Injection Of Bacterial Lipopolysaccharide Research Articles

In Drosophila Hemolymph, Serine Proteases Are the Major Gelatinases and Caseinases.

After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges.

Treatment of intrauterine adhesions in rats with hypoxia-cultured BMSC-derived exosomes

Objective: To perform intrauterine adhesion modeling, and to investigate the repair effect of hypoxic treated bone marrow mesenchymal stem cells (BMSC) and their derived exosomes (BMSC-exo) on endometrial injury. Methods: BMSC and their exosomes BMSC-exo extracted from rats' femur were cultured under conventional oxygen condition (21%O2) or hypoxia condition (1%O2). Intrauterine adhesion modeling was performed on 40 healthy female SD rats by intrauterine injection of bacterial lipopolysaccharide after curettage. On the 28th day of modeling, 40 rat models were randomly divided into five groups, and interventions were performed: (1) NC group: 0.2 ml phosphate buffered solution was injected into each uterine cavity; (2) BMSC group: 0.2 ml BMSC (1×106/ml) with conventional oxygen culture was injected intrauterine; (3) L-BMSC group: 0.2 ml of hypoxic cultured BMSC (1×106/ml) was injected intrauterine; (4) BMSC-exo group: 0.2 ml of BMSC-exo cultured with conventional oxygen at a concentration of 500 μg/ml was injected into the uterine cavity; (5) L-BMSC-exo group: 0.2 ml hypoxic cultured BMSC-exo (500 μg/ml) was injected intrauterine. On the 14th and 28th day of treatment, four rats in each group were sacrificed by cervical dislocation after anesthesia, and endometrial tissues were collected. Then HE and Masson staining were used to observe and calculate the number of glands and fibrosis area in the endometrium. The expressions of angiogenesis related cytokines [vascular endothelial growth factor A (VEGFA) and CD31], and fibrosis-related proteins [collagen-Ⅰ, collagen-Ⅲ, smooth muscle actin α (α-SMA), and transforming growth factor β1 (TGF-β1)] in endometrial tissues were detected by western blot. Results: (1) HE and Masson staining showed that the number of endometrial glands in L-BMSC group, BMSC-exo group and L-BMSC-exo group increased and the fibrosis area decreased compared with NC group on the 14th and 28th day of treatment (all P<0.05). Noteworthily, the changes of L-BMSC-exo group were more significant than those of BMSC-exo group (all P<0.05), and the changes of BMSC-exo group were greater than those of BMSC group (all P<0.05). (2) Western blot analysis showed that, compared with NC group, the expressions of collagen-Ⅲ and TGF-β1 in BMSC group, L-BMSC group, BMSC-exo group and L-BMSC-exo group decreased on the 14th and 28th day of treatment (all P<0.05). As the treatment time went on, the expressions of fibrosis-related proteins were different. Compared with BMSC group, the expressions of collagen-Ⅲ, α-SMA and TGF-β1 in the BMSC-exo group and L-BMSC group decreased on the 28th day (all P<0.05). Moreover, the expressions of collagen-Ⅲ and TGF-β1 in L-BMSC-exo group were lower than those in BMSC-exo group on the 28th day (all P<0.05). And the expressions of collagen-Ⅰ, α-SMA and TGF-β1 in L-BMSC-exo group were lower than those in L-BMSC group on the 28th day (all P<0.05). (3) The results of western blot analysis of VEGFA and CD31 showed that, the expressions of VEGFA and CD31 in BMSC group, L-BMSC group, BMSC-exo group and L-BMSC-exo group increased on the 14th and 28th day of treatment compared with NC group (all P<0.05). Treatment for 28 days, the expressions of VEGFA and CD31 in BMSC-exo group and CD31 in L-BMSC group were higher than those in BMSC group (all P<0.05). Moreover, the expressions of VEGFA and CD31 in L-BMSC-exo group were higher than those in BMSC-exo group and L-BMSC group on the 28th day (all P<0.05). Conclusions: Treatment of BMSC and their exosomes BMSC-exo with hypoxia could promote endometrial gland hyperplasia, inhibit tissue fibrosis, and further repair the damaged endometrium in rats with intrauterine adhesion. Importantly, hypoxic treatment of BMSC-exo is the most effective in intrauterine adhesion rats.

Deficiency of hydrogen sulfide production and pregnancy rate in an experimental model: Association with preterm delivery.

Pro-inflammatory phenomena drive preterm delivery (PTD). Hydrogen sulfide is a gasotransmitter with anti-inflammatory properties produced through the activity of the enzyme cystathionine-γ-lyase (CSE), and its impact was studied in models of normal delivery and PTD in mice. Female CSE+/+ and CSE-/- mice were mated with male CSE+/+ mice; mating was done with drinking water unsupplemented and supplemented with cysteine. The pregnancy rate was monitored. PTD was induced by the intraperitoneal injection of bacterial lipopolysaccharide (LPS) on day 14.5 of pregnancy. Mice were sacrificed for tissue collection and splenocyte isolation after 6 and 12h. Isolated splenocytes were stimulated for the production of tumor necrosis factor-alpha (TNFα), interleukin (IL)-10 and interferon-gamma (IFNγ); TNFα and vascular endothelial growth factor (VEGF) were measured in the fetuses and the placenta. The successful pregnancy rate was lower in CSE-/- mice and it was restored with cysteine supplementation. CSE deficiency was associated with higher tissue concentrations of TNFα in the fetuses, attenuated IL-10 responses and higher IFNγ production from splenocytes. CSE deficiency was not associated with PTD. Following PTD induction, CSE-/- mice did not show attenuated IL-10 responses but the production of TNFα and IFNγ was lowered over-time; placental VEGF was also increased over-time. CSE deficiency has an unfavorable impact on pregnancy. H2 S deficiency through CSE does not drive PTD but mediates pro-inflammatory phenomena in fetuses.

Autoregulation of blood flow drives early hypotension in a rat model of systemic inflammation induced by bacterial lipopolysaccharide.

Uncontrolled vasodilation is known to account for hypotension in the advanced stages of sepsis and other systemic inflammatory conditions, but the mechanisms of hypotension in earlier stages of such conditions are not clear. By monitoring hemodynamics with the highest temporal resolution in unanesthetized rats, in combination with ex-vivo assessment of vascular function, we found that early development of hypotension following injection of bacterial lipopolysaccharide is brought about by a fall in vascular resistance when arterioles are still fully responsive to vasoactive agents. This approach further uncovered that the early development of hypotension stabilized blood flow. We thus hypothesized that prioritization of the local mechanisms of blood flow regulation (tissue autoregulation) over the brain-driven mechanisms of pressure regulation (baroreflex) underscored the early development of hypotension in this model. Consistent with this hypothesis, an assessment of squared coherence and partial-directed coherence revealed that, at the onset of hypotension, the flow-pressure relationship was strengthened at frequencies (<0.2 Hz) known to be associated with autoregulation. The autoregulatory escape to phenylephrine-induced vasoconstriction, another proxy of autoregulation, was also strengthened in this phase. The competitive demand that drives prioritization of flow over pressure regulation could be edema-associated hypovolemia, as this became detectable at the onset of hypotension. Accordingly, blood transfusion aimed at preventing hypovolemia brought the autoregulation proxies back to normal and prevented the fall in vascular resistance. This novel hypothesis opens a new avenue of investigation into the mechanisms that can drive hypotension in systemic inflammation.

Comparison of effects of quercetin and ascorbic acid on inflammatory cytokines and antioxidant biomarkers in infant rats using an experimental sepsis model.

There is ongoing research on treatments that promote antioxidant and anti-inflammatory mechanisms, which will reduce mortality in sepsis. In this study, we compared the anti-inflammatory and antioxidant activities of quercetin and ascorbic acid using a sepsis model induced in infant rats. A total of 28 infant rats 21-days-old that had just completed the lactation period were divided into four groups: control, sepsis, sepsis + quercetin, and sepsis + ascorbic acid. The sepsis model was created with an intraperitoneal injection of bacterial lipopolysaccharide. After 24 hours, blood samples were collected for analysis of serum levels of inflammatory cytokines (IL-1β, IL-6, TNF-α, and CRP) and antioxidants (CAT, GPx, SOD, and GST). The superoxide dismutase levels were significantly higher in the sepsis + ascorbic acid group compared to the sepsis and sepsis + quercetin groups. The levels of the most active cytokines in sepsis were significantly lower in the serum samples of the septic subjects who received quercetin and ascorbic acid. The antioxidant activity, which is impaired in sepsis, was increased by both molecules. We observed that these two molecules, which are free of side effects, have a positive influence on the progression of sepsis to severe and fatal sepsis in childhood (Tab. 2, Ref. 38).

Reduction in vasopressin cells in the suprachiasmatic nucleus in mice increases anxiety and alters fluid intake

Central vasopressin (AVP) has been implicated in the control of multiple behaviors, including social behavior, anxiety-like behavior, and sickness behavior. The extent to which the different AVP-producing cell groups contribute to regulating these behaviors has not been extensively investigated. Here we test the role of AVP cells in the suprachiasmatic nucleus (SCN) in these behaviors by ablating these cells using viral-mediated, Cre-dependent caspase in male and female AVP-Cre + mice and Cre-controls. We compared anxiety and social behaviors, as well as sickness behaviors (lethargy, anhedonia (indexed by sucrose consumption), and changes in anxiety-like- and social behavior) induced via injection of bacterial lipopolysaccharide (LPS). We found that SCN AVP cell ablation increased anxiety-like behavior and sucrose consumption in both sexes, as well as increased urine marking by males in a non-social context, but did not alter behavioral responses to sickness. Our data suggest that SCN AVP does not strongly affect LPS-induced behavioral changes, but may contribute to anxiety-like behavior, and may play a role in ingestive reward/motivation and fluid intake.

Removal of vasopressin cells from the paraventricular nucleus of the hypothalamus enhances lipopolysaccharide-induced sickness behaviour in mice.

Vasopressin (AVP) cells in the paraventricular nucleus of the hypothalamus (PVN) are activated during sickness and project to multiple nuclei responsible for the anxiety, social and motivated behaviours affected during sickness, suggesting that these cells may play a role in sickness behaviours, typically expressed as reduced mobility, increased anxiety, anhedonia and social withdrawal. In the present study, we selectively ablated AVP neurones in the PVN of male and female mice (Mus musculus) and induced sickness behaviour via injection of bacterial lipopolysaccharide (LPS). We found that PVN AVP ablation increased the effects of LPS, specifically by further decreasing sucrose preference in males and females and decreasing the social preference of males, monitored within 24 hours of LPS injection. These results suggest that PVN AVP contributes to the change in motivated behaviours during sickness and may help promote recovery from infection..

Expression of osteoclastogenic and anti-osteoclastogenic cytokines differs in mouse gingiva injected with lipopolysaccharide, peptidoglycan, or both

ObjectiveBacterial substances in subgingival biofilm evoke alveolar bone resorption. We previously reported that gingival injection of bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) induced alveolar bone resorption in mice. However, the mechanism by which LPS and PGN induce osteoclast formation has not been investigated. The aim of this study is to clarify the role of osteoclastogenic and anti-osteoclastogenic cytokines in the alveolar bone resorption induced by LPS and PGN. MaterialsLPS from Escherichia coli, PGN from Staphylococcus aureus, or both were injected into the gingiva of mice every 48 h for a total of 13 times. Alveolar bone resorption was assessed histochemically by tartrate-resistant acid phosphatase staining. Expression of the receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor (TNF)-α, interleukin (IL)-17, and IL-10 were analyzed by immunostaining. To analyze the role of these cytokines, RANKL-pretreated mouse bone marrow macrophages were stimulated with LPS, PGN, or LPS + PGN with or without anti-TNF-α antibody, IL-17, or IL-10. ResultsAlveolar bone resorption was induced by both LPS and PGN and exacerbated by LPS + PGN. LPS induced higher RANKL expression than PGN. Expression of TNF-α and IL-10 was correlated with bone resorption. PGN injections induced the strongest expression of IL-17, followed by LPS + PGN and LPS. In an in vitro osteoclastogenesis assay, anti-TNF-α antibody and IL-10 inhibited osteoclast formation, but IL-17 promoted it. ConclusionLPS, PGN, or LPS + PGN injections induce distinctive expression of TNF-α, IL-10, and IL-17, suggesting that the composition of these bacterial ligands in dental plaque is critical for alveolar bone resorption.

Innate immune system function following systemic RNA-interference of the Fragile X Mental Retardation 1 gene in the cricket Acheta domesticus

Fragile X syndrome (FXS), caused by a mutation in the Fragile X Mental Retardation 1 (FMR1) gene, is a common form of inherited mental retardation. Mutation of the gene leads to a loss of the gene product Fragile X Mental Retardation Protein (FMRP). While a loss of FMRP has been primarily associated with neural and cognitive deficits, it has also been reported to lead to immune system dysfunction in both humans and flies. We used the Acheta domesticus transcriptome to identify a highly conserved cricket ortholog of FMR1 (adfmr1). We cloned a partial cDNA of adfmr1, used systemic RNA interference (RNAi) to knockdown adfmr1 expression, and examined the impact of this knockdown (KD) on the cellular and humoral responses of the insect innate immune system. Following RNAi, both male and female crickets exhibited an increase in the number of circulating hemocytes, a decrease in total hemolymph phenoloxidase (PO) activity, and an increase in fat body lysozyme expression. Despite similar changes in these immune parameters in both sexes, male and female crickets responded differently to an immune challenge. Most KD males failed to survive an intra-abdominal injection of bacterial lipopolysaccharide, while KD females were just as likely as control females to survive this challenge. Our results support that decreased fmr1 expression can alter the cellular and humoral defenses of the insect innate immune system, and may lead to a decrease in male, but not female, immunocompetence.

Deficit of Long-Term Potentiation Induction, but Not Maintenance, in the Juvenile Hippocampus after Neonatal Proinflammatory Stress

CA3-CA1 long-term potentiation (LTP) in the hippocampal slices from juvenile Wistar rats was studied to reveal factors potentially contributing to different sensitivity to neonatal proinflammatory stress (NPS). NPS was induced by intra­peritoneal injections of bacterial lipopolysaccharide (LPS) to neonatal rats (two injections of LPS, or saline in the control group, consecutively on postnatal days 3 and 5 [PND3 and PND5]). In females, a significant effect of NPS on hippocampus development was associated with modifications of long-term synaptic plasticity, the synapses becoming more resistant to LTP induction. LTP deficit in the slices of the NPS group was not associated with a decrease in LTP maintenance, since late LTP generally corresponded to early LTP magnitude, similar in all groups. Moreover, partial correlation revealed significantly higher residual LTP 1 h after high-frequency stimulation in the NPS groups compared to the corresponding value of early LTP in the control groups, suggesting improved consolidation. Both effects were evident in NPS females. A number of males responded to NPS similarly to females, while others were relatively resistant to NPS exposure, a significant increase in variability of LTP magnitude being revealed in NPS males compared to respective females and the control groups. We suggest that postnatal development of long-term plasticity after NPS is similar in animals of both sexes; however, additional specific factor(s) may promote a relative resistance of the male brain.

Antimicrobial Peptides from Rat-Tailed Maggots of the Drone Fly Eristalis tenax Show Potent Activity against Multidrug-Resistant Gram-Negative Bacteria.

The spread of multidrug-resistant Gram-negative bacteria is an increasing threat to human health, because novel compound classes for the development of antibiotics have not been discovered for decades. Antimicrobial peptides (AMPs) may provide a much-needed breakthrough because these immunity-related defense molecules protect many eukaryotes against Gram-negative pathogens. Recent concepts in evolutionary immunology predict the presence of potent AMPs in insects that have adapted to survive in habitats with extreme microbial contamination. For example, the saprophagous and coprophagous maggots of the drone fly Eristalis tenax (Diptera) can flourish in polluted aquatic habitats, such as sewage tanks and farmyard liquid manure storage pits. We used next-generation sequencing to screen the E. tenax immunity-related transcriptome for AMPs that are synthesized in response to the injection of bacterial lipopolysaccharide. We identified 22 AMPs and selected nine for larger-scale synthesis to test their activity against a broad spectrum of pathogens, including multidrug-resistant Gram-negative bacteria. Two cecropin-like peptides (EtCec1-a and EtCec2-a) and a diptericin-like peptide (EtDip) displayed strong activity against the pathogens, even under simulated physiological conditions, and also achieved a good therapeutic window. Therefore, these AMPs could be used as leads for the development of novel antibiotics.

Association of modulation of pro-inflammatory responses by dectin-2 with preterm delivery: An experimental model.

Pro-inflammatory responses of pathogen recognition receptors (PRR) are implicated in preterm delivery (PTD). Dectin-2 is one PRR recognizing unselective carbohydrate structures; its participation in PTD has never been studied before. In an experimental model, PTD was induced in female pregnant wild-type (WT) mice and mice with homologous deficiency for dectin-2 by the intraperitoneal injection of bacterial lipopolysaccharide (LPS) on day 14 of pregnancy. Time to delivery and fetal mortality were recorded. Challenged mice were killed for tissue collection and splenocyte isolation 6hours later. Concentrations of tumour necrosis factor-alpha (TNFα), interleukin (IL)-1α, and IL-1β were measured. Delivery was induced significantly earlier in WT than dectin-2-/- mice; however, fetal mortality was higher among dectin-2-/- mice. Candida albicans challenge could not lead to these changes. Sacrifice experiments showed that LPS challenge led to significant increase of TNFα, IL-1α, and IL-1β in maternal tissues of WT; this was further enhanced for TNFα and IL-1β in dectin-2-/- mice. Pre-treatment with the prostaglandin inhibitor diclofenac delayed time to delivery of WT mice, but not of dectin2-/- mice. TNFα stimulation of splenocytes of dectin2-/- mice was enhanced with the addition of anti-TLR4 and decreased in the presence of lipid A. Dectin-2 delays LPS-induced PTD by enhancing the production of pro-inflammatory cytokines.

Neurons and astrocytes of the chicken hypothalamus directly respond to lipopolysaccharide and chicken interleukin-6

In 4–5-month-old chicken, intravenous injections of bacterial lipopolysaccharide (LPS) induced a dose-dependent fever response and a pronounced increase of circulating interleukin-6 (IL-6). To assess a possible role for IL-6 in the brain of birds, a hypothalamic neuro-glial primary culture from 1-day-old chicken was established. Each well of cultured hypothalamic cells contained some 615 neurons, 1350 astrocytes, and 580 microglial cells on average. Incubation of chicken hypothalamic primary cultures with 10 or 100 µg/ml LPS induced a dose-dependent release of bioactive IL-6 into the supernatant. Populations of hypothalamic neurons (4%) and astrocytes (12%) directly responded to superfusion with buffer containing 10 µg/ml LPS with a transient increase of intracellular calcium, a sign of direct cellular activation. Stimulation of hypothalamic cultures with buffer containing 50 ng/ml chicken IL-6 induced calcium signaling in 11% of neurons and 22% of astrocytes investigated. These results demonstrate that IL-6 is produced in the periphery and in the hypothalamus in response to LPS in chicken. The observed cellular responses of hypothalamic cells to chicken IL-6 indicate that this cytokine may readily be involved in the manifestation of fever in the avian hypothalamus.

TLR4/NF-kappaB p65 signaling pathway mediates protective effect of triptolide on endothelium in rats with endotoxemia

The aim of this paper was to observe the effect of triptolide( TP) on cardiovascular function and its possible mechanism by intraperitoneal injection of bacterial lipopolysaccharide in rats with endotoxemia. Sixty male Sprague-Dawley rats were randomly divided intonormal group( NC group),endotoxemia model group( LPS group),TP low concentration intervention group( LPS + TP-L group,25 μg·kg~(-1)),TP middle concentration intervention group( LPS+TP-M group,50 μg·kg~(-1)),TP high concentration intervention group( LPS+TP-H group,100 μg·kg~(-1)) and polymyxin B group( LPS+PMX-B group,0. 2 mg·kg~(-1)). 10 mg·kg~(-1) LPS was injected intraperitoneally for 6 h to replicate the endotoxemia rat model. The rats in TP intervention groups were pre-treated 15 min before intraperitoneal injection of LPS. Rats in each group underwent total arterial intubation to measure hemodynamic parameters: heart rate( HR),left ventricular diastolic pressure( LVDP),the maximum rate of the increase/decrease of left ventricular pressure( ±dp/dtmax). The levels of BNP,CK-MB and c Tn-Ⅰ in serum and levels of TNF-α and IL-6 in plasma were detected by ELISA. The contents of p65 protein in myocardium and contents of p65,TLR4,i NOS and e NOS protein in thoracic aorta were detected by Western blot. As compared with NC group,the hemodynamic indexes in LPS group were significantly decreased; the contents of BNP,CK-MB and c Tn-Ⅰ in serum,TNF-α and IL-6 in plasma,p65 in myocardium,i NOS,e NOS,TLR4 and p65 in vascular tissues were significantly increased. As compared with LPS group,the hemodynamic indexes were significantly improved in LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group; the contents of BNP,CK-MB and c Tn-Ⅰ in serum,TNF-α and IL-6 in plasma,p65 in myocardium,i NOS,e NOS,TLR4 and p65 in vascular tissues were significantly decreased in each treatment group. Triptolide has a protective effect on cardiovascular damage in a dose-dependent manner in endotoxemia rats,probably through TLR4/NF-κB p65 signaling pathway to improve endothelial function.

Evaluation of Neutrophil Gelatinase-Associated Lipocalin as a Predictor of Glomerular Filtration Rate and Amikacin Clearance During Early Rat Endotoxemia: Comparison with Traditional Endogenous and Exogenous Biomarkers.

Renal elimination of amikacin and other aminoglycosides is slowed down in sepsis-induced acute kidney injury increasing the risk of adverse effects. Since neutrophil gelatinase-associated lipocalin (NGAL) and aminoglycosides share the mechanisms for renal excretion, the predictive power of NGAL was examined towards the changes in amikacin pharmacokinetics during early endotoxemia in anesthetized Wistar rats. Endogenous biomarkers of inflammation and acute kidney injury were assessed including NGAL in saline-injected controls and two groups of rats challenged with an intravenous injection of bacterial lipopolysaccharide (5mg/kg)-a fluid-resuscitated group (LPS) and a fluid-resuscitated group infused intravenously with 8μg/kg/h terlipressin (LPS-T). Sinistrin and amikacin were infused to measure glomerular filtration rate (GFR) and amikacin clearance (CLam). The investigations included blood gas analysis, chemistry and hematology tests and assessment of urine output, creatinine clearance (CLcr) and sinistrin clearance (CLsini). Within 3h of injection, systemic and renal inflammatory responses were induced by lipopolysaccharide. Gene and protein expression of NGAL was increased in the kidneys and the concentrations of NGAL in the plasma (pNGAL) and urine rose 4- to 38-fold (P < 0.01). The decreases in CLam and the GFR markers (CLcr, CLsini) were proportional, reflecting the extent to which endotoxemia impaired the major elimination mechanism for the drug. Terlipressin attenuated lipopolysaccharide-induced renal dysfunction (urine output, CLcr, CLsini) and accelerated CLam. The pNGAL showed a strong association with the CLsini (rs = - 0.77, P < 0.0005). Concerning prediction of CLam, pNGAL was comparable to CLcr (mean error - 24%) and inferior to CLsini (mean error - 6.4%), while the measurement of NGAL in urine gave unsatisfactory results. During early endotoxemia in the rat, pNGAL has a moderate predictive ability towards CLam. Clinical studies should verify whether pNGAL can support individualized dosing of aminoglycosides to septic patients.

Anti-Inflammatory Activity of Combined Aerodisperse System in the Conditions of Experimental Cervical Lymphadenite

: Objective: The aim of this study was to assess the presence of an antiinflammatory activity of a new combined aerodisperse system under conditions experimental lymphadenite. Materials and methods: Lymphadenite was reproduced by injection of bacterial lipopolysaccharide into the lymph node. The test spray and comparison spray (Tantum Verde®) were introduced by the irrigation of the posterior pharyngeal wall for 3 days after the induction of inflammation in doses: 0.5 mg / kg; 1 mg / kg; 1.5 mg / kg, 2 mg / kg. After this time estimate the concentration of CRP, TNF-α, leukocytes in the blood and the severity of the inflammatory edema of the lymph node. Results: test spray (all doses) provide reduce the concentration of CRP, TNF-α and leukocytes in the blood, and in doses of 0.5 mg / kg and 1 mg / kg – inflammatory edema of the lymph nodes by 12.9% (p <0.05) and 9.3% (p<0.05), respectively. In this case, the anti-inflammatory effect of the test spray in doses of 1 mg / kg is comparable to that of benzidamine. Conclusion: The test spray has an anti-inflammatory effect comparable to the pharmacological effect with the benzidamine, and may be included in further development, with the goal of creating a drug for the treatment of URT diseases. Since the increase in the dose of the test spray does not increase the pharmacological activity, the optimal dose of the test spray can be considered 1 mg, which is equivalent to 2 sprayings.Key words: Inflammation, Respiratory tract, Eucalyptus, Sage.

MicroRNAs downregulated following immune activation of rat testis

Little is known about how infection and the response to inflammation affect the microRNA (miRNA) profile of the male reproductive tract. We hypothesized that expression of inflammatory-related miRNAs would be altered following immune activation of rat testis. Testis total RNA was purified from Sprague-Dawley rats 3 or 6 hours after receiving a 5mg/kg intraperitoneal injection of bacterial lipopolysaccharide (LPS) and examined by qPCR using an 84-panel miRNA array. Five inflammatory-related miRNAs showed a greater than twofold downregulation (P<.05) in the 3-hour group (rno-let-7f-5p, rno-miR-200c-3p, rno-miR-23a-3p, rno-miR-23b-3p, rno-miR-98-5p) and five from the 6-hour group (rno-miR-17-5p, rno-miR-19a-3p, rno-miR-34a-5p, rno-miR-34c-5p, rno-miR-449a-5p). Review of the literature has revealed that these miRNAs also play important roles in the maintenance of fertility, formation and elimination of cancer, and development of the male reproductive tract. Further study will lead to a greater understanding of male reproductive immunology and related health issues.

Electric stimulation of the vagus nerve reduced mouse neuroinflammation induced by lipopolysaccharide.

BackgroundNeuroinflammation (NI) is a key feature in the pathogenesis and progression of infectious and non-infectious neuropathologies, and its amelioration usually improves the patient outcome. Peripheral inflammation may promote NI through microglia and astrocytes activation, an increased expression of inflammatory mediators and vascular permeability that may lead to neurodegeneration. Several anti-inflammatory strategies have been proposed to control peripheral inflammation. Among them, electrical stimulation of the vagus nerve (VNS) recently emerged as an alternative to effectively attenuate peripheral inflammation in a variety of pathological conditions with few side effects.Considering that NI underlies several neurologic pathologies we explored herein the possibility that electrically VNS can also exert anti-inflammatory effects in the brain.MethodsNI was experimentally induced by intraperitoneal injection of bacterial lipopolysaccharide (LPS) in C57BL/6 male mice; VNS with constant voltage (5 Hz, 0.75 mA, 2 ms) was applied for 30 s, 48 or 72 h after lipopolysaccharide injection. Twenty four hours later, pro-inflammatory cytokines (IL-1β, IL-6, TNFα) levels were measured by ELISA in brain and spleen extracts and total brain cells were isolated and microglia and macrophage proliferation and activation was assessed by flow cytometry. The level of ionized calcium binding adaptor molecule (Iba-1) and glial fibrillary acidic protein (GFAP) were estimated in whole brain extracts and in histologic slides by Western blot and immunohistochemistry, respectively.ResultsVNS significantly reduced the central levels of pro-inflammatory cytokines and the percentage of microglia (CD11b/CD45low) and macrophages (CD11b/CD45high), 24 h after the electrical stimulus in LPS stimulated mice. A significantly reduced level of Iba-1 expression was also observed in whole brain extracts and in the hippocampus, suggesting a reduction in activated microglia.ConclusionsVNS is a feasible therapeutic tool to attenuate the NI reaction. Considering that NI accompanies different neuropathologies VNS is a relevant alternative to modulate NI, of particular interest for chronic neurological diseases.

The role of carbohydrate component of recombinant α7 nicotinic acetylcholine receptor extracellular domain in its immunogenicity and functional effects of resulting antibodies

Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) attenuate the inflammatory cytokines production by macrophages and are involved in pathogenesis of Alzheimer disease by directly influencing the processing of amyloid-beta (Aβ) precursor protein in the brain. Previously we found that regular injections of bacterial lipopolysaccharide (LPS) decreased the level of α7 nAChRs and stimulated accumulation of Aβ peptide (1–42) in the brain of mice resulting in memory impairment. Similar effects were observed in mice immunized with recombinant extracellular domain (1–208) of α7 nAChR subunit. However, the mechanism of inflammation-like effect of α7-specific antibodies remained unclear. The aim of the present study was to reveal the impact of carbohydrate component of recombinant α7(1–208) produced in yeast in the functional effect of resulting antibodies. For this purpose, C57Bl/6 mice were immunized with either initial α7(1–208) or with that pre-treated with endoglycosidase. Control groups of mice obtained injections of either LPS or complete Freund’s adjuvant. Mice were tested for memory performance, their blood sera were examined for the presence and fine specificity of α7(1–208)-specific antibodies and the brain preparations were studied for the levels of α7 nAChR, Aβ(1–42) and interleukin-6. It was found that the original α7(1–208) was more immunogenic than the deglycosylated one, and their epitopes were recognized with different efficiency. In contrast to LPS and original α7(1–208), deglycosylated α7(1–208) did not stimulate interleukin-6 elevation in the brain, i.e. had no pro-inflammatory effect. Nevertheless, immunizations with either the original or deglycosylated α7(1–208) resulted in similar decrease of α7 nAChRs, accumulation of Aβ(1–42) in the brain and significant episodic memory decline, comparable to those exerted by LPS injections. We conclude that the decrease of α7 nAChR density, caused by α7(1–208)-specific antibody, is critical for Aβ(1–42) accumulation and episodic memory impairment, while pro-inflammatory capacity of α7(1–208)-specific antibody plays a secondary role for the development of Alzheimer-like symptoms.

Study of the neuronal response to olfactory stimuli in control and LPS-stimulated mice by functional magnetic resonance imaging

Olfactory perception plays the key role in the interaction of animals with biotic factors of the species- specific econiche. Identification of odorants informs nocturnal animals about social environment, presence of predators, or infected food. Olfactory efficiency depends on physiological conditions; in particular, odor sensitiveity can be changed by infection. This work considers use of fMRI in the study of the influence of innate immunity activation on the neuronal response during perception and differentiation of socially significant (2,5-dimethylpyrazine, 2-heptanon) and socially insignificant (1-hexanol and isoprene) olfactory stimuli by CD-1 mice. We stimulated innate immunity by intraperitoneal injection of bacterial lipopolysaccharide (LPS) in a dose of 500 μg/kg three hours before tomography. Urethane anesthesia was used during the MRI trail. Odor was stimulated with a lab-made metering unit for supplying standard doses of volatile organic compounds. The supply of olfactory stimuli induced the activation of neurons in the primary perceptual center and the centers of secondary processing of olfactory information. The olfactory stimulus type affected the neuronal response rate in an olfactory bulb but did not affect the response parameters in the other brain regions studied. This increase in neuronal activity is likely to be of adaptive significance as a mechanism supporting an increase in olfactory sensitivity, which plays the key role in the identification of potential infection sources.