Eva Tavares

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.

Folic acid intestinal absorption in newborn rats at 21 day postpartum: effects of maternal ethanol consumption.

This study was designed to examine the effects of prenatal and postnatal exposure of ethanol in the in vivo absorption of free folic acid in the small intestine in pups rats at the 21st day after birth. The rats were accustomed to increasing amounts of ethanol (5 to 20%, vol/vol) in tap water for 1 month. During pregnancy and suckling period, ethanol-fed dams were assigned again to ethanol 20% in drinking water. Two sets of experiments were performed. In the first set, jejunal free folic acid absorption in control group and litters nursed by dams receiving ethanol showed a gradual increase along with the increase of perfusion time at all the assayed concentrations. In general, in litters of ethanol-fed dams, jejunal free folic acid absorption expressed as nmol/intestinal surface, nmol/g tissue wet weight and nmol/g tissue dry weight were higher than in control animals. In the second set of experiments, in distal ileum loops, free folic acid absorption did not occur in control pups, but appeared in litters exposed to ethanol. Milk folic acid levels are significantly decreased in ethanol-treated dams. However, only a slight decrease in the serum folic acid levels occurs in litters of ethanol-fed dams. In conclusion, the results obtained in the present work suggested a different pattern of free folic acid absorption in distal ileum for the two groups. The exposure of rats to ethanol during the pregnancy and suckling period, can affect postnatal development of intestinal functions and could play a role in the genesis of malnutrition observed in the infant.

RANTES: a new prostaglandin dependent endogenous pyrogen in the rat

Fever, a hallmark of disease, is a highly complex process initiated by the action of a number of endogenous pyrogens on the thermosensitive cells of the brain. We describe the activity of RANTES, a chemotactic cytokine, as intrinsically pyrogenic in the rat, when it is delivered directly to the thermosensitive region of the rats anterior hypothalamic, pre-optic area (AH/POA). RANTES, microinjected into the AH/POA in a dose of 1, 5, 10, 15, 25 or 50 pg, produces an immediate and intense dose-related fever following injection. Increasing the dose to 100 pg did not result in a further increase in the febrile response. No significant change in body temperature was produced by heat-inactivated RANTES. The intrahypothalamic injection of antibodies against RANTES (2.0 microg, 15 min prior to RANTES) significantly blocked the fever induced by this chemokine. Pretreatment with ibuprofen blocked the fever induced by RANTES. In order of potency, the magnitude of the febrile response induced by RANTES was greater than that produced with equipotent doses of either macrophage inflammatory protein-1beta or interleukin-6. The results thus demonstrate that RANTES is the most potent endopyrogen discovered thus far and exerts its action directly on pyrogen-sensitive cells of the AH/POA through a prostaglandin-dependent pathway.

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)-1β. 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-1β 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.

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

Macrophage inflammatory protein (MIP)‐1β 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‐1β 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‐1β or RANTES into the POA induced a rapid onset; monophasic fever in rats that persisted for a long period. The microinjection of 2.0 μg specific neutralizing antibodies against CCR5 (anti‐CCR5) into the POA fails to affect the effects on body temperature induced by MIP‐1β. 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‐1β. 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.

Macrophage inflammatory protein-1β induces dexamethasone-unresponsive fever in rats

IT has been hypothesized that endogenous glucocorticoids represent an important negative feedback system involved in the modulation of cytokine-induced fever through the inhibition of prostaglandins (PG) production in the preoptic anterior hypothalamus (AH/POA). The purpose of this study was to determinate whether glucocorticoids modulate the PG-independent febrile response induced by macrophage inflammatory protein-1β (MIP-1β) in a manner similar to other pyrogenic cytokines. Subcutaneous pretreatment with dexamethasone (1, 2 and 4 mg/kg; 1 h) had no effect on fever induced by microinjection of 50 pg MIP-1β into the rats AH/POA. It is demonstrated for the first time that, unlike other cytokines, fever induced by MIP-1β is independent of glucocorticoid modulation. Finally, these results offer new perspectives about the pathogenesis of glucocorticoid-unresponsive pyrexia.

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.

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.