CD25(+) T cell depletion impairs murine squamous cell carcinoma development via modulation of antitumor immune responses

Squamous cell carcinoma (SCC) constitutes a microenvironment that could modulate the antitumor immune response. Also, tumor-infiltrating lymphocytes are believed to play complex regulatory roles in antitumor immunity against SCC. The presence of regulatory T cells (Tregs) has been associated with the suppression of tumor-reactive T cells. However, the underlying mechanism for this T cell dysfunction is not clear. We used a multistage model of SCC to examine the role of Treg cells during tumor development. 7,12-dimethylbenz[a]-anthracene/phorbol 12-myristate 13-acetate treatment and systemic depletion of Treg cells using an anti-CD25 monoclonal antibody (PC61) resulted in a decrease in the number and incidence of papilloma. Furthermore, CD25 depletion increased the proportion of CD8(+) and CD4(+) T cells that were isolated from tumor lesions. The levels of interleukin (IL)-1 beta, IL-10, IL-12, IL-13, interferon-gamma, transforming growth factor-beta and tumor necrosis factor-alpha, but not IL-17, were increased in the tumor microenvironment after Treg depletion. Therefore, our results indicated involvement of CD25(+) T cells in SCC development and in the suppression of the inflammatory immune response.

KCNQ Channels Determine Serotonergic Modulation of Ventral Surface Chemoreceptors and Respiratory Drive

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H+ changes. Their activity is also sensitive to neuromodulatory inputs from multiple respiratory centers, and thus they serve as a key nexus of respiratory control. However, molecular mechanisms that control their activity and susceptibility to neuromodulation are unknown. Here, we show in vitro and in vivo that KCNQ channels are critical determinants of RTN neural activity. In particular, we find that pharmacological block of KCNQ channels (XE991, 10 mu M) increased basal activity and CO2 responsiveness of RTN neurons in rat brain slices, whereas KCNQ channel activation (retigabine, 2-40 mu M) silenced these neurons. Interestingly, we also find that KCNQ and apamin-sensitive SK channels act synergistically to regulate firing rate of RTN chemoreceptors; simultaneous blockade of both channels led to a increase in CO2 responsiveness. Furthermore, we also show that KCNQ channels but not SK channels are downstream effectors of serotonin modulation of RTN activity in vitro. In contrast, inhibition of KCNQ channel did not prevent modulation of RTN activity by Substance P or thyrotropin-releasing hormone, previously identified neuromodulators of RTN chemoreception. Importantly, we also show that KCNQ channels are critical for RTN activity in vivo. Inhibition of KCNQ channels lowered the CO2 threshold for phrenic nerve discharge in anesthetized rats and decreased the ventilatory response to serotonin in awake and anesthetized animals. Given that serotonergic dysfunction may contribute to respiratory failure, our findings suggest KCNQ channels as a new therapeutic avenue for respiratory complications associated with multiple neurological disorders.

Transcriptome Analysis of Renal Ischemia/Reperfusion Injury and Its Modulation by Ischemic Pre-Conditioning or Hemin Treatment

Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.

Thyroid hormones are involved in 5 '-nucleotidase modulation in soluble fraction of cardiac tissue

Aims: To investigate the role of TH (thyroid hormones) in 5'-nucleotidase activity and expression in cardiac soluble fraction (SF). Main methods: Male Wistar rats received daily injections of 14 (10, 25 or 50 mu g T4/100 g body weight) for 14 days to develop a hyperthyroidism condition. Thyroidectomy was performed in other animals to mimic hypothyroidism, and 14 days after surgery they were submitted to TH replacement therapy. Key findings: T4 reduced the 5'-nucleotidase activity (T4-25. P<0.05 and 14-50, P<0.01) in the SF. Conversely, hypothyroidism significantly increased the 5'-nucleotidase activity in this fraction (P<0.001) and TH replacement therapy reversed the latter result (P<0.001 compared to hypothyroid group). The analysis of protein expression in the SF showed that 5'-nucleotidase was more expressed in hypothyroid than in the control group and that the phosphorylated state of PKC observed in this condition may contribute to a possible mechanism of 5'-nucleotidase modulation by thyroid status. Significance: Taken together, these data reveal that TH can influence adenosine production by modulating 5'-nucleotidase activity and expression, which may contribute to the cardioprotective effect and the maintenance of cardiac function under TH privation. (C) 2012 Elsevier Inc. All rights reserved.

High levels of C-reactive protein are associated with reduced vagal modulation and low physical activity in young adults

The purpose of this study was to examine the relationship between cardiac autonomic control derived from heart rate variability (HRV), high-sensitivity C-reactive protein (hs-CRP) and physical activity (PA) levels measured using accelerometers. A total of 80 healthy university students volunteered to participate in this study (20.56 +/- 0.82 years, 1.36 +/- 1.5 mg/L of hs-CRP). The participants were divided into groups based on tertiles of hs-CRP. Analysis of covariance adjusted to PA was used to assess group differences in HRV. Associations between hs-CRP, HRV indices and PA were analyzed using Pearson's correlation. The participants at the highest tertile of hs-CRP (tertile 3) had lower cardiac vagal modulation (SDNN, tertile 1=78.05 +/- 5.9,tertile 2=82.43 +/- 5.9,tertile 3=56.03 +/- 6.1; SD1, tertile 1=61.27 +/- 5.3, tertile 2=62.93 +/- 5.4, tertile 3=40.03 +/- 5.5). In addition, vagal indices were inversely correlated with hs-CRP but positively correlated with PA (SDNN r=-0.320, SD1 r=-0.377; SDNN r=0.304, SD1 r=0.299; P<0.05). Furthermore, the most physically active subjects had lower levels of hs-CRP and the highest levels of vagal modulation.

Opposing Roles for Cannabinoid Receptor Type-1 (CB1) and Transient Receptor Potential Vanilloid Type-1 Channel (TRPV1) on the Modulation of Panic-Like Responses in Rats

The midbrain dorsal periaqueductal gray (dPAG) has an important role in orchestrating anxiety-and panic-related responses. Given the cellular and behavioral evidence suggesting opposite functions for cannabinoid type 1 receptor (CB1) and transient receptor potential vanilloid type-1 channel (TRPV1), we hypothesized that they could differentially influence panic-like reactions induced by electrical stimulation of the dPAG. Drugs were injected locally and the expression of CB1 and TRPV1 in this structure was assessed by immunofluorescence and confocal microscopy. The CB1-selective agonist, ACEA (0.01, 0.05 and 0.5 pmol) increased the threshold for the induction of panic-like responses solely at the intermediary dose, an effect prevented by the CB1-selective antagonist, AM251 (75 pmol). Panicolytic-like effects of ACEA at the higher dose were unmasked by pre-treatment with the TRPV1 antagonist capsazepine (0.1 nmol). Similarly to ACEA, capsazepine (1 and 10 nmol) raised the threshold for triggering panic-like reactions, an effect mimicked by another TRPV1 antagonist, SB366791 (1 nmol). Remarkably, the effects of both capsazepine and SB366791 were prevented by AM251 (75 pmol). These pharmacological data suggest that a common endogenous agonist may have opposite functions at a given synapse. Supporting this view, we observed that several neurons in the dPAG co-expressed CB1 and TRPV1. Thus, the present work provides evidence that an endogenous substance, possibly anandamide, may exert both panicolytic and panicogenic effects via its actions at CB1 receptors and TRPV1 channels, respectively. This tripartite set-point system might be exploited for the pharmacotherapy of panic attacks and anxiety-related disorders. Neuropsychopharmacology (2012) 37, 478-486; doi:10.1038/npp.2011.207; published online 21 September 2011

Neutrophil dysfunction induced by hyperglycemia: modulation of myeloperoxidase activity

Our data suggest that impaired activity of myeloperoxidase (MPO) may play an important role in the dysfunction of neutrophils from hyperglycemic rats. Neutrophil biochemical pathways include the NADPH oxidase system and the MPO enzyme. They both play important role in the killing function of neutrophils. The effect of hyperglycemia on the activity of these enzymes and the consequences with regard to Candida albicans phagocytosis and the microbicidal property of rat peritoneal neutrophils is evaluated here. The NADPH oxidase system activity was measured using chemiluminescence and cytochrome C reduction assays. MPO activity was measured by monitoring HOCl production, and MPO protein expression was analysed using Western blot and immunofluorescence. C. albicans phagocytosis and death were evaluated by optical microscopy using the MayGrunwaldGiemsa staining method. ROS generation kinetic was slightly delayed in the diabetic group. MPO expression levels were higher in diabetic neutrophils; however, MPO activity was decreased in these same neutrophils compared with the controls. C. albicans phagocytosis and killing were lower in the diabetic neutrophils. Based on our experimental model, the phagocytic and killing functions of neutrophil phagocytosis are impaired in diabetic rats because of the decreased production of HOCl, highlighting the importance of MPO in the microbicidal function of neutrophils. Copyright (c) 2012 John Wiley & Sons, Ltd.

Muscarinic and Nicotinic Modulation of Thalamo-Prefrontal Cortex Synaptic Pasticity In Vivo

The mediodorsal nucleus of the thalamus (MD) is a rich source of afferents to the medial prefrontal cortex (mPFC). Dysfunctions in the thalamo-prefrontal connections can impair networks implicated in working memory, some of which are affected in Alzheimer disease and schizophrenia. Considering the importance of the cholinergic system to cortical functioning, our study aimed to investigate the effects of global cholinergic activation of the brain on MD-mPFC synaptic plasticity by measuring the dynamics of long-term potentiation (LTP) and depression (LTD) in vivo. Therefore, rats received intraventricular injections either of the muscarinic agonist pilocarpine (PILO; 40 nmol/mu L), the nicotinic agonist nicotine (NIC; 320 nmol/mu L), or vehicle. The injections were administered prior to either thalamic high-frequency (HFS) or low-frequency stimulation (LFS). Test pulses were applied to MD for 30 min during baseline and 240 min after HFS or LFS, while field postsynaptic potentials were recorded in the mPFC. The transient oscillatory effects of PILO and NIC were monitored through recording of thalamic and cortical local field potentials. Our results show that HFS did not affect mPFC responses in vehicle-injected rats, but induced a delayed-onset LTP with distinct effects when applied following PILO or NIC. Conversely, LFS induced a stable LTD in control subjects, but was unable to induce LTD when applied after PILO or NIC. Taken together, our findings show distinct modulatory effects of each cholinergic brain activation on MD-mPFC plasticity following HFS and LFS. The LTP-inducing action and long-lasting suppression of cortical LTD induced by PILO and NIC might implicate differential modulation of thalamo-prefrontal functions under low and high input drive.

Induction of Psychosis by Delta 9-Tetrahydrocannabinol Reflects Modulation of Prefrontal and Striatal Function During Attentional Salience Processing

Context: The aberrant processing of salience is thought to be a fundamental factor underlying psychosis. Cannabis can induce acute psychotic symptoms, and its chronic use may increase the risk of schizophrenia. We investigated whether its psychotic effects are mediated through an influence on attentional salience processing. Objective: To examine the effects of Delta 9-tetrahydrocannabinol (Delta 9-THC) and cannabidiol (CBD) on regional brain function during salience processing. Design: Volunteers were studied using event-related functional magnetic resonance imaging on 3 occasions after administration of Delta 9-THC, CBD, or placebo while performing a visual oddball detection paradigm that involved allocation of attention to infrequent (oddball) stimuli within a string of frequent (standard) stimuli. Setting: University center. Participants: Fifteen healthy men with minimal previous cannabis use. Main Outcome Measures: Symptom ratings, task performance, and regional brain activation. Results: During the processing of oddball stimuli, relative to placebo, Delta 9-THC attenuated activation in the right caudate but augmented it in the right prefrontal cortex. Delta 9-Tetrahydrocannabinol also reduced the response latency to standard relative to oddball stimuli. The effect of Delta 9-THC in the right caudate was negatively correlated with the severity of the psychotic symptoms it induced and its effect on response latency. The effects of CBD on task-related activation were in the opposite direction of those of Delta 9-THC; relative to placebo, CBD augmented left caudate and hippocampal activation but attenuated right prefrontal activation. Conclusions: Delta 9-Tetrahydrocannabinol and CBD differentially modulate prefrontal, striatal, and hippocampal function during attentional salience processing. These effects may contribute to the effects of cannabis on psychotic symptoms and on the risk of psychotic disorders.

Simplified approach to low-cost multiparameter monitoring based on low frequency polarization modulation

We experimentally revisit a technique of low-cost multiparameter monitor for optical performance monitoring based on low frequency polarization modulation. A simplified calibration procedure, which significantly reduces the mathematical complexity and processing effort is proposed. Validation is achieved by carrying out relative optical power, wavelength, and differential group delay measurements. (C) 2012 Wiley Periodicals, Inc. Microwave Opt Technol Lett 54:18201824, 2012; View this article online at wileyonlinelibrary.com. DOI 10.1002/mop.26956

Cancer cell proliferation is inhibited by specific modulation frequencies

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology. British Journal of Cancer (2012) 106, 307-313. doi:10.1038/bjc.2011.523 www.bjcancer.com Published online 1 December 2011 (C) 2012 Cancer Research UK

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-alpha, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this beta-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.

Induction of chronic non-inflammatory widespread pain increases cardiac sympathetic modulation in rats

Fibromyalgia (FM) is characterized by chronic non-inflammatory widespread pain (CWP) and changes in sympathetic function. In attempt to elucidate the pathophysiological mechanisms of FM we used a well-established CWP animal model. We aimed to evaluate changes in cardiac autonomic balance and baroreflex function in response to CWP induction in rats. CWP was induced by two injections of acidic saline (pH 4.0, n = 8) five days apart into the left gastrocnemius muscle. Control animals were injected twice with normal saline (pH 7.2, n = 6). One day after the second injection of acidic saline or normal saline, the animals had pulse interval (PI) and systolic arterial pressure (SAP) variability, and spontaneous baroreflex sensitivity (BRS) evaluated. After induction of CWP, there was an increase of power in the low frequency (LF) band of PI spectrum (12.75 +/- 1.04 nu), a decrease in the high frequency (HF) band (87.25 +/- 1.04 nu) and an increase of LF/HF ratio (0.16 +/- 0.01), when compared to control animals (7.83 +/- 1.13 nu LF; 92.16 +/- 1.13 nu HF; 0.08 +/- 0.01 LF/HF). In addition, there was an increase of power in the LF band of SAP spectrum (7.93 +/- 1.39 mmHg(2)) when compared to control animals (2.97 +/- 0.61 mmHg(2)). BRS was lower in acidic saline injected rats (0.59 +/- 0.06 ms/mmHg) when compared to control animals (0.71 +/- 0.03 ms/mmHg). Our results showed that induction of CWP in rats shifts cardiac sympathovagal balance towards sympathetic predominance and decreases BRS. These data corroborate findings in humans with FM. (C) 2011 Elsevier B.V. All rights reserved.

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.

Modulation of Lung Allergic Response by Renal Ischemia and Reperfusion Injury

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors. Copyright (C) 2012 S. Karger AG, Basel