Description and Analysis of a Novel Subtype of the Anti-Synthetase Syndrome Characterized by Frequent Attacks of Fever and Systemic Inflammation in a Single-Center Cohort Study.

Objectives: The aim of this study was to investigate anti-synthetase syndrome (ASyS) patients who presented with recurrent episodes of fever and systemic inflammation. Methods: A retrospective cohort of Chinese ASyS patients (n=126) in our center (between January 2013 and January 2020) was included. Patients presenting with concomitant autoimmune rheumatic diseases or malignancies were subsequently excluded. The number of non-infectious fever attacks and attack frequency were recorded and calculated. Patients with two or more attacks and within the upper three quartiles of attack frequency were defined as high-inflammation group. Univariate and multivariate analyses were carried out to characterize the high-inflammation subtype. Results: Out of 113 eligible patients with an average of 5 years follow up, 25 patients were defined as the high-inflammation group (16 for anti-Jo1, 9 for anti-PL7), with an average of 1.12 attack/patient-year. Compared to low-inflammation group (0-1 attack only and a frequency lower than 0.5 attack/patient-year), the high-inflammation group had higher occurrence of fever and rapid progressive interstitial lung disease (RPILD) as the first presentation (84% vs. 21% and 40% vs. 9%, respectively, both p<0.01). Anti-PL-7 was related to the more inflammatory phenotype (p=0.014). Cumulative disease-modifying agent exposures (>=3) were much higher in the high-inflammation group (60% vs. 26%), while biological agents, i.e., rituximab and tocilizumab, showed better "drug survival" for Jo-1+ and PL-7+ ASyS patients with high inflammation, respectively, in our cohort. Conclusions: ASyS with recurrent systemic inflammatory episodes reflects a subtype of more aggressive and refractory disease in the spectrum of ASyS. Increased awareness of this subtype might lead to more appropriate management.

Loss of hypothermic and anti-pyretic action of paracetamol in cyclooxygenase-1 knockout mice is indicative of inhibition of cyclooxygenase-1 variant enzymes.

Paracetamol (acetaminophen), is a centrally-acting antipyretic analgesic drug, which can also lower body temperature. Despite a century of clinical use, its mechanism of pharmacological action has not been completely elucidated. Previously, we demonstrated significant attenuation in the paracetamol induced hypothermia in parallel with its inhibitory action on the synthesis of brain prostaglandin E2 (PGE2) in cyclooxygenase-1 (COX-1) knockout mice in comparison to wild-type mice. The above reported pharmacological actions by paracetamol were completely retained in COX-2 knockout mice. We thus concluded that the mechanism of hypothermic action of paracetamol is dependent on inhibition of a COX-1 gene-derived enzyme. In the current investigation, we provide further support for this notion by demonstrating that the paracetamol-induced hypothermia is not mediated through inhibition of COX-1 as neither the COX-1 selective inhibitor, SC560, nor the COX-1/COX-2 dual inhibitor, indomethacin, induced hypothermia at pharmacologically active doses in mice. In addition, using a COX-2-dependent and PGE2-mediated model of endotoxin-induced fever, paracetamol induced anti-pyretic and hypothermic actions in COX-1 wild-type mice. These effects were fully or partially attenuated in COX-1 knockout mice after prophylactic or therapeutic administration, respectively. Therapeutically-administered paracetamol also reduced hypothalamic PGE2 biosynthesis in febrile COX-1 wild-type mice, but not in febrile COX-1 knockout mice. In conclusion, we provide further evidence which suggests that the hypothermic and now anti-pyretic actions of paracetamol are mediated through inhibition of a COX-1 variant enzyme.

Role of leucocyte caspase-1 activity in epidural-related maternal fever: a single-centre, observational, mechanistic cohort study.

BACKGROUND: Epidural-related maternal fever (ERMF) has been reported in ∼26% of labouring women. The underlying mechanisms remain unclear. We hypothesised that ERMF is promoted by bupivacaine disrupting cytokine production/release from mononuclear leucocytes [mononuclear fraction (MNF)]. We examined whether bupivacaine (i) reduces caspase-1 activity and release of the anti-pyrogenic cytokine interleukin (IL)-1 receptor antagonist (IL-1ra), and (ii) is pro-inflammatory through mitochondrial injury/IL-1ß. METHODS: In labouring women, blood samples were obtained before/after epidural analgesia was implemented. Maternal temperature was recorded hourly for the first 4 h of epidural analgesia. Time-matched samples/temperatures were obtained from labouring women without epidural analgesia, pregnant non-labouring, and non-pregnant women. The primary clinical outcome was change in maternal temperature over 4 h after the onset of siting epidural catheter/enrolment. The secondary clinical outcome was development of ERMF (temperature ≥ 38°C). The effect of bupivacaine/saline on apoptosis, caspase-1 activity, intracellular IL-1ra, and plasma IL-1ra/IL-1ß ratio was quantified in MNF from labouring women or THP-1 monocytes (using flow cytometry, respirometry, or enzyme-linked immunosorbent assay). RESULTS: Maternal temperature increased by 0.06°C h-1 [95% confidence interval (CI): 0.03-0.09; P=0.003; n=38] after labour epidural placement. ERMF only occurred in women receiving epidural analgesia (five of 38; 13.2%). Bupivacaine did not alter MNF or THP-1 apoptosis compared with saline control, but reduced caspase-1 activity by 11% (95% CI: 5-17; n=10) in MNF from women in established labour. Bupivacaine increased intracellular MNF IL-1ra by 25% (95% CI: 10-41; P<0.001; n=10) compared with saline-control. Epidural analgesia reduced plasma IL-1ra/IL-1ß ratio (mean reduction: 14; 95% CI: 7-30; n=30) compared with women without epidural analgesia. CONCLUSIONS: Impaired release of anti-pyrogenic IL-1ra might explain ERMF mechanistically. Immunomodulation by bupivacaine during labour could promote ERMF.

FAAH inhibition attenuates TLR3-mediated hyperthermia, nociceptive- and anxiety-like behaviour in female rats.

Aberrant activation of toll-like receptor (TLR)s results in persistent and prolonged neuroinflammation and has been implicated in the pathogenesis and exacerbation of psychiatric and neurodegenerative disorders. TLR3 coordinates the innate immune response to viral infection and recent data have demonstrated that inhibiting fatty acid amide hydrolase (FAAH), the enzyme that primarily metabolizes anandamide, modulates TLR3-mediated neuroinflammation. However, the physiological and behavioural consequences of such modulation are unknown. The present study examined the effect of URB597, a selective FAAH inhibitor, on neuroinflammation, physiological and behavioural alterations following administration of the TLR3 agonist and viral mimetic poly I:C to female rats. URB597 attenuated TLR3-mediated fever, mechanical and cold allodynia, and anxiety-like behaviour in the elevated plus maze and open field arena. There was no effect of URB597 on TLR3-mediated decreases in body weight and no effect in the sucrose preference or forced swim tests. URB597 attenuated the TLR3-mediated increase in the expression of CD11b and CD68, markers of microglia/macrophage activation. In summary, these data demonstrate that enhancing FAAH substrate levels suppresses TLR3-mediated microglia/macrophage activation and associated changes in fever, nociceptive responding and anxiety-related behaviour. These data provide further support for FAAH as a novel therapeutic target for neuroinflammatory disorders.

Acute Hyperthermia Inhibits TGF-ß1-induced Cardiac Fibroblast Activation via Suppression of Akt Signaling.

Transforming growth factor-ß1 (TGF-ß1) induces phenotypic changes in fibroblasts to become myofibroblasts with increased production of extracellular matrix (ECM) components and cytokines. It is also known that excessive activation of myofibroblasts accelerates cardiac fibrosis, remodeling, and thus cardiac dysfunction. However, no effective therapy has been established to prevent this process although recent clinical studies have demonstrated the effectiveness of hyperthermia in cardiac dysfunction. The aim of this study was to examine the molecular mechanism of hyperthermia on TGF-ß1-mediated phenotypic changes in cardiac fibroblasts. TGF-ß1 increased the expression of IL-6, α-smooth muscle actin (α-SMA), and collagen in human cardiac fibroblasts (HCFs). Hyperthermia (42 °C) significantly prevented these changes, i.e., increases in IL-6, α-SMA, and collagen, as induced by TGF-ß1 in a time-dependent manner. Immunoblotting showed that hyperthermia decreased Akt/S6K signaling, but did not affect Smad2 and Smad3 signaling. Pharmacological inhibition of Akt signaling mimicked these effects of hyperthermia. Furthermore, hyperthermia treatment prevented cardiac fibrosis in Ang II infusion mice model. Putting together, our findings suggest that hyperthermia directly inhibits TGF-ß-mediated activation of HCFs via suppressing Akt/S6K signaling.

Clinical usefulness of serum 2'-5'-oligoadenylate synthetase for early diagnosis of viral infections among febrile adult patients.

BACKGROUND: This study aimed to evaluate the clinical usefulness of serum 2'-5'-oligoadenylate synthetase (2-5AS) levels in diagnosing viral infection. METHODS: 2-5AS, procalcitonin (PCT) and C-reactive protein (CRP) serum levels and leukocyte counts were evaluated in 104 febrile patients (26 viral and 55 bacterial infections, 23 non-infectious diseases). RESULTS: Assessment of areas under the receiver-operator characteristic curves revealed that 2-5AS and CRP levels and the age of the patient differentiated between viral and bacterial infections, 2-5AS levels differentiated between viral infection and non-infectious disease and PCT levels differentiated between bacterial infection and non-infectious disease. The decision tree constructed according to the results correctly classified the origin of fever in 88 of 104 patients (85%). CONCLUSIONS: 2-5AS is a useful diagnostic biomarker for viral infections and combining it with the PCT and CRP levels and leukocyte counts improves determination of causes of fever.

Monoacylglycerol Lipase Regulates Fever Response.

Cyclooxygenase inhibitors such as ibuprofen have been used for decades to control fever through reducing the levels of the pyrogenic lipid transmitter prostaglandin E2 (PGE2). Historically, phospholipases have been considered to be the primary generator of the arachidonic acid (AA) precursor pool for generating PGE2 and other eicosanoids. However, recent studies have demonstrated that monoacyglycerol lipase (MAGL), through hydrolysis of the endocannabinoid 2-arachidonoylglycerol, provides a major source of AA for PGE2 synthesis in the mammalian brain under basal and neuroinflammatory states. We show here that either genetic or pharmacological ablation of MAGL leads to significantly reduced fever responses in both centrally or peripherally-administered lipopolysaccharide or interleukin-1ß-induced fever models in mice. We also show that a cannabinoid CB1 receptor antagonist does not attenuate these anti-pyrogenic effects of MAGL inhibitors. Thus, much like traditional nonsteroidal anti-inflammatory drugs, MAGL inhibitors can control fever, but appear to do so through restricted control over prostaglandin production in the nervous system.

Glucose-6-phosphate dehydrogenase deficiency A- variant in febrile patients in Haiti.

Haiti is one of two remaining malaria-endemic countries in the Caribbean. To decrease malaria transmission in Haiti, primaquine was recently added to the malaria treatment public health policy. One limitation of primaquine is that, at certain doses, primaquine can cause hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd). In this study, we genotyped two mutations (A376G and G202A), which confer the most common G6PDd variant in West African populations, G6PDd A-. We estimated the frequency of G6PDd A- in a sample of febrile patients enrolled in an on-going malaria study who represent a potential target population for a primaquine mass drug administration. We found that 33 of 168 individuals carried the G6PDd A- allele (includes A- hemizygous males, A- homozygous or heterozygous females) and could experience toxicity if treated with primaquine. These data inform discussions on safe and effective primaquine dosing and future malaria elimination strategies for Haiti.

Human procaspase-1 variants with decreased enzymatic activity are associated with febrile episodes and may contribute to inflammation via RIP2 and NF-κB signaling.

The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1ß production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti-RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

Glucocorticoids downregulate systemic nitric oxide synthesis and counteract overexpression of hepatic heme oxygenase-1 during endotoxin tolerance.

Heme oxygenase (HO)-1 has antioxidant and cytoprotective properties if properly expressed, whereas nitric oxide (NO) impairs tissue perfusion when greatly increased in the blood circulation. Here we hypothesized that the NO and HO-1 systems are altered during lipopolysaccharide (LPS) tolerance, and that glucocorticoids are crucial modulators of systemic NO production and hepatic HO-1 expression during this intriguing phenomenon of cellular reprogramming. Adrenalectomized (ADX) rats with or without administration of dexamethasone (DEX) were challenged with LPS for 3 consecutive days. The plasma levels of corticosterone and nitrate (NOx), and expression of HO-1 protein were assessed. During tolerance, corticosterone levels were elevated, NOx reduced, and HO-1 overexpressed. ADX rats challenged with LPS for 3 consecutive days exhibited a ~9-fold increase in NOx and a ~6-fold increase in HO-1, reverted by DEX. Our findings strongly support the fact that glucocorticoids downregulate systemic NO synthesis and counteract hepatic HO-1 overexpression during LPS tolerance.

Acetaminophen reduces lipopolysaccharide-induced fever by inhibiting cyclooxygenase-2.

Acetaminophen is one of the world's most commonly used drugs to treat fever and pain, yet its mechanism of action has remained unclear. Here we tested the hypothesis that acetaminophen blocks fever through inhibition of cyclooxygenase-2 (Cox-2), by monitoring lipopolysaccharide induced fever in mice with genetic manipulations of enzymes in the prostaglandin cascade. We exploited the fact that lowered levels of a specific enzyme make the system more sensitive to any further inhibition of the same enzyme. Mice were immune challenged by an intraperitoneal injection of bacterial wall lipopolysaccharide and their body temperature recorded by telemetry. We found that mice heterozygous for Cox-2, but not for microsomal prostaglandin E synthase-1 (mPGES-1), displayed attenuated fever, indicating a rate limiting role of Cox-2. We then titrated a dose of acetaminophen that did not inhibit the lipopolysaccharide-induced fever in wild-type mice. However, when the same dose of acetaminophen was given to Cox-2 heterozygous mice, the febrile response to lipopolysaccharide was strongly attenuated, resulting in an almost normalized temperature curve, whereas no difference was seen between wild-type and heterozygous mPGES-1 mice. Furthermore, the fever to intracerebrally injected prostaglandin E2 was unaffected by acetaminophen treatment. These findings reveal that acetaminophen, similar to aspirin and other non-steroidal anti-inflammatory drugs, is antipyretic by inhibiting cyclooxygenase-2, and not by inhibiting mPGES-1 or signaling cascades downstream of prostaglandin E2.

Association between serum levels of MASP-2 and neutropenic febrile attacks in children with leukemia.

BACKGROUND: Infectious complications are a major etiology of morbidity and mortality in febrile neutropenic patients. Low serum mannose-binding lectin (MBL)-associated serine protease-2 (MASP-2) concentration may represent a risk factor for infection in leukemia patients receiving chemotherapy. This study evaluates the relationship between serum levels of MASP-2 with neutropenic febrile attacks in children with leukemia. METHOD: This prospective cohort study conducted between 2009-2010, we measured baseline serum MASP-2 levels by enzyme-linked immunosorbent assay (ELISA) prior to chemotherapy in leukemia patients less than 14 years of age. The relationship of febrile neutropenia (FN) episodes and duration of hospitalization with MASP-2 concentration was analyzed. RESULTS: We evaluated 75 children [38 girls (51%), 37 boys (49%); mean age, 61.6 ± 43.7 months]. There were 8 (10.7%) children with MASP-2 deficiency (< 200 ng/mL). Mean MASP-2 was 673.2 ± 288.7 ng/mL (range: 116-1112). Eight patients had no FN episodes. Of the 129 FN episodes recorded, 19 (average 2.4 times) were from the MASP-2deficient group and 110 (average 1.6 times) were in the normal group. There was a significant difference between the mean MASP-2 concentration and FN episodes (P = 0.043). There was an inverse relationship between FN episodes (r = -0.332, P = 0.004) and the duration of hospitalization (r = -0.334, P = 0.005) with MASP-2 concentration. MASP-2 deficient patients were hospitalized longer than the normal group, which was strongly significant (P < 0.001). CONCLUSION: Our study confirmed the results of several previous studies. MASP-2 deficiency in leukemic children treated with chemotherapy was associated with an increased risk of FN episodes, prolonged cumulative duration of hospitalization, and intravenous antimicrobial therapy.

IL-2-inducible T-cell kinase deficiency with pulmonary manifestations due to disseminated Epstein-Barr virus infection.

IL-2-inducible T-cell kinase (ITK) deficiency is a rare inherited immunodeficiency disease characterized by homozygous mutations in the ITK gene and the inability to control Epstein-Barr virus (EBV) infection leading to EBV-associated lymphoproliferative disorders of B cell origin. Many aspects of its clinical presentation and immunologic phenotype are still unclear to clinicians. We report on a 14-year-old female patient with complaints of an 8-month history of cough and fever. Imaging studies revealed diffuse pulmonary nodules and mediastinal lymphadenopathy. Transbronchial lung biopsy showed nonmalignant polyclonal B cell proliferation. High titers of EBV DNA were detected by PCR analysis in bronchoalveolar lavage fluid, bone marrow, and blood. Genomic analysis revealed a homozygous single base pair deletion in exon 5 of the ITK gene (c.468delT) in this patient. Treatment with rituximab (anti-CD20 mab) resulted in complete clinical remission with resolution of pulmonary lesions and a negative EBV titer in serum. All patients with EBV-associated lymphoproliferative disorders should be analyzed for mutations in ITK.

Inhibition of checkpoint kinase 1 abrogates G2/M checkpoint activation and promotes apoptosis under heat stress.

Hyperthermia induced by heat stress (HS) inhibits the proliferation of cancer cells and induces their apoptosis. However, the mechanism underlying HS-induced apoptosis remains elusive. Here, we demonstrated a novel evidence that checkpoint kinase 1 (Chk1) plays crucial roles in the apoptosis and regulation of cell cycle progression in cells under HS. In human leukemia Jurkat cells, interestingly, the ataxia telangiectasia and Rad-3 related (ATR)-Chk1 pathway was preferentially activated rather than the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway under HS. The selective inhibitors of ATR or Chk1 abrogated HS-induced apoptosis in human leukemia Jurkat cells whereas the inhibition of ATM or Chk2 caused only marginal effects. Inhibition of ATR and Chk1 also abrogated G2/M checkpoint activation by HS in Jurkat cells. The effects of small interfering RNA targeting Chk1 were similar to those of the selective inhibitor of Chk1. In addition, the efficiencies of Chk1 inhibition on G2/M checkpoint abrogation and apoptosis induction were confirmed in the adherent cancer cell lines HeLa, HSC3, and PC3, suggesting that the targeting of Chk1 can be effective in solid tumors cells. In conclusion, these findings indicate a novel molecular basis of G2/M checkpoint activation and apoptosis in cells exposed to HS.

TAK1 in brain endothelial cells mediates fever and lethargy.

Systemic inflammation affects the brain, resulting in fever, anorexia, lethargy, and activation of the hypothalamus-pituitary-adrenal axis. How peripheral inflammatory signals reach the brain is still a matter of debate. One possibility is that, in response to inflammatory stimuli, brain endothelial cells in proximity to the thermoregulatory centers produce cyclooxygenase 2 (COX-2) and release prostaglandin E2, causing fever and sickness behavior. We show that expression of the MAP kinase kinase kinase TAK1 in brain endothelial cells is needed for interleukin 1ß (IL-1ß)-induced COX-2 production. Exploiting the selective expression of the thyroxine transporter Slco1c1 in brain endothelial cells, we generated a mouse line allowing inducible deletion of Tak1 specifically in brain endothelium. Mice lacking the Tak1 gene in brain endothelial cells showed a blunted fever response and reduced lethargy upon intravenous injection of the endogenous pyrogen IL-1ß. In conclusion, we demonstrate that TAK1 in brain endothelial cells induces COX-2, most likely by activating p38 MAPK and c-Jun, and is necessary for fever and sickness behavior.

The influence of natural variation at the foraging gene on thermotolerance in adult Drosophila in a narrow temperature range.

Poikilothermic organisms such as insects have mechanisms to protect neural function under high temperature stress. Natural variation at the foraging (for) locus of the fruit fly, Drosophila melanogaster, encoding a cGMP-dependent protein kinase (PKG), influences neural thermotolerance in Drosophila larvae. The current study re-examines thermotolerance of adult flies to account for inconsistencies in the documented role of for during hyperthermia. We found that adult for (R) (rover) flies with high PKG activity were incapacitated faster under hyperthermic conditions of 39°C compared to their lower PKG activity counterparts for (s) and for (s2) (sitters), but not at higher temperatures. This indicates that lowered PKG activity promotes tolerance to heat stress, and that the for gene influences thermotolerance for a narrow range of temperatures in adult flies.

Matrix metalloproteinase-9 (mmp-9) in children with dengue virus infection.

The purpose of this study was to investigate the role of matrix metalloproteinase-9 (MMP-9) in the pathogenesis of vascular leakage in patients with dengue virus infection. Serum samples from 24 children with serologically confirmed dengue virus infection (dengue fever [DF], 16; dengue hemorrhagic fever [DHF], 8; age, 9.5+/-2.4 years; 67% male] were analyzed for MMP-9 during the febrile and toxic stages and at follow-up. Serum samples obtained from 7 healthy children were used as controls. Serum MMP-9 levels in patients with dengue virus infection were found to be lower at the febrile (227.0+/-186.9 ng/ml) and toxic stages (150.9+/-151.7 ng/ml) than at follow-up (424.5+/-227.8 ng/ml) or in the control group (393.3+/-125.9 ng/ml, P<0.001 by one-way ANOVA). There was no significant difference between MMP-9 levels in patients with DHF and those with DF at any stage of the disease. In conclusion, MMP-9 levels are reduced during the febrile and toxic stages of dengue virus infection.

Inhibition of nitric oxide synthase in the paraventricular nucleus prevents the hyperthermia-induced reduction of mesenteric blood flow in rats.

Increasing body core temperature reflexly decreases mesenteric blood flow (MBF), and the hypothalamic paraventricular nucleus (PVN) plays an essential role in this response. Nitric oxide (NO) is involved in temperature regulation and is concentrated within the PVN. The present study investigated whether NO in the PVN contributes to the cardiovascular responses elicited by hyperthermia. Anesthetized rats were microinjected bilaterally in the PVN (100 nl/side) with saline or N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (100 or 200 nmol/100 nl) (n = 5/group). Body core temperature was then elevated from 37 degrees C to 39 degrees C, and blood pressure (BP), heart rate (HR), MBF, and mesenteric vascular conductance (MVC) were monitored. In separate groups, L-NAME (200 nmol) (n = 5) or saline (n = 5) was microinjected in the PVN, but body core temperature was not elevated. In control rats, increasing body core temperature resulted in no marked change of BP but an increase in HR and significant decreases in MBF (15%) and MVC. Pretreatment with 100 nmol L-NAME did not affect the responses. In contrast, 200 nmol L-NAME prevented the normal reduction in MBF and MVC but did not significantly affect the BP and HR responses. In rats in which body core temperature was not increased, L-NAME reduced MBF by 19%. The present results suggest that endogenous NO in the PVN is important in mediating the reduction of MBF induced by hyperthermia. In the absence of hyperthermia, however, endogenous NO in the PVN may play a role in maintaining mesenteric vasodilation.

Inhibition of protein kinase G activity protects neonatal mouse respiratory network from hyperthermic and hypoxic stress.

In spite of considerable research attention focused on clarifying the mechanisms by which the mammalian respiratory rhythm is generated, little attention has been given to examining how this neuronal circuit can be protected from heat stress. Hyperthermia has a profound effect on neuronal circuits including the circuit that generates breathing in mammals. As temperature of the brainstem increases, respiratory frequency concomitantly rises. If temperature continues to increase respiratory arrest (apnea) and death can occur. Previous research has implicated protein kinase G (PKG) activity in regulating neuronal thermosensitivity of neuronal circuits in invertebrates. Here we examine if pharmacological manipulation of PKG activity in a brainstem slice preparation could alter the thermosensitivity of the fictive neonatal mouse respiratory rhythm. We report a striking effect following alteration of PKG activity in the brainstem such that slices treated with the PKG inhibitor KT5823 recovered fictive respiratory rhythm generation significantly faster than control slices and slices treated with a PKG activator (8-Br-cGMP). Furthermore, slices treated with 8-Br-cGMP arrested fictive respiration at a significantly lower temperature than all other treatment groups. In a separate set of experiments we examined if altered PKG activity could regulate the response of slices to hypoxia by altering the protective switch to fictive gasping. Slices treated with 8-Br-cGMP did not switch to the fictive gasp-like pattern following exposure to hypoxia whereas slices treated with KT5823 did display fictive gasping. We propose that PKG activity inversely regulates the amount of stress the neonatal mammalian respiratory rhythm can endure.

Atypical protein kinase C activity in the hypothalamus is required for lipopolysaccharide-mediated sickness responses.

By activating the Toll-like receptor 4-nuclear factor-kappaB signal transduction pathway, the bacterial endotoxin lipopolysaccharide (LPS) induces anorexia, weight loss, fever, and other components of the sickness response. By comparison, the hormones leptin and insulin cause anorexia without sickness via a central mechanism involving the phosphatidylinositol-3 kinase signaling pathway. In the current study, we investigated whether a common Toll-like receptor 4 and phosphatidylinositol-3 kinase signaling intermediate, atypical protein kinase Czeta/lambda (aPKC), contributes to changes of energy balance induced by these stimuli. Immunohistochemistry analysis revealed that aPKC is expressed in the arcuate and paraventricular nuclei of the hypothalamus, key sites of leptin, insulin, and LPS action. Although administration of LPS, insulin, and leptin each acutely increased hypothalamic aPKC activity at doses that also reduce food intake, LPS treatment caused over 10-fold greater activation of hypothalamic a PKC signaling than that induced by leptin or insulin. Intracerebroventricular pretreatment with an aPKC inhibitor blocked anorexia induced by LPS but not insulin or leptin. Similarly, LPS-induced hypothalamic inflammation (as judged by induction of proinflammatory cytokine gene expression) and neuronal activation in the paraventricular nucleus (as judged by c-fos induction) were reduced by central aPKC inhibition. Although intracerebroventricular aPKC inhibitor administration also abolished LPS-induced fever, it had no effect on sickness-related hypoactivity or weight loss. We conclude that although hypothalamic aPKC signaling is not required for food intake inhibition by insulin or leptin, it plays a key role in inflammatory anorexia and fever induced by LPS.