Pyrogen-free Saline Research Articles

Putative antihyperpyretic factor induced by LPS in spleen of guinea pigs

We reported previously that the onset of LPS-induced fever, irrespective of its route of administration, is temporally correlated with the appearance of LPS in the liver and that splenectomy significantly increases both the febrile response to LPS and the uptake of LPS by Kupffer cells (KC). To further evaluate the role of the spleen in LPS fever production, we ligated the splenic vein and, 7 and 30 days later, monitored the core temperature changes over 6 h after intraperitoneal (ip) injection of LPS (2 microg/kg). Both the febrile response and the uptake of LPS by KC were significantly augmented. Like splenectomy, splenic vein ligation (SVL) increased the febrile response and LPS uptake by KC until the collateral circulation developed, suggesting that the spleen may normally contribute an inhibitory factor that limits KC uptake of LPS and thus affects the febrile response. Subsequently, to verify the presence of this factor, we prepared splenic extracts from guinea pigs pretreated with LPS (8 microg/kg ip) or pyrogen-free saline, homogenized and ultrafiltered them, and injected them intravenously into splenectomized (Splex) guinea pigs pretreated with LPS (8 microg/kg ip). The results confirmed our presumption that the splenic extract from LPS-treated guinea pigs inhibits the exaggerated febrile response and the LPS uptake by the liver of Splex guinea pigs, indicating the presence of a putative splenic inhibitory factor, confirming the participation of the spleen in LPS-induced fever, and suggesting the existence of a novel antihyperpyretic mechanism. Preliminary data indicate that this factor is a lipid.

The Effects of a Brief Light Pulse and Intracerebroventricular Injection on Sleep-Wake Activity in Rats

Objectives The light/dark (L/D) cycle is an important regulator of the circadian rhythm of sleep-wake activity and neuroendocrine fluctuation. We previously reported that the intracerebroventricular (ICV) injection of corticotropin-releasing hormone (CRH) antisense oligodeoxynucleotide during the middle of the dark period altered spontaneous waking; however, it is believed that either the light pulse during the ICV injection or the injection per se in the middle of the L/D period may disturb circadian rhythm of rats. The purpose of this study was to further elucidate the effects of ICV injection and the light pulse given during the dark period and during the light period on sleep-wake activity. Methods. Male Sprague-Dawley rats were implanted with electroencephalogram (EEG) electrodes and an ICV guide cannula. Rats were accustomed to a 12/12h L/D cycle. Na rats received a 5-mm period of light flash either in the middle of the light period or in the middle of the dark period, whereas the well-habituated rats received a light pulse and ICV injection of pyrogen-free saline (PFS) either in the middle of the light period or in the middle of the dark period. Results In naive rats, the 5-mm period of light pulse given during the dark period of the L/D cycle significantly enhanced slow wave sleep (SWS) and decreased rapid eye movement sleep (REMS) during the hour when the light flashed. Sleep was not altered when the light pulse occurred during the light period. There was no change in brain temperature. In well-habituated rats, the light pulse with ICV injection of PFS decreased SWS and REMS during the hour when the manipulation was given; during the subsequent 12-h period, the SWS and REMS significantly rebounded. Brain temperature increased after ICV injection. Conclusions. Our results indicate that injection and handling, which are considered to be stressors to rats, either during the middle of light period or during the middle of the dark period, mask the effect of light pulse on sleep-wake activities.

Expression of Stromal Cell-Derived Factor-1 and of Its Receptor CXCR4 in Liver Regeneration from Oval Cells in Rat

Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.

Surfactant protein D expression in normal and pneumonic ovine lung.

Surfactant protein D (SP-D) is a collagenous calcium-dependent lectin constitutively expressed by alveolar type II pneumocytes and non-ciliated bronchiolar epithelial (Clara) cells. It binds to surface glycoconjugates expressed by a wide variety of microorganisms such as Gram-negative bacteria, influenza A virus, and various fungi, leading to pathogen inactivation or enhanced neutrophil and macrophage activity. Since a hallmark of bronchopneumonia is the initiation of inflammation in the bronchi and bronchoalveolar junction, we chose a classic ruminant model of bronchopneumonia caused by Mannheimia haemolytica to study the expression of SP-D within the bronchioles of infected lambs. Healthy weaned lambs were inoculated with either pyrogen-free saline (controls) or M. haemolytica intrabronchially using a fiber-optic bronchoscope. SP-D protein and mRNA expression in lung was detected by immunohistochemistry (IHC) and fluorogenic real-time relative quantitative reverse transcriptase polymerase chain reaction (real-time RT-PCR), respectively, during acute (1 day), subacute (15 days), and chronic (45 days) bronchopneumonia. At 15 and 45 days post-inoculation, areas of lung had peribronchiolar inflammatory cell infiltrate, epithelial cell hyperplasia, tortuosity of the airway lumens, and decreased intensity of SP-D protein staining and number of positive cells. The levels of SP-D mRNA were not increased or significantly altered by M. haemolytica infection when compared to control animals. In conclusion, cell-associated SP-D protein expression significantly decreases within hyperplastic epithelium of lungs from infected animals during chronic bronchopneumonia. Exhaustion of SP-D protein reserves and absence of SP-D gene upregulation during the progression of bacterial pneumonia into chronicity may result in failure to clear the pathogen from the lung and/or cause animals to be more susceptible to re-infection.

Changes in quinolinic acid production and its related enzymes following d-galactosamine and lipopolysaccharide-induced hepatic injury

Increases in quinolinic acid (QUIN), a neurotoxic l-tryptophan metabolite, have been observed in human serum and cerebrospinal fluid and in animal models of severe hepatic injury. The aim of this study was to evaluate the changes in QUIN accumulation and its related enzymes after acute hepatic injury induced by d-galactosamine and endotoxin. Gerbils were given an intraperitoneal injection of pyrogen-free saline alone as control, lipopolysaccharide (LPS) alone (150 ng/kg), d-galactosamine alone (500 mg/kg) or a combination of d-galactosamine with LPS. Concentrations of QUIN, its related metabolites, and related enzyme activities were determined. d-Galactosamine treatment significantly decreased activities of hepatic aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) resulting in increased QUIN concentrations in serum and tissues. The magnitude of QUIN responses was markedly increased by endotoxin due to the increased availability of l-kynurenine, a rate-limiting substrate for QUIN synthesis. Further, infiltration of monocytes/macrophages, which is a possible major source of QUIN production in the liver, was shown by immunohistochemistry after hepatic injury induced by d-galactosamine and endotoxin. Increased serum QUIN concentrations are probably due to the increased substrate availability and the decreased activity of aminocarboxymuconate-semialdehyde decarboxylase in the liver, accompanying the increased monocyte/macrophage infiltration into the liver after hepatic injury.

Long-term alterations in neuroimmune responses after neonatal exposure to lipopolysaccharide.

Fever is an integral part of the host's defense to infection that is orchestrated by the brain. A reduced febrile response is associated with reduced survival. Consequently, we have asked if early life immune exposure will alter febrile and neurochemical responses to immune stress in adulthood. Fourteen-day-old neonatal male rats were given Escherichia coli lipopolysaccharide (LPS) that caused either fever or hypothermia depending on ambient temperature. Control rats were given pyrogen-free saline. Regardless of the presence of neonatal fever, adult animals that had been neonatally exposed to LPS displayed attenuated fevers in response to intraperitoneal LPS but unaltered responses to intraperitoneal interleukin 1beta or intracerebroventricular prostaglandin E(2). The characteristic reduction in activity that accompanies fever was unaltered, however, as a function of neonatal LPS exposure. Treatment of neonates with an antigenically dissimilar LPS (Salmonella enteritidis) was equally effective in reducing adult responses to E. coli LPS, indicating an alteration in the innate immune response. In adults treated as neonates with LPS, basal levels of hypothalamic cyclooxygenase 2 (COX-2), determined by semiquantitative Western blot analysis, were significantly elevated compared with controls. In addition, whereas adult controls responded to LPS with the expected induction of COX-2, adults pretreated neonatally with LPS responded to LPS with a reduction in COX-2. Thus, neonatal LPS can alter CNS-mediated inflammatory responses in adult rats.

Interleukin-8 promotes non-rapid eye movement sleep in rabbits and rats.

Interleukin-8 (IL-8) is a cytokine found in the brain. In this study, the ability of IL-8 to induce sleep in rabbits and rats was investigated. Twenty-seven Sprague-Dawley rats and 16 male New Zealand White rabbits were provided electroencephalographic (EEG) electrodes, a brain thermistor, and a lateral intracerebroventricular cannula. The animals were injected intracerebroventricularly (i.c.v.) with pyrogen-free saline and, one of the following doses of IL-8 on a separate day: 1.25 or 12.5 ng in rabbits and 10, 50, or 100 ng in rats. EEG, brain temperature, and motor activity were recorded for 23 h after the i.c.v. injections. IL-8 increased time spent in non-rapid eye movement sleep (NREMS) without affecting rapid eye movement sleep (REMS). In rabbits, both doses of IL-8 promoted NREMS. In rats, the 10 and 50 ng doses of IL-8 failed to affect sleep, but the 100 ng dose of IL-8 enhanced NREMS. EEG slow-wave activity during NREMS was increased after the high dose of IL-8 in rabbits. IL-8 also induced fever in rabbits but not rats. Heat inactivated IL-8 did not alter any of the parameters measured. Current results support the notion that the brain cytokine network plays a role in sleep regulation.

Environmental stress and encystment of Pneumocystis carinii.

The structural elements and molecular events associated with encystment of Pneumocystis carinii are poorly understood. The cyst form of P. carinii is present in the lungs of infected hosts in low numbers as compared to the trophozoite; as few as 1/100 of the total organism number in severely infected animals are cysts [3]. It is not known if the cyst form is infectious or the degree to which it participates in eliciting immune responses from the host. Studies using drugs, that specifically prevent formation or destabilization of the cyst wall result in lower numbers of both trophozoites and cysts, suggesting that the cyst is an integral life cycle stage of P. carinii [1,4]. The environmental or organism signals that trigger encystment of P. carinii are unknown. Cysts appear late in the infection [3], suggesting that damage done to the host pulmonary system may create an inhospitable environment or reduce nutritional resources for the trophozoite, thereby triggering formation of the more environmental resistant and quiescent form, the cyst. This study sought to determine if a specific source of stress in the trophozoites’ environment is associated with encystment. MATERIALS AND METHODS Female Sprague Dawley rats (colony 202) were obtained from Harlan (Indianapolis, Ind.) and housed in an AAALAC-approved facility. Rats were immunosuppressed with dexamethasone (Dex) (0.36 mg/liter) in their drinking water for seven days. Then P. carinii organisms were introduced into rat lungs by transtracheal inoculation as previously described [2]. Broncheoalveolar lavage (BAL) fluid collection was performed as described previously [3]. Briefly, the trachea was exposed by blunt dissection. The thoracic cavity was opened by incisions through the ribcage and removing the ribcage to allow full expansion of the lungs during lavage. A 14-gauge Angiocath (Becton Dickinson, Infusion Therapy Systems, Sandy, UT) with a 10-mL syringe was inserted into the trachea, and the lungs were lavaged with aliquots of sterile, pyrogen-free saline solution at 378C, pH 7.3. The saline was recovered from the lung by withdrawing the plunger of the syringe in a deliberate manner. In some cases, only 5 mL of lavage fluid was instilled and recovered. Some lavage samples for pH determination were obtained from animals that were incubated at 48C for 24 hr after cardiac exsanguination. In these cases, lavage was performed as described above, with special attention to slow instillation and removal of lavage fluid to account for the increased friability of the tissue. Severity of P. carinii infection was determined by scoring numbers of organisms on histochemically stained impression smears of lung tissue using the semi-logarithmic scale of Barlett et al. [2]. The smears were stained with Wright’s-Giemsa stain for both trophozoite and cysts and with Grocott methenamine-silver nitrate stain for cyst forms [2]. For culture experiments, human embryonic lung (HEL 299) cells of fibroblast morphology (CCL-137; ATCC, Manassas, VA) were grown as monolayers on 24-well culture dishes using minimum essential medium (MEM) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 10% fetal calf serum (FCS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Prior to inoculation of monolayers with P. carinii, the pH of the MEM was adjusted using 1 N HCl to 7.4, 7.3, 7.2, 7.1, 7.0, or 6.9. P. carinii samples were diluted to ten trophozoites per 10003 field to seed wells in the different pH media. Every other day, four individual wells were mixed using a Pasteur pipet, and 10-lL aliquots were

Staphylococcus aureus exosecretions and bovine mastitis.

The role of Staphylococcus aureus and coagulase-negative staphylococcal exosecretions in bovine udder infection was tested by monitoring the cows' response to in vivo inoculation of bacterial exosecretions into udder quarters. Twenty Israeli-Holstein dairy cows were included in the study; two or three of the udder quarters of each cow were intracisternally inoculated with 0.04-0.05 mg/quarter (total proteins) of the various sterile bacterial exosecretions in a sterile pyrogen-free saline. Each udder was inoculated with two or three different bacterial exosecretions or placebo (Columbia Broth). Cows were monitored for 96 h post-inoculation for rectal temperature, heart and respiratory rates, alimentary tract activity (rumen contraction), udder temperature, pain, oedema and udder size. Milk samples were examined bacteriologically and for somatic cell count, N-acetyl-D-glucosaminidase (NAGase) activity and somatic cell differentiation. No enterotoxins (beta-G) or toxic shock syndrome toxin-1 were detected in response to any of the bacteria tested. Control quarters or those inoculated with Columbia Broth, showed similar NAGase and somatic cell count values throughout the experiment. Twelve of the 18 strains tested, induced inflammation in the inoculated quarters while six did not. Of the 12 strains causing local inflammation, only six were found significantly different from the control and were considered as high response (group 1). The other six that caused a local inflammation did not differ significantly from the control, and were considered to be moderate response (group 2). The six S. aureus isolates that did not cause an inflammatory response were considered to have low response (group 3). In all quarters inoculated with S. aureus bacterial exosecretions belonging to groups 1 and 2, the polymorphonuclear cells and macrophages were proportionally increased while CD4+ and CD8+ T-lymphocyte populations decreased. One-dimensional NuPAGE (7%) Tris-acetate gel electrophoresisof the bacterial exosecretions revealed four different bands appearing between 36 and 31 kDa, marked from top to bottom as A, B, C and D. An association was found between the combinations of expressed bands and the cow responses: the majority of the cases could be linked to the expression of bands B and C.

Discrete electrolytic lesion of the preoptic area prevents LPS-induced behavioral fever in toads.

The preoptic area (POA) plays an important role in fever in mammals, but the role of this region in fever in ectothermic vertebrates has never been assessed. Toads, like all ectotherms, regulate their body temperature (T(b)) primarily by behavior and develop behavioral fever when injected with lipopolysaccharide (LPS). Therefore, we tested the hypothesis that the POA plays a role in the behavioral fever induced by LPS in the toad Bufo paracnemis. We made electrolytic lesions in the POA of toads (0.3 mA, 8 s) and measured preferred T(b) using a thermal gradient. After a period of 24h inside the gradient chamber, control, sham-operated and lesioned toads were systemically injected with LPS (200 micro g kg(-1)) or pyrogen-free saline. There was no significant effect of POA lesion in animals at their normal preferred T(b). LPS caused a significant increase in preferred T(b) of control and sham-operated toads, but lesions in the POA abolished this response. These results indicate that the POA is an important site in the central nervous system of toads, and perhaps of all vertebrates, involved in the development of fever.

Cellular distribution of anionic antimicrobial peptide in normal lung and during acute pulmonary inflammation.

Anionic peptides (APs) are small antimicrobial peptides present in human and ovine lung. In this study APs were also detected in bovine lung, and production of APs in lungs with acute inflammation induced by various stimuli was determined. The distribution and intensity of APs were determined by immunohistochemistry in lungs of 1) neonatal calves (1-3 days of age) inoculated with Mannheimia (Pasteurella) haemolytica, a known inducer of the bovine beta-defensin lingual antimicrobial peptide (LAP) or pyrogen-free saline (PFS), and 2) growing calves (3 months of age) similarly inoculated with M. haemolytica, a lipopolysaccharide (LPS) from M. haemolytica, an LPS-associated protein from M. haemolytica, or PFS. APs were also detected by western blots with the same antibody in lungs of the calves above, as well as in calves inoculated with Pseudomonas aeruginosa, and an adult cow. Anionic peptide (AP) immunoreactivity was detected in bands (approximate weights) in the western blots of lung at 28-30 (strongest signal), 31, 45, and 52-60 kd regardless of inoculum. The adult cow lacked bands at 45 kd, but it had additional bands at 64 (inconsistently) and 35-38 kd. All these band sizes are consistent with those of the western blots of human and ovine lung. The cellular distribution of APs in lung of neonatal and growing cattle was similar to that in lung of human and sheep. In lungs with acute inflammation induced by live bacteria, LPS, or protein, AP distribution and intensity were similar to those in control (PFS-inoculated) lungs and slightly decreased in bronchioles. This work demonstrates that AP is present in lung of cattle and is thereby conserved among two ruminant species and man. Distribution and intensity of AP production are not enhanced by infection or acute inflammation and are decreased in bronchioles, which suggests that AP is not induced like beta-defensins such as LAP, but, instead, is produced constitutively.

Thermal Response to Zymosan: The Differential Role of Complement

Objectives: This study was designed to determine whether the complement (C) system may be involved in the febrile response to zymosan (Zym), a glycan derived from yeast cell walls. Methods: Cobra venom factor (CVF) at 100 U/animal or its vehicle, pyrogen-free saline (PFS), was injected intravenously (i.v.) into guinea pigs to deplete serum C. Eighteen hours later, a low or high dose of Zym or its vehicle, PFS, was administered i.v. or intraperitoneally (i.p.) to these animals. The core temperature (T_c) was measured continuously by thermocouples. Serum C levels were determined by sheep erythrocyte hemolytic assay. Results: Zym at 1 mg/kg caused a 1°C T_c rise that was not significantly affected by CVF pretreatment. However, CVF-induced hypocomplementation converted the T_c fall (∼1.2°C) produced by 100 mg/kg of Zym i.p. into a 1°C T_c rise. Similarly, CVF pretreatment did not affect the T_c rise caused by 0.5 mg/kg of Zym i.v., but converted the T_c fall induced by 25 mg/kg i.v. into a 1°C T_c rise. A separate experiment showed that 25, but not 0.5 mg/kg of Zym i.v., decreased serum C by 34% in 15 min; C did not recover over the next 6 h. A second i.v. injection of 25 mg Zym/kg 210 min later, when the T_c had recovered but the serum C had not, yielded a smaller and briefer T_c fall. Conclusion: These results suggest that Zym is inherently pyrogenic, but this effect is manifested only when the dose of zymosan is too small to activate C or when C has been reduced by prior activation.

Protective Effect of a New C5a Receptor Antagonist against Ischemia–Reperfusion Injury in the Rat Small Intestine

Background. The complement system is a major contributor to the pathogenesis of intestinal ischemia–reperfusion (I/R) injury. We have studied the action of an orally active complement factor 5a (C5a) receptor antagonist, the cyclic peptide AcF-(OPdChaWR) [Ac-Phe(Orn-Pro-d-cyclohexylalanine-Trp-Arg)] against local and remote intestinal I/R injuries in rats.Materials and methods. Anesthetized rats were administered with AcF-(OPdChaWR) at doses of 1 mg/kg intravenously or 0.3, 1, or 10 mg/kg orally with pyrogen-free saline for sham control animals. The superior mesenteric artery was occluded for 30 min and the intestine reperfused for 120 min. Changes associated with tissue injury were assessed by neutropenia, intestinal edema, serum tumor necrosis factor-α, serum haptoglobin, plasma aspartate aminotransferase, and histopathology.Results. Pretreatment with either a single intravenous dose (1 mg/kg), or a single oral dose (10 mg/kg) of AcF-(OPdChaWR) significantly inhibited I/R induced neutropenia, the elevated serum levels of tumor necrosis factor-α, haptoglobin, and plasma aspartate aminotransferase, as well as intestinal edema. Histological analysis of AcF-(OPdChaWR)-treated I/R animals showed markedly reduced mucosal layer damage compared to that of untreated rats.Conclusions. These results indicate that a potent antagonist of C5a receptors on human cells protects the rat small intestine from I/R injury after oral or intravenous administration. Small molecule C5a antagonists may have some therapeutic utility in reperfusion injury.

Increased anionic peptide distribution and intensity during progression and resolution of bacterial pneumonia.

Anionic peptides (APs) are small anionic antimicrobial peptides composed of 7 aspartic acid residues and are produced in the lungs of humans, sheep, and cattle. Although expression by epithelial cells of some antimicrobial peptides (e.g., beta-defensins) of humans and ruminants is increased in response to acute infection, AP expression is not increased during acute infection, which suggests that the expression of the latter peptide is constitutive. In this study, the degree of AP expression during the progression (acute, subacute, and chronic) of bronchopneumonia was determined. Mannheimia (Pasteurella) haemolytica, a known inducer of bovinebeta-defensins, was inoculated intrabronchially with a fiber-optic bronchoscope in nine 3-month-old sheep, and tissues were collected at 1, 15, and 45 days postinoculation (p.i.); nine control animals received pyrogen-free saline by the same procedure and were killed at the same time points. In the acute group (1 day p.i.), the lungs had lesions typical of bronchopneumonia and the distribution and intensity of AP immunoreactivity (AP-IR) were similar to those of previous studies (minimal intensity and distribution of AP-IR in bronchiolar epithelial cells). In the subacute group (15 days p.i.), there was prominent hyperplasia of bronchiolar and alveolar epithelial cells, and the chronic group (45 days p.i.) had yet more pronounced hyperplasia. In the subacute and chronic groups, the intensity and distribution of AP-IR in the cytoplasm of hyperplastic bronchiolar and type II alveolar cells were significantly increased compared to those of saline-inoculated and contralateral (noninoculated) lung lobes. Although AP expression appears constitutive, the constitutive production of AP is higher in hyperplastic, less differentiated cells than in fully differentiated, mature cells of the respiratory airways. The increased intensity and distribution of AP-IR in immature (hyperplastic) epithelial cells may be a mechanism by which production of a noninducible antimicrobial is increased temporarily during lesion progression and repair. This increased production of AP by hyperplastic cells may protect the lung against further infection until new, fully differentiated epithelial cells are capable of expressing their own inducible array of antimicrobial peptides.

A Mutant Form of JAB/SOCS1 Augments the Cytokine-induced JAK/STAT Pathway by Accelerating Degradation of Wild-type JAB/CIS Family Proteins through the SOCS-box

Cytokines exert biological functions by activating Janus tyrosine kinases (JAKs), and JAK inhibitors JAB (also referred to as SOCS1 and SSI1) and CIS3 (SOCS3) play an essential role in the negative regulation of cytokine signaling. We have found that transgenic (Tg) mice expressing a mutant JAB (F59D-JAB) exhibited a more potent STAT3 activation and a more severe colitis than did wild-type littermates after treatment with dextran sulfate sodium. We now find that there is a prolonged activation of JAKs and STATs in response to a number of cytokines in T cells from Tg mice with lck promoter-driven F59D-JAB. Overexpression of F59D-JAB also sustained activation of JAK2 in Ba/F3 cells. These data suggested that F59D-JAB up-regulated STAT activity by sustaining JAK activation. To elucidate molecular mechanisms related to F59D-JAB, we analyzed the effects of F59D-JAB on the JAK/STAT pathway using the 293 cell transient expression system. We found that the C-terminal SOCS-box played an essential role in augmenting cytokine signaling by F59D-JAB. The SOCS-box interacted with the Elongin BC complex, and this interaction stabilized JAB. F59D-JAB induced destabilization of wild-type JAB, whereas overexpression of Elongin BC canceled this effect. Levels of endogenous JAB and CIS3 in T cells from F59D-JAB Tg-mouse were lower than in wild-type mice. We propose that F59D-JAB destabilizes wild-type, endogenous JAB and CIS3 by chelating the Elongin BC complex, thereby sustaining JAK activation.

Glial cell line-derived neurotrophic factor promotes sleep in rats and rabbits.

Various growth factors (e.g., growth hormone-releasing hormone, acidic fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, and interleukin-1) are implicated in sleep regulation. It is hypothesized that neuronal activity enhances the production of such growth factors, and they in turn form part of the sleep regulatory mechanism. Glial cell line-derived neurotrophic factor (GDNF) promotes development, differentiation, maintenance, and regeneration of neurons, and its production is induced by well-characterized sleep regulatory substances such as interleukin-1 and tumor necrosis factor. Therefore, we investigated whether GDNF would promote sleep. Twenty-six male Sprague-Dawley rats and 30 male New Zealand White rabbits were surgically implanted with electroencephalogram (EEG) and electromyogram (EMG; rats only) electrodes, a brain thermistor, and a lateral intracerebroventricular cannula. The animals were injected intracerebroventricularly with pyrogen-free saline and on a separate day with one of the following doses of GDNF: 5, 50, and 500 ng in rabbits and 50 and 500 ng in rats. The EEG, brain temperature, EMG (in rats), and motor activity (in rabbits) were recorded for 23 h after the intracerebroventricular injection. GDNF (500-ng dose) increased the time spent in nonrapid eye movement sleep in both rats and rabbits. Rapid eye movement sleep was not affected by the lower doses of GDNF but was inhibited in rabbits after the high dose. EEG slow-wave activity was not affected by GDNF. The current results provide further evidence that various growth factors are involved in sleep regulation.

Leptin-Induced Nuclear Translocation of STAT3 Immunoreactivity in Hypothalamic Nuclei Involved in Body Weight Regulation

Leptin is involved in the hypothalamic control of food intake and body weight. Fos immunohistochemistry has been used to functionally map leptin target neurons involved in these regulatory processes. However, only a subset of hypothalamic neurons expressing the long form of the leptin receptor (Ob-Rb) also coexpress the neuronal activation marker Fos after leptin stimulation. To functionally map all leptin target neurons, regardless of whether leptin-mediated neuronal activation or inhibition occurs, we immunohistochemically investigated the leptin-induced nuclear translocation of the signal transducer and activator of transcription molecule STAT3, which represents a crucial step in the regulation of leptin-dependent gene expression. As proven by colocalization studies with the nuclear 4',6-diamidino-2-phenylindole dilactate stain, intracerebroventricular leptin treatment, but not intracerebroventricular application of pyrogen-free saline, induced a time-dependent nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei, with strong nuclear STAT3 signals detectable in the arcuate nucleus, the lateral hypothalamus, and the ventromedial and dorsomedial hypothalamic nuclei. This leptin-induced STAT3 translocation pattern proved to be distinct from that induced by interleukin-6, another cytokine using STAT3 in its signaling pathway. Combined immunohistochemical STAT3 and Fos detection after leptin treatment revealed a higher number of STAT3-positive than Fos-positive cell nuclei in the aforementioned hypothalamic structures and showed that Fos immunoreactivity colocalized only in a subset of all leptin-responsive STAT3 nuclei. These results suggest that the detection of nuclear STAT3 immunoreactivity represents a new neuroanatomical tool to functionally map central leptin actions. They further support the importance of ventrally located caudal hypothalamic structures representing the main leptin targets involved in body weight regulation.

Estradiol inhibits astrocytic GFAP expression in an animal model of neuroinflammation

There are many animal models for studying different aspects of neurodegeneration. Lipopolysaccharide (LPS) injected in rats intracerebroventricularly induces neuroinflammation quite similar to the inflammatory component of chronic neurodegenerative conditions such as Alzheimer's disease. We used this model to examine the effect of estradiol on neuroinflammation. LPS or pyrogen-free saline were injected intracerebroventricularly (i.c.v.) into the lateral ventricle of male Wistar rats and estradiol was administered (200 micrograms/kg s.c.) 48 h before or 24 h after LPS injection. LPS-induced body weight loss was partially postponed by the treatment, especially in the rats pretreated with estradiol. When analyzing GFAP glial cell morphology in the CA3c area of the hippocampus and corpus callosum, as well as the number of astroglial cells in CA3c and CA1, GFAP expression was found to be reduced. This was true especially in the animals pretreated with estradiol and to a much lesser extent in the posttreated ones. The data support the possible existence of a neuroimmunomodulatory effect of estradiol administration in neurodegenerative conditions, which influences the inflammatory component.

Ethanol gastric lesion aggravated by lung injury in rat. Therapy effect of antiulcer agents

Hemorrhagic mucosal lesions in the stomach in the rat induced by an intragastrical application of 1 ml of 50 or 75% ethanol were aggravated by preceding lung damage provoked by an intratracheal instillation of pyrogen-free saline or HCl (pH 1.75) or 50-h exposure to 100% oxygen. Due to the particular preceding aggravating circumstances, these lesions were taken to be of a special kind, rather than ordinary. So far, it is not known whether and how antiulcer agents may influence these lesions. Rats received an intratracheal (i.t.) HCl instillation [1.5 ml/kg HCl (pH 1.75)] (lung-lesion), and an intragastric instillation of 96% ethanol (gastric lesion; 1 ml/rat, 24 h after i.t. HCl instillation), and were sacrificed 1 h after ethanol. Basically, in lung injured rats, the subsequent ethanol-gastric lesion was markedly aggravated. This aggravation, however, in turn, did not affect the severity of the lung lesions in the further period, at least for a 1-h observation. Taking intratracheal HCl-instillation as time 0, a gastric pentadecapeptide, GEPPPGKPADDAGLV, M.W.1419, coded BPC 157 (PL-10, PLD-116; 10 μg, 10 ng, 10 pg), ranitidine (10 mg), atropine (10 mg), omeprazole (10 mg), were given [/kg, intraperitoneally (i.p.)] (1) once, only prophylactically [as a pre-treatment (at −1 h), or as a co-treatment (at 0)], or only therapeutically (at +18 h or +24 h); (2) repeatedly, combining prophylactic/therapeutic regimens [(−1 h)+(+24 h) or (0)+(+24 h)], or therapeutic/therapeutic regimens [(+18 h)+(+24 h)]. In general, the antiulcer agents did protect against ethanol gastric lesions regardless of the presence of the severe lung injury, in all of the used regimens. Of note, combining their prophylactic and salutary regimens (at −1 h/+24 h, or at 0/+24 h) may increase the antiulcer potential, and the effect that had been not seen already with single application, became prominent after repeated treatment.

Subdiaphragmatic vagotomy does not block intraperitoneal lipopolysaccharide-induced fever

Several recent findings, including the inability of subdiaphragmatic vagotomy to block lipopolysaccharide (LPS)-induced interleukin-1β (IL-1β) protein in brain, have made it necessary to reexamine the role of the subdiaphragmatic vagal afferents in immune-to-brain communication. In this study, we examined the effects of intraperitoneal (i.p.) injections of LPS on core body temperature in control and subdiaphragmatically vagotomized rats. Vagotomized and sham-operated male Sprague–Dawley rats were injected i.p. with vehicle (pyrogen-free saline) on the control day and LPS (1, 10 or 50 μg/kg) on the experimental day, and core body temperature was monitored by telemetry for 6 h after the injection. At this time, rats were sacrificed, and serum, liver, and pituitary samples were collected. The i.p. injection of LPS increased core body temperature in both sham-operated and vagotomized rats compared to the saline injection. In addition, LPS significantly increased IL-1β levels in serum, liver, and pituitary compared to saline-injected controls. There were no significant differences in the magnitude of the fever or in the levels of IL-1β in serum, liver, or pituitary between sham-operated and vagotomized rats. Thus, the current data indicate that, at the doses tested, subdiaphragmatic vagal afferents are not crucial for i.p. LPS-induced fever. Because several effects of vagotomy have been shown to be dependent on dose, we are currently investigating whether vagal afferents are involved in lower-dose i.p. LPS-induced fever.