections became uniformly mature 7 days after infection and were functionally impaired as they were unable to endocytose latex particles, secrete IL-12, or present OVA to transgenic OTII T cells. These changes coincided with a peak in levels of systemic TNF-alpha. Because TNF-alpha is known to mature DCs, we used TNF-KO mice to determine the role of this cytokine in the loss of DC function. In the TNF-KO mice, phenotype, Ag presentation, and IL-12 secretion by DCs were restored to normal following both lethal infections. This study shows that the systemic production of TNF-alpha contributes to poor DC function during lethal infections. These studies may explain, at least in part, immunosuppression that is associated with malaria.     

Hydrogen peroxide release by rat peritoneal macrophages in the presence and absence of tumor cells

The ability of mineral oil-elicited rat peritoneal macrophages to release hydrogen peroxide (H₂O₂) to the extracellular medium was measured in the presence and absence of rat lymphoma cells grown in tissue culture, and in the presence of phorbol myristate acetate (PMA). Horseradish peroxidase (HRP)-catalyzed oxidation of scopoletin or phenol red was used to measure H₂O₂ release during incubation of cells in monolayer culture for periods up to 24 h. Macrophages appeared to release H₂O₂ with or without PMA, although PMA greatly increased the amount of H₂O₂ released in short (1 to 4 h) incubations. Tumor cells did not replace PMA as a triggering agent for H₂O₂ release. Instead, tumor cells inhibited H₂O₂ release. The probable basis for inhibition was competition between macrophages and tumor cells for the supply of oxygen (O₂). Tumor cells did not inhibit H₂O₂ release when the O₂ concentration was held constant. The rates at which macrophages took up O₂ and released H₂O₂ were proportional to the O₂ concentration, as measured with the O₂ electrode. Rates of H₂O₂ release could be calculated from the difference in the rate constants for O₂ uptake measured in the presence of two different extracellular H₂O₂-consuming systems (HRP-scopoletin vs catalase). PMA-stimulated uptake of O₂ and release of H₂O₂ were highest in a small subpopulation of macrophages, obtained at the lowest-density position on gradients of bovine serum albumin. These cells also released H₂O₂ in the absence of PMA. Tumor cells had no effect on the rate constants for O₂ uptake and H₂O₂ release by the unfractionated macrophages or the macrophage subpopulations.     

Sialoadenectomy alters liver cell turn-over and function in mice

In rodents, submandibular salivary glands accumulate a number of biologically active peptides, and release some of them to both saliva and the bloodstream. Surgical removal of these glands (sialoadenectomy) alters the ability of the liver to regenerate after partial hepatectomy. We show here that 5 weeks after surgery, the liver of sialoadenectomized mice contained 40% fewer hepatocytes than the liver of sham-operated mice. We did not obtain evidence of necrotic cell death after surgery. In contrast, sialoadenectomy transiently increased apoptotic hepatocyte death, as revealed by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis was determined in vivo by the incorporation of bromo-deoxyuridine (BrdU) into hepatocyte nuclei. BrdU-labeling progressively increased after sialoadenectomy. We conclude that sialoadenectomy induced a transient wave of apoptotic cell death followed by a rise in DNA synthesis but not by cell division. This reduced cell number but increased mean cell volume. In spite of these alterations in cellularity, the liver responded adequately to several stressful conditions, as judged by the lack of any differential effect of sialoadenectomy on liver glycogen and plasma glucose concentration after immobilization, aggressive encounter, or fasting. However, the liver of sialoadenectomized mice was more sensitive to the effect of a non-lethal dose of bacterial lipopolysaccharide (LPS) combined with d-galactosamine, as shown by the enhanced rise in plasma alanine aminotransferase and aspartate aminotransferase, and liver myeloperoxidase (MPO) activities. All these results indicate that a submandibular salivary glands-liver axis is involved in the maintenance of liver structure in mice. A disturbance of this axis induces an adaptive response that preserves the metabolic function of the liver but renders it more sensitive to bacterial endotoxins.     

Impaired B cell development and function in the absence of IkappaBNS

IκBNS has been identified as a member of the IκB family of NF-κB inhibitors, which undergoes induction upon TCR signaling. Mice carrying a targeted gene disruption of IκBNS demonstrate dysregulation of cytokines in T cells, macrophages, and dendritic cells. IκBNS mediates both positive and negative gene regulation, depending on individual cell type and/or cytokine. In this study, we demonstrate an additional role for IκBNS in the B cell lineage. B cells from IκBNS knockout (KO) mice were impaired in proliferative responses to LPS and anti-CD40. IgM and IgG3 Igs were drastically reduced in the serum of IκBNS KO mice, although IκBNS KO B cells exhibited a higher level of surface IgM than that found in wild-type mice. Switching to IgG3 was significantly reduced in IκBNS KO B cells. The in vitro induction of plasma cell development demonstrated that progression to Ab-secreting cells was impaired in IκBNS KO B cells. In agreement with this finding, the number of Ab-secreting cells in the spleens of IκBNS KO mice was reduced and production of Ag-specific Igs was lower in IκBNS KO mice after influenza infection as compared with wild-type mice. Additionally, IκBNS KO mice lacked B1 B cells and exhibited a reduction in marginal zone B cells. Thus, IκBNS significantly impacts the development and functions of B cells and plasma cells.     

Cell K activity in frog skin in the presence and absence of cell current

Summary Cell K activity,a _k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO₃ Ringer on the apical surface.Under control conditionsa _k ^r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm²). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina _k ^r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E _k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa _k ^r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.     

Tumor-induced modulation of dendritic cell function

Dendritic cells (DC) are specialized antigen presenting cells that acquire, process, and present tumor-associated antigens to T cells for the induction of antigen-specific tumor immune responses. DC have been shown to infiltrate many tumors but both, circulating and tumor-infiltrating DC from cancer patients, appear to be phenotypically and functionally defective. Several tumor-derived factors such as VEGF, IL-6, IL-10, M-CSF, and STAT-3 have been shown to be responsible for systemic and local DC defects. Furthermore, tumor metabolites such as lactic acid may also critically contribute to DC dysfunction and tumor immune escape. The correction of abnormal DC function might be a requirement for successful vaccine approaches against cancer.     

Varicella-zoster virus productively infects mature dendritic cells and alters their immune function

Mature dendritic cells (DCs) are potent antigen-presenting cells essential for initiating successful antiviral immune responses and would therefore serve as an ideal target for viruses seeking to evade or delay the immune response by disrupting their function. We have previously reported that VZV productively infects immature DCs (A. Abendroth, G. Morrow, A. L. Cunningham, and B. Slobedman, J. Virol. 75:6183-6192, 2001), and in the present study we assessed the ability of VZV to infect mature DCs. Mature DCs were generated from immature monocyte-derived DCs by lipopolysaccharide treatment before being exposed to VZV-infected fibroblasts. On day 4 postexposure, flow cytometry analysis revealed that 15 to 45% of mature DCs were VZV antigen positive, and immunofluorescent staining together with infectious-center assays demonstrated that these cells were fully permissive for the complete VZV replicative cycle. VZV infection of mature DCs resulted in a selective downregulation of cell surface expression of the functionally important immune molecules major histocompatibility complex (MHC) class I, CD80, CD83, and CD86 but did not alter MHC class II expression. Immunofluorescent staining showed that the downregulation of cell surface CD83 was concomitant with a retention of CD83 in cytoplasmic vesicles. Importantly, VZV infection of mature DCs significantly reduced their ability to stimulate the proliferation of allogeneic T lymphocytes. These data demonstrate that mature DCs are permissive for VZV and that infection of these cells reduces their ability to function properly. Thus, VZV has evolved yet another immune evasion strategy that would likely impair immunosurveillance and enhance the chances for lifelong persistence in the human population.     

MicroRNAs regulate dendritic cell differentiation and function

MicroRNAs (miRNAs) are an important class of cellular regulators that modulate gene expression and thereby influence cell fate and function. In the immune system, miRNAs act at checkpoints during hematopoietic development and cell subset differentiation, they modulate effector cell function, and they are implicated in the maintenance of homeostasis. Dendritic cells (DCs), the professional APCs involved in the coordination of adaptive immune responses, are also regulated by miRNAs. Some DC-relevant miRNAs, including miR-155 and miR-146a, are shared with other immune cells, whereas others have been newly identified. In this review, we summarize the current understanding of where miRNAs are active during DC development from myeloid precursors and differentiation into specialized subsets, and which miRNAs play roles in DC function.     

IL-7 administration alters the CD4:CD8 ratio, increases T cell numbers, and increases T cell function in the absence of activation

IL-7 is vital for the development of the immune system and profoundly enhances the function of mature T cells. Chronic administration of IL-7 to mice markedly increases T cell numbers, especially CD8(+) T cells, and enhances T cell functional potential. However, the mechanism by which these effects occur remains unclear. This report demonstrates that only 2 days of IL-7 treatment is needed for maximal enhancement of T cell function, as measured by proliferation, with a 6- to 12-fold increase in the proportion of CD4(+) and CD8(+) T cells in cell cycle by 18 h of ex vivo stimulation. Moreover, a 2-day administration of IL-7 in vivo increases basal proliferation by 4- and 14-fold in CD4(+) and CD8(+) T cells, respectively. These effects occur in the absence of cytokine production, increases in most activation markers, and changes in memory markers. This enhanced basal proliferation is the basis for the increase in T cell numbers in that IL-7 induces an additional 60% and 85% of resting CD4(+) and CD8(+) T cells, respectively, to enter cell cycle in mice given IL-7 for 7 days. These results demonstrate that in vivo administration of IL-7 increases T cell numbers and functional potential via a homeostatic, nonactivating process. These findings may suggest a unique clinical niche for IL-7 in that IL-7 therapy may increase T cell numbers and enhance responses to specific antigenic targets while avoiding a general, nonspecific activation of the T cell population.