Rosario Maldonado

Circulating Inflammatory Mediators during Start of Fever in Differential Diagnosis of Gram-Negative and Gram-Positive Infections in Leukopenic Rats

ABSTRACT Gram-negative and gram-positive infections have been considered the most important causes of morbidity and mortality in patients with leukopenia following chemotherapy. However, discrimination between bacterial infections and harmless fever episodes is difficult. Because classical inflammatory signs of infection are often absent and fever is frequently the only sign of infection, the aim of this study was to assess the significance of serum interleukin-6 (IL-6), IL-10, macrophage inflammatory protein-2 (MIP-2), procalcitonin (PCT), and C-reactive protein (CRP) patterns in identifying bacterial infections during start of fever in normal and cyclophosphamide-treated (leukopenic) rats following an injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP) as a model for gram-negative and gram-positive bacterial infections. We found that, compared to normal rats, immunosuppressed animals exhibited significantly higher fevers and lesser production of all mediators, except IL-6, after toxin challenge. Moreover, compared to rats that received MDP, both groups of animals that received an equivalent dose of LPS showed significantly higher fevers and greater increase in serum cytokine levels. Furthermore, in contrast to those in immunocompetent rats, serum levels of IL-6 and MIP-2 were not significantly changed in leukopenic animals after MDP injection. Other serum markers such as PCT and CRP failed to discriminate between bacterial stimuli in both groups of animals. These results suggest that the use of the analyzed serum markers at an early stage of fever could give useful information for the clinician for excluding gram-negative from gram-positive infections.

Identification and localization of procalcitonin-like immunoreactivity in the rat hypothalamus

Procalcitonin (PCT) is a 116-amino acid polypeptide physiologically produced, as the precursor protein of calcitonin (CT), in the parafollicular cells of the thyroid gland, but physiological functions and other major sources of PCT remains unclear. The distribution of PCT-like immunoreactivity (PCT-LI) in the rat hypothalamus was examined by immunohistochemistry using a monoclonal antibody raised against the mid-region of human PCT (60-77-amino acid fragment). This antibody cross-reacts well with rat PCT and immature CT, but it cross-react poorly with free mature CT. Abundant expression of PCT-LI was found in zones at the interface between brain and cerebrospinal fluid (CSF) such as the ependymal layer and ventral glia limitans (VGL). Double labeling of PCT and glial fibrillary acidic protein (GFAP) identified this population of small cells as astrocytes, possibly tanycytes, a type of specialized glial cell that interacts in neuroendocrine functional dynamics. The fibers of these cells extend to circumventricular organs (CVOs) and to astrocytes located inside the parenchyma of key autonomic regulatory hypothalamic areas, with highest densities in the supraoptic nucleus (SO), arcuate nucleus (Arc), area postrema (AP), median eminence (ME), medial preoptic nucleus, tuber cinereum, and accessory neurosecretory nuclei. No strongly labeled cells were found in the paraventricular nucleus. The wide distribution of PCT-LI in the hypothalamus, in close correspondence with previous mapping of CT receptors in the rat brain, suggests that PCT may influence a multitude of biological activities associated with the hypothalamic-pituitary axis.

Endotoxin fever in granulocytopenic rats: evidence that brain cyclooxygenase‐2 is more important than circulating prostaglandin E2

PGE2 is a recognized mediator of many fevers, and cyclooxygenase (COX) is the major therapeutic target for antipyretic therapy. The source, as well as the site of action of PGE2, as an endogenous pyrogen, is widely accepted as being central, but PGE2 in the circulation, possibly from leukocytes, may also contribute to the development of fever. However, bacterial infections are important causes of high fever in patients receiving myelosuppressive chemotherapy, and such fevers persist despite the use of COX inhibitors. In the study reported here, the febrile response to bacterial LPS was measured in rats made leukopenic by cyclophosphamide. A striking increase in LPS fever occurred in these granulocytopenic rats when compared with febrile responses in normal animals. Unlike LPS fever in normal rats, fever in granulocytopenic rats was neither accompanied by an increase in blood PGE2 nor inhibited by ibuprofen. Both leukopenic and normal rats showed LPS‐induced COX‐2‐immunoreactivity in cells associated with brain blood vessels. Furthermore, LPS induced an increase of PGE2 in cerebrospinal fluid. Induction of COX‐2‐expression and PGE2 production was inhibited by ibuprofen in normal but not in leukopenic rats. Although the results presented are, in part, confirmatory, they add new information to this field and open a number of important questions as yet unresolved. Overall, the present results indicate that, in contrast to immunocompetent rats, leukocytes and/or other mechanisms other than PGE2 are implicated in the mechanisms restricting and reducing the enhanced febrile response to endotoxin in immunosuppressed hosts.

Aminoprocalcitonin-mediated suppression of feeding involves the hypothalamic melanocortin system

Aminoprocalcitonin (N-PCT), a neuroendocrine peptide encoded by the calcitonin-I (CALC-I) gene, suppresses food intake when administered centrally in rats. However, the neural pathways underlying this effect remain unclear. N-PCT and calcitonin receptors (CT-R) have been identified in hypothalamic regions involved in energy homeostasis, including the arcuate nucleus (ARC). Here, we hypothesized an involvement of the hypothalamic ARC in mediating the anorexic effects of central N-PCT based on its content of peptidergic neurons involved in feeding and its expression of N-PCT and CT-R. Fasting strongly reduced expression of the N-PCT precursor gene CALC-I in the ARC, and central immunoneutralization of endogenous N-PCT increased food intake. Intracerebroventricular administration of N-PCT reduced food intake in fed and fasted rats, and its effect was attenuated by a neutralizing anti-N-PCT antibody. Immunohistochemistry for N-PCT showed that it is expressed in astrocytes and neurons in the ARC and is colocalized with anorexigenic proopiomelanocortin (POMC) neurons. Fasting reduced coexpression of N-PCT and POMC, and N-PCT administration activated hypothalamic neurons, including rostral POMC neurons. We also found that N-PCT stimulates POMC mRNA expression in fed and fasted rats, whereas it reduced the expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP) only in fasted rats in which those mRNAs are normally elevated. Finally, we showed that the melanocortin-3/4 receptor antagonist SHU 9119 attenuates the intake-suppressive effect of N-PCT. These data demonstrate that hypothalamic N-PCT is involved in control of energy balance and that its anorexigenic effects are mediated through the melanocortin system.

Immunoneutralization of Endogenous Aminoprocalcitonin Attenuates Sepsis-Induced Acute Lung Injury and Mortality in Rats

Acute lung injury (ALI) secondary to sepsis is a complex syndrome associated with high morbidity and mortality. We report that aminoprocalcitonin (NPCT), an endogenous peptide derived from the prohormone procalcitonin, plays a critical role in the development of ALI during severe sepsis and is a suggested risk factor for sepsis morbidity and mortality. Lethal sepsis was induced in rats by cecal ligation and puncture (CLP). Two hours after CLP, an i.p. injection of 200 μg/kg of anti-rat NPCT antibody was followed by continuous infusion of anti-NPCT (16 μg per hour) via a minipump for 18 hours. Samples were harvested 20 hours after CLP. High expressions of the CALCA gene, procalcitonin, and NPCT were detected in the lung tissue of rats with severe sepsis. Immunoneutralization of NPCT decreased pulmonary levels of CALCA, procalcitonin, and NPCT; reduced lung inflammation and injury, neutrophil infiltration, and bacterial invasion; and improved survival in sepsis. Anti-NPCT treatment also suppressed sepsis-induced inflammatory cytokine expression, cytoplasmic degradation of the inhibitor of NF-κB, IκBα, and nuclear NF-κB translocation in lung tissues. Therapeutic benefits of anti-NPCT were also associated with increased pulmonary levels of the anti-inflammatory cytokine IL-10. These data support a pathogenic role for NPCT in sepsis and suggest NPCT as a potential new target for clinical prevention and treatment of ALI in severe sepsis.

Central Administration of Aminoprocalcitonin Inhibits Food Intake and Stimulates the Hypothalamic-Pituitary-Adrenal Axis in Rats via the Corticotrophin-Releasing Factor System

Aminoprocalcitonin (N‐PCT), a neuroendocrine peptide derived from procalcitonin, reduces food intake and body weight when administered centrally in rats. We have recently shown that N‐PCT is expressed in brain areas known to be involved in energy homeostasis, including the paraventricular nucleus (PVN) of the hypothalamus, which contains a prominent population of corticotrophin‐releasing factor (CRF)‐synthesising neurones. CRF plays a pivotal role in the regulation of the hypothalamic‐pituitary adrenal (HPA) axis and food intake. However, little is known about functional interactions of N‐PCT and CRF. In the present study, we found endogenous N‐PCT protein in the rat PVN. We also showed N‐PCT immunoreactivity in PVN co‐localised with NeuN, a neuronal marker, or glial fibrillary acidic protein, an astrocyte marker. Double staining immunohistochemistry revealed that N‐PCT co‐localised with CRF in parvocellular neurones of the PVN. Intracerebroventricular N‐PCT administration increased CRF mRNA and content in the hypothalamus, suggesting that N‐PCT stimulates the HPA axis and suppresses food intake and body weight via CRF‐dependent pathways. In keeping with this, i.c.v. co‐injection of D‐Phe‐CRF12‐41, a CRF receptor antagonist, significantly attenuated N‐PCT‐induced reduction in food intake and body weight in a dose‐dependent manner. Furthermore, i.c.v. administration of N‐PCT increased plasma adrenocorticotrophic hormone and corticosterone concentrations and induced the expression of Fos protein, a marker of neuronal activity, in parvocellular CRF neurones. These data collectively support the hypothesis that N‐PCT inhibits food intake and body weight and stimulates the HPA axis via CRF‐mediated pathways.

Obesity and neuroinflammatory phenotype in mice lacking endothelial megalin.

BackgroundThe multiligand receptor megalin controls the brain uptake of a number of ligands, including insulin and leptin. Despite the role of megalin in the transport of these metabolically relevant hormones, the role of megalin at the blood–brain-barrier (BBB) has not yet been explored in the context of metabolic regulation.MethodsHere we investigate the role of brain endothelial megalin in energy metabolism and leptin signaling using an endothelial cell-specific megalin deficient (EMD) mouse model.ResultsWe found megalin is important to protect mice from developing obesity and metabolic syndrome when mice are fed a normal chow diet. EMD mice developed neuroinflammation, by triggering several pro-inflammatory cytokines, displayed reduced neurogenesis and mitochondrial deregulation.ConclusionsThese results implicate brain endothelial megalin expression in obesity-related metabolic changes through the leptin signaling pathway proposing a potential link between obesity and neurodegeneration.

Role of endogenous macrophage inflammatory protein-2 in regulating fever induced by bacterial endotoxin in normal and immunosuppressed rats

1. During myelosuppressive chemotherapy, Gram‐negative bacterial infection with consequent exposure to lipopolysaccharide (LPS) is one of the most important causes of persistent fever.