Potential Role of Aminoprocalcitonin in the Pathogenesis of Alzheimer Disease.

Increasing evidence suggests that inflammatory responses cause brain atrophy and play a prominent and early role in the progression of Alzheimer disease. Recent findings show that the neuroendocrine peptide aminoprocalcitonin (NPCT) plays a critical role in the development of systemic inflammatory response; however, the presence, possible function, regulation, and mechanisms by which NPCT may be involved in Alzheimer disease neuropathology remain unknown. We explored the expression of NPCT and its interaction with amyloid-ß (Aß), and proinflammatory and neurogenic effects. By using brain samples of Alzheimer disease patients and APP/PS1 transgenic mice, we evaluated the potential role of NPCT on Aß-related pathology. We found that NPCT is expressed in hippocampal and cortical neurons and Aß-induced up-regulation of NPCT expression. Peripherally administered antibodies against NPCT decreased microglial activation, decreased circulating levels of proinflammatory cytokines, and prevented Aß-induced neurotoxicity in experimental models of Alzheimer disease. Remarkably, anti-NPTC therapy resulted in a significant improvement in the behavioral status of APP/PS1 mice. Our results indicate a central role of NPCT in Alzheimer disease pathogenesis and suggest NPCT as a potential biomarker and therapeutic target.

THDP17 decreases ammonia production through glutaminase inhibition. A new drug for hepatic encephalopathy therapy.

Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing Vmax and Km values of 384.62 µmol min(-1), 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.

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.

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 the aminoprocalcitonin peptide of procalcitonin protects rats from lethal endotoxaemia: neuroendocrine and systemic studies.

Severe sepsis and septic shock are an important cause of mortality and morbidity. These illnesses can be triggered by the bacterial endotoxin LPS (lipopolysaccharide) and pro-inflammatory cytokines, particularly TNF-α (tumour necrosis factor-α) and IL (interleukin)-1ß. Severity and mortality of sepsis have also been associated with high concentrations of N-PCT (aminoprocalcitonin), a 57-amino-acid neuroendocrine peptide derived from ProCT (procalcitonin). Previous studies in a lethal model of porcine polymicrobial sepsis have revealed that immunoneutralization with IgG that is reactive to porcine N-PCT significantly improves short-term survival. To explore further the pathophysiological role of N-PCT in sepsis, we developed an antibody raised against a highly conserved amino acid sequence of human N-PCT [N-PCT-(44-57)]. This sequence differs by only one amino acid from rat N-PCT. First, we demonstrated the specificity of this antibody in a well-proven model of anorexia induced in rats by central administration of human N-PCT-(1-57). Next we explored further the therapeutic potential of anti-N-PCT-(44-57) in a rat model of lethal endotoxaemia and determined how this immunoneutralization affected LPS-induced lethality and cytokine production. We show that this specific antibody inhibited the LPS-induced early release of TNF-α and IL-1ß and increased survival, even if treatment began after the cytokine response had occurred. In addition, anti-N-PCT-(44-57) may increase long-term survival in LPS-treated rats by up-regulating the late production of counter-regulatory anti-inflammatory mediators such as ACTH (adrenocorticotropic hormone) and IL-10. In conclusion, these results support N-PCT as a pro-inflammatory factor in both the early and the late stages of lethal endotoxaemia, and suggest anti-N-PCT as a candidate for septic shock therapy.

Procalcitonin N-terminal peptide causes catabolic effects via the hypothalamus and prostaglandin-dependent pathways.

Recent evidence suggests that the free amino-terminal fragment of procalcitonin (N-PCT) plays a role in the central control of feeding behavior and energy homeostasis. However, little is known about the mechanisms through which N-PCT works. Here we report that intracerebroventricular administration of N-PCT to free-feeding male rats induced a significant decrease of longer-term food intake and body weight gain. Conversely, N-PCT increased body temperature. We also show that intracerebroventricular administration of N-PCT induced a marked neuronal activation in key thermoregulatory and feeding areas of the hypothalamus. We further show that N-PCT increases the responsiveness of proopiomelanocortin anorexigenic neurons in the arcuate nucleus of the hypothalamus, and that stimulation of the de novo synthesis of prostaglandins is crucial for the central effects induced by N-PCT. Results support the role of N-PCT to the central control of feeding behavior and suggest that N-PCT, acting probably through the eicosanoid cyclooxygenase pathway, may act as a signaling molecule in the hypothalamus by regulating the activity of anorexigenic neurons in the hypothalamus.

N-procalcitonin: central effects on feeding and energy homeostasis in rats.

Procalcitonin (PCT), the precursor of calcitonin (CT), is a 116-amino-acid peptide, but PCT itself has no known activity. However, although the C cells of the thyroid gland are the dominant source of circulating CT, PCT and its free bioactive amino-terminal fragment (N-PCT) have been localized in adipocytes and neuroendocrine cells as well as in some hypothalamic regions of primary importance in the regulation of feeding and energy balance. These findings together with the coelaboration of N-PCT and CT, and N-PCT's sequence conservation during evolution, suggest that N-PCT has a critical, and as yet undefined, physiological function. We demonstrate here that intracerebroventricular administration of N-PCT significantly decreased food intake and body weight gain for at least 48 h in conscious, freely moving, and unstressed rats fed ad libitum. These effects were accompanied by a transitory increase in body temperature and a decrease in locomotor activity. Moreover, after intracerebroventricular N-PCT administration, Fos protein, a marker of neuronal activation, was found in regions of primary importance in the integration of hormonal signals for energy homeostasis and feeding. In contrast, ip administration of N-PCT did not elicit any anorectic or catabolic effects. Furthermore, PCT was found in key feeding areas such as the arcuate nucleus of free-feeding rats, and its level was significantly reduced after fasting. These results suggest that N-PCT can function as an endogenous ligand for the CT receptor and may act as a catabolic signaling molecule in the central regulation of feeding behavior and energy homeostasis.

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 E(2).

PGE(2) 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 PGE(2), as an endogenous pyrogen, is widely accepted as being central, but PGE(2) 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 PGE(2) 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 PGE(2) in cerebrospinal fluid. Induction of COX-2-expression and PGE(2) 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 PGE(2) are implicated in the mechanisms restricting and reducing the enhanced febrile response to endotoxin in immunosuppressed hosts.

5-HT1A receptor activation counteracts c-Fos immunoreactivity induced in serotonin neurons of the raphe nuclei after immobilization stress in the male rat.

The serotoninergic system and the 5-HT1A receptors have been involved in the brain response to acute stress. The aim of our study was evaluate the role of the 5-HT1A receptors in serotoninergic cells of rostral and caudal raphe nuclei under acute immobilization in rats. Double immunocytochemical staining of 5-hydroxy-tryptamine and c-Fos protein and stereology techniques were used to study the specific cell activation in the raphe nuclei neurons in five groups (control group, immobilization group (immobilization lasting 1 h), DPAT group (8-OH-DPAT 0.3 mg/kg, s.c.), DPAT+IMMO group (8-OH-DPAT 0.3 mg/kg, s.c., 30' prior acute immobilization) and WAY+DPAT+IMMO group (WAY-100635 0.3 mg/kg, s.c. and 8-OH-DPAT 0.3 mg/kg, s.c., 45' and 30', respectively, before immobilization). Our results showed an increase in the number of c-Fos immunoreactive nuclei in serotoninergic cells in both dorsal and median raphe nuclei in the immobilized group. The 8-OH-DPAT pretreatment counteracted the excitatory effects of the acute immobilization in these brain regions. In addition, WAY-100635 administration reduced the effect of 8-OH-DPAT injection, suggesting a selective 5-HT1A receptor role. Raphe pallidus and raphe obscurus did not show any differences among experimental groups. We suggest that somatodendritic 5-HT1A receptors in rostral raphe nuclei may play a crucial role in both mediating the consequences of uncontrollable stress and the possible beneficial effects of treatment with 5-HT1A receptor agonists.

Circulating inflammatory mediators during start of fever in differential diagnosis of gram-negative and gram-positive infections in leukopenic rats.

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.

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

During myelosuppressive chemotherapy, Gram-negative bacterial infection with consequent exposure to lipopolysaccharide (LPS) is one of the most important causes of persistent fever. The classical model of the pathogenesis of fever suggests that pro-inflammatory cytokines, produced by leucocytes in the bloodstream in response to exogenous pyrogens such as bacterial LPS, represent the distal mediators of the febrile response. Neutrophils are the first effectors cells and the most prominent leucocyte population involved in acute bacterial infection. Macrophage inflammatory protein (MIP)-2 plays a crucial role in influencing early cell trafficking and neutrophil activation during pathophysiological processes and serves the same chemotactic function as human interleukin-8. In the present study, we investigated the role of MIP-2 in the development of a febrile response induced by LPS in immunocompetent and leukopenic rats. Intraperitoneal injection of LPS in leukopenic rats elicited a biphasic febrile response of rapid onset, the magnitude and duration of which were significantly greater than in immunocompetent animals. The febrile responses to LPS were accompanied by a pronounced induction of serum MIP-2 levels at 1, 2 and 4 h compared with their respective controls. In both normal and leukopenic rats, neutralization of endogenous MIP-2 bioactivity by systemic administration of antirat MIP-2 antibody caused a significant attenuation of the early phase of LPS fever. However, in contrast with normal rats, the second phase of fever was unimpaired by anti-MIP-2 in leukopenic rats. These findings suggest that circulating MIP-2 is involved in the generation of the early phase of LPS fever that contributes to the maintenance of the later phase of fever in immunocompetent, but not leukopenic, rats. Our data support a regulatory role for endogenous MIP-2 in initiating the fever responses to LPS. Furthermore, these results provide evidence that different cellular and humoral mechanisms are implicated in the development of a febrile response triggered by Gram-negative bacterial infections in leukopenic hosts.

Differential sensitivities of pyrogenic chemokine fevers to CC chemokine receptor 5 antibodies.

Macrophage inflammatory protein (MIP)-1beta and RANTES (regulated on activation, normal T-cells expressed and secreted) are members of the CC-family of chemokines. Although these two peptides are structurally and functionally related to one another, each exhibits distinct features, which allows it to independently regulate specific aspects of the host inflammatory response. They evoked intense and functionally different febrile responses when applied directly on pyrogen-sensitive cells located in the in the preoptic area of the anterior hypothalamus (POA). The present experiments were carried out to test the central role of CCR5, a functional receptor for MIP-1beta and RANTES, in the febrile responses induced by these chemokines when injected directly into the POA. The microinjection of an equimolecular dose (50 pg) of either MIP-1beta or RANTES into the POA induced a rapid onset; monophasic fever in rats that persisted for a long period. The microinjection of 2.0 microg specific neutralizing antibodies against CCR5 (anti-CCR5) into the POA fails to affect the effects on body temperature induced by MIP-1beta. However, pretreatment with the same dose of anti-CCR5 suppressed the febrile response induced by RANTES given at the same site. The microinjection of control IgG or anti-CCR5 does not affect basal temperature, when administered alone at the same hypothalamic site. The present experiments show that hypothalamic CCR5 are functionally involved in the febrile response induced by RANTES, but not by MIP-1beta. They also suggest the existence of functionally different components in the presumptive primary locus of the thermoregulatory controller, in which both chemotactic cytokines, together other mediators, could play a relevant role in the complex process of fever pathogenesis.

Differential sensitivities of pyrogenic chemokine fevers to cyclooxygenase isozymes antibodies.

It has been proposed that prostaglandin (PG)E(2) production via a process catalyzed by the inducible isoform of cyclooxygenase (COX)-2 and activation of specific PGE(2) receptor subtypes within the preoptic/anterior hypothalamus (AH/POA) is the last step and unique pathway in the induction of a fever. However, many data support the existence of a PG-independent pathway. That is, other more rapid mechanisms, which involve the constitutive COX-1 isozyme, may be more critical for a PG-dependent fever. Thus, we examined the role of both COX isoforms in the AH/POA in fevers induced by macrophage inflammatory protein (MIP)-1beta, a PG-independent pyrogen, and RANTES (regulated on activation, normal T-cells expressed and secreted), a PG-dependent pyrogen. In freely moving rats, two independent polyclonal antibodies were used which neutralize COX-1 and COX-2. The microinjection of either MIP-1beta or RANTES into the pyrogen-sensitive region of the AH/POA induced an intense fever of rapid onset. Peripheral pretreatment with an antipyretic dose of dexamethasone which prevents COX-2 expression, or the microinjections into the AH/POA of either anti-COX-1 or anti-COX-2, blocked the febrile response induced by RANTES but not that induced by MIP-1beta. These results provide strong evidence for the existence of rapid mechanisms in the AH/POA which involve both COX isozymes during the fever induced by RANTES, and further support the existence of an alternative PG-independent pathway in the febrile response.