Cytokine phenotype,genotype, and renal outcomes at cardiac surgery

BackgroundCardiac surgery modulates pro- and anti-inflammatory cytokine balance involving plasma tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) together with urinary transforming growth factor beta-1 (TGFβ1), interleukin-1 receptor antagonist (IL1ra) and tumour necrosis factor soluble receptor-2 (TNFsr2). Effects on post-operative renal function are unclear. We investigated if following cardiac surgery there is a relationship between cytokine (a) phenotype and renal outcome; (b) genotype and phenotype and (c) genotype and renal outcome. Since angiotensin-2 (AG2), modulates TGFβ1 production, we determined whether angiotensin converting enzyme insertion/deletion (ACE I/D) genotype affects urinary TGFβ1 phenotype as well as renal outcome.MethodsIn 408 elective cardiac surgery patients we measured pre- and 24 h post-operative urinary TGFβ-1, IL1ra and TNFsr2 and pre- and 2 h post-operative plasma TNFα and IL-10. Post-operative responses were compared for each cytokine in patients grouped according to presence or absence of renal dysfunction defined as a drop from baseline eGFR of greater than 25% (as calculated by the method of modification of diet in renal disease (MDRD)) occurring (1) within the first 24 and (2) 48 postoperative hours (early renal dysfunction), (3) on the fifth postoperative day (late renal dysfunction) or (4) at any time throughout the 5 day postoperative period (early and late combined). Patient genotype was determined for TNF/G-308A, TGFβ1-509 C/T, IL10/G-1082A and ACE I/D.ResultsPost-operative plasma IL-10 and urinary TGFβ1 responses were significantly higher in patients who developed early renal dysfunction. IL1ra and TNFsr2 responses were significantly lower 24 h post-operatively in patients who developed late renal dysfunction. Genotype did not alter cytokine phenotype or outcome.Conclusions/inferencesCytokine profiling may help predict early and late renal dysfunction. Genotypes studied did not alter phenotype or outcome.     

Aging is associated with circulating cytokine dysregulation

PurposeAging is accompanied by a progressive increase in pro-inflammatory cytokine status. However, little is known about the development of age-dependent modifications in other circulating cytokines. The aim of this study was to investigate in vivo the influence of age on circulating cytokine production in healthy subjects (HC).MethodsCirculating cytokines were measured by CBA and ELISA in 73 HC. Intracellular cytokine production was assessed in CD3+ and CD14+ cells by flow cytometry. Production of cytokines in cell culture supernatants was also studied after polyclonal stimulation.ResultsSubjects were divided into three different groups according to age: 28 young HC (<30 years, 26.2 ± 2.4), 24 middle age HC (30–60 years, 44.7 ± 8.4) and 21 elderly HC (>60 years, 70.6 ± 7.9). Age was positively correlated with the circulating levels of IL-12p70, IL-1β, TNFα, IL-6, and IL-10. Age had a negative correlation with circulating levels of IL-17. Besides, age was positively correlated with spontaneous intracellular expression of proinflammatory cytokines in circulating monocytes. No correlation was found with other intracellular cytokine expression or with the production of cytokines in cell culture supernatants after in vitro stimulation. Gender had a marginal effect on the circulating cytokine profile.ConclusionAging has a significant impact on the production of circulating cytokines in healthy individuals. The circulating cytokine milieu may contribute to the development of age-restricted conditions.     

Cytokine expression in placenta-derived mesenchymal stem cells in patients with pre-eclampsia and normal pregnancies

Objectives: The aim of this study was to investigate the levels of cytokines in placenta-derived mesenchymal stem cells (MSCs) in normal pregnancies and those with pre-eclampsia. Materials and methods: C5a, CD40 Ligand, G-CSF, GM-CSF, GROα, I-309, sICAM-1, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32α, IP-10, I-TAC, MCP-1, MIF, MIP-1α, MIP-1β, Serpin E1, RANTES, SDF-1, TNFα, and sTREM-1 were measured in mesenchymal stem cells using the human cytokine array panel A. The soluble intracellular adhesion molecule-1 (sICAM-1), stromal-derived factor-1 (SDF-1) and monocyte chemotactic protein-1 (MCP-1) were measured by real-time PCR and confirmed by Western blot analysis. Results: MSCs derived from the deciduas of normal pregnancies had significantly elevated levels of sICAM (p = 0.000) and SDF-1 (p = 0.011), compared to the pregnancies with pre-eclampsia. The level of MCP-1 in the decidua-derived MSCs was not significantly different. No significant difference was observed between normal and pre-eclamptic pregnancies for the amnion-derived MSCs. Conclusions: The decreased levels of sICAM and SDF-1 found in the decidua-derived MSCs from pre-eclamptic pregnancies might be associated with some of the immunological alterations in pre-eclampsia.     

Expression of IL-32 modulates NF-κB and p38 MAP kinase pathways in human esophageal cancer

BackgroundEsophageal cancer is the seventh leading cause of cancer death in males in USA, and there is a strong link has been demonstrated between inflammation and esophageal cancer, interleukin (IL)-32 is a recently described pro-inflammatory cytokine characterized by the induction of nuclear factor NF-κB activation, the p38MAPK also plays an important role in key cellular processes related to inflammation and cancer. We investigated whether the IL-32 expression may be involved in esophageal carcinogenesis through modulates the activity of NF-κB and p-p38 MAPK.MethodMalignant esophageal tissue and blood samples were obtained from 65 operated untreated patients, normal samples was obtained from 35 patients operated for other reasons as control. IL-32 expression visualized by immunohistochemistry, Real time RT–PCR for IL-32 mRNA expression, NF-κB phosphorylation and phosphorylated p38mapk were analyzed by immunoblotting, ELISA for further detection IL-32 and cytokines (TNF-α, IL-1β, IL-6 and IL-8) concentration in the patient’s sera.ResultsIL-32 expression was increased in immunohistochemical staining for malignant esophageal tissue and it’s correlated with the relative expression level of IL-32 mRNA P = 0.007, the P-NF-κB level elevated in tumor tissue compared with control and no difference in the total NF-κB level P = 0.003 while the IL-32 up-regulated the P-pNF-κB in the esophageal tumor P = 0.005. There is increase in p-p38MAPK activation underlying IL-32 expression in tumor P = 0.004, but no change in total p38 MAPK in malignant esophagus. The plasma level of IL-32 expression was increased in malignant esophageal patients P = 0.01, with increased in the levels of the cytokines TNF-α, IL-6, and IL-1β P<0.05.ConclusionsUnderstanding the pathway of IL-32 expression to stimulate the secretion cytokines via the activation of NF-κB and up-regulation of p-p38MAPK may or may not prove to be a therapeutic target, or a biomarker, and future studies will finally answer this hypothesis generated.     

Th1/Th2 cytokine profile in childhood-onset systemic lupus erythematosus

ObjectiveTo determine the serum levels of Th1 (IL-12, IFN-γ,TNF-α) and Th2 (IL-5, IL-6 and IL-10) cytokines in childhood-onset SLE, first-degree relatives and healthy controls. To elucidate their association with disease activity, laboratory and treatment features.MethodsWe included 60 consecutive childhood-onset SLE patients [median age 18 years (range 10–37)], 64 first-degree relatives [median 40 (range 28–52)] and 57 healthy [median age 19 years (range 6–30 years)] controls. Controls were age and sex-matched to SLE patients. SLE patients were assessed for clinical and laboratory SLE manifestations, disease activity (SLEDAI), damage (SDI) and current drug exposures. Mood and anxiety disorders were determined through Becks Depression (BDI) and Anxiety Inventory (BAI). Th1 (IL-12, IFN-γ,TNF-α) and Th2 (IL-5, IL-6 and IL-10) cytokines levels were measured by ELISA and compared by non-parametric tests.ResultsSerum TNF-α (p = 0.004), IL-6 (p = 0.007) and IL-10 (p = 0.03) levels were increased in childhood-onset SLE patients when compared to first-degree relatives and healthy controls. TNF-α levels were significantly increased in patients with active disease (p = 0.014) and correlated directly with SLEDAI scores (r = 0.39; p = 0.002). IL-12 (p = 0.042) and TNF-α (p = 0.009) levels were significantly increased in patients with nephritis and TNF-α in patients with depression (p = 0.001). No association between cytokine levels and SDI scores or medication was observed.ConclusionTh1 cytokines may play a role in the pathogenesis of neuropsychiatric and renal manifestations in childhood-onset SLE. The correlation with SLEDAI suggests that TNF-α may be a useful biomarker for disease activity in childhood-onset SLE, however longitudinal studies are necessary to determine if increase of this cytokine may predict flares in childhood-onset SLE.     

Macrophage colony-stimulating factor (CSF-1) induces pro-inflammatory gene expression and enhances antimicrobial responses of goldfish (Carassius auratus L.) macrophages

We report on the regulation of pro-inflammatory functions of goldfish macrophages and induction of gene expression by recombinant goldfish CSF-1 (rgCSF-1). Recombinant goldfish TNFα-2 (rg TNFα-2), rgIFNγ but not rgTGFβ induced time-dependent increase of CSF-1 expression in macrophages. Treatment of goldfish macrophages with rgCSF-1 increased expression of several immune genes including CXCL-8 (= IL-8), CCL-1, TNFα-1, TNFα-2, IL-1β-1, IL-1β-2, IL-12-p35, IL-12-p40, IFN, IL-10 and iNOS A and B. The rgCSF-1 treatment did not significantly alter the mRNA levels of TGFβ and NRAMP in macrophages up to 48 h post treatment. However, at 72 h post treatment, the expression of TGFβ increased whereas that of NRAMP decreased. The treatment of macrophages with rgCSF-1 enhanced their respiratory burst and nitric oxide responses that were abrogated after addition of soluble CSF-1 receptor (sCSF-1R) to cell cultures. Macrophages exhibited a concentration-dependent chemotactic response toward rgCSF-1 as well as an increase in phagocytic activity that was abrogated after addition of sCSF-1R to cell cultures. Our results indicate that in addition to being an important growth factor of goldfish macrophages, rgCSF-1 also plays a central role in the regulation of their pro-inflammatory responses.     

Macrophage secretions modulate the steroidogenesis of polycystic ovary in rats: effect of testosterone on macrophage pro-inflammatory cytokines

AimsThe macrophage secretions' effect on ovarian steroidogenesis is investigated in a polycystic ovary syndrome rat model (PCO rat). The influence of testosterone environment on the expression of macrophage pro-inflammatory cytokines that participate in ovarian steroidogenesis is studied.Main methodsPCO rats were induced by estradiol valerate. Spleen macrophages were cultured with and without testosterone (10^? 6 M) and their secretions were used to stimulate ovaries from PCO and control rats. Ovarian hormones released and ovary mRNA levels of P450 aromatase and 3β-hydroxysteroid dehydrogenase were measured by radioimmunoassay and RT-PCR, respectively. The tumor necrosis factor alpha (TNFα) and nitric oxide (NO) levels in macrophage culture medium, along with the TNFα, interleukin (IL)-6, IL-10 and androgen receptors (AR) mRNA levels in macrophage cells were determined.Key findingsMacrophages from PCO rats released more TNFα and NO, expressed higher TNFα and IL-6, lower AR, and no change in IL-10 mRNA levels than control macrophages. TNFα, IL-6 and AR changes were greater after macrophage testosterone treatment. Macrophage secretions from PCO rats stimulated androstenedione and decreased estradiol release and ovarian mRNA P450 aromatase expression in PCO rats compared to macrophage secretions from control rats.These effects were greater when macrophages from PCO rats were treated with testosterone. Ovarian progesterone response was unchanged.SignificanceThe differential steroidogenic ability of macrophage secretions from PCO rats is associated to the in vitro testosterone environment. Testosterone, probably acting on macrophage AR, induces a greater release of TNFα, modifying ovarian response by increasing androstenedione and slightly decreasing estradiol without affecting progesterone.     

Antifungal saponins from bulbs of white onion,Allium cepa L.

Three saponins, named ceposide A, ceposide B, and ceposide C were isolated from the bulbs of white onion, Allium cepa L. Elucidation of their structure was carried out by comprehensive spectroscopic analyses, including 2D NMR spectroscopy and mass spectrometry, and chemical evidences. The structures of the compounds were identified as (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-β-d-xylopyranosyl 26-O-α-d-rhamnoyranosyl-(1 → 2)-O-β-d-galactopyranoside (ceposide A), (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-β-d-xylopyranosyl 26-O-α-d-rhamnoyranosyl-(1 → 2)-O-β-d-glucopyranoside (ceposide B), and (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-β-d-galactopyranosyl 26-O-α-d-rhamnoyranosyl-(1 → 2)-O-β-d-galactopyranoside (ceposide C). The isolated compounds, alone and in combinations, were evaluated for their antimicrobial activity on ten fungal species. Antifungal activity of all three saponins increased with their concentration and varied with the following rank: ceposide B > ceposide A–ceposide C. We found a significant synergism in the antifungal activity of the three ceposides against Botrytis cinerea and Trichoderma atroviride, because growth of these fungi was strongly inhibited when the three saponins were applied in combination. In contrast, Fusarium oxysporum f. sp. lycopersici, Sclerotium cepivorum and Rhizoctonia solani were very little affected by saponins.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 3

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological activities were evaluated. The derivatives were found to have potent inhibitory activities on the production of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most promising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition of p38α, p38β, p38γ, and p38δ MAP kinases: IC₅₀ = 0.034, 0.572, >10, and >10 μM, respectively; (ii) inhibition of TNFα, IL-1β, IL-6, and IL-8 production in human whole blood: IC₅₀ = 0.026, 0.020, 0.88, and 0.016 μM, respectively; (iii) inhibition of LPS induced TNFα, IL-1β and IL-6 production in mice: ID₅₀ = 0.93, 8.63, and 0.11 mg/kg, po, respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice: ID₅₀ = 2.22 mg/kg, po; (v) inhibition of collagen-induced arthritis in mice: ID₅₀ = 2.38 mg/kg, po; (vi) prophylactic effect on adjuvant-induced arthritis in rats: ID₅₀ = 3.1 mg/kg, po; (vii) therapeutic effect on adjuvant-induced arthritis in rats: ID₅₀ = 4.9 mg/kg, po; (viii) analgesic effect on adjuvant-induced arthritic pain in rats: ID₅₀ = 2.9 mg/kg, po). As a result, compound 4a was chosen as a candidate for further pre-clinical studies.     

Gout prophylactic constituents from the flower buds of Lonicera japonica

Two new sesquiterpene glycosides (R)-dehydroxyabscisic alcohol β-d-apiofuranosyl-(1″ → 6′)-β-d-glucopyranoside (1) and (−)-(1S,2R,6R,7R)-1,2,6-trimethyl-8-hydroxy methyltricyclic[5.3.1.0^2,6]-undec-8-en-10-one β-d-apiofuranosyl-(1″ → 6′)-β-d-glucopyranoside (2), were isolated from the flower buds of Lonicera japonica. Their structures were determined by spectroscopic and chemical methods. Compound 2 could significantly decrease monosodium urate-mediated cytokine production from activated macrophage through lowering IL-1β and TNFα.     

Discovery of benzo[g]indol-3-carboxylates as potent inhibitors of microsomal prostaglandin E2 synthase-1

Selective inhibition of pro-inflammatory prostaglandin (PG)E₂ formation via microsomal PGE₂ synthase-1 (mPGES-1) might be superior over inhibition of all cyclooxygenase (COX)-derived products by non-steroidal anti-inflammatory drugs (NSAIDs) and coxibs. We recently showed that benzo[g]indol-3-carboxylates potently suppress leukotriene biosynthesis by inhibiting 5-lipoxygenase. Here, we describe the discovery of benzo[g]indol-3-carboxylates as a novel class of potent mPGES-1 inhibitors (IC₅₀ ? 0.1 μM). Ethyl 2-(3-chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (compound 7a) inhibits human mPGES-1 in a cell-free assay (IC₅₀ = 0.6 μM) as well as in intact A549 cells (IC₅₀ = 2 μM), and suppressed PGE₂ pleural levels in rat carrageenan-induced pleurisy. Inhibition of cellular COX-1/2 activity was significantly less pronounced. Compound 7a significantly reduced inflammatory reactions in the carrageenan-induced mouse paw edema and rat pleurisy. Together, based on the select and potent inhibition of mPGES-1 and 5-lipoxygenase, benzo[g]indol-3-carboxylates possess potential as novel anti-inflammatory drugs with a valuable pharmacological profile.     

Pyrogenic effects of cytokines (IL-1β, IL- 6, TNF-α) and their mode of action on thermoregulatory centers and functions

The objective of this study was to compare the thermoregulatory responses of rabbits to fever-inducing doses of various cytokines (IL-1β, IL-6, TNF-α) injected peripherally (i.v.), or centrally (i.h.).TNF-α (1 μg kg⁻¹), when applied i.v. at thermoneutral conditions, induced a long lasting increase in hypothalamic temperature, which reached maximal values within 50 min and then persisted for at least 3 h. This monophasic fever-like response was due to extensive and long lasting attenuation of panting and due to transient vasoconstriction. Metabolic rate tended to increase during the early phase of the fever, only. On the other hand, IL-6 when applied i.v. in the same dose (1 μg kg⁻¹) and IL-1β, in a much lower dose (60 ng kg⁻¹), induced a short lasting monophasic hyperthermia due to transient attenuation of panting and due to transient vasoconstriction. No significant increase in metabolic rate was observed. The immediate attenuation of panting and induction of vasoconstriction rather resembled reflex (shock) responses than specific thermoregulatory reactions. Thirty minutes after i.v. administration of TNF-α, temperature thresholds for induction of cold thermogenesis, panting and vasomotion were shifted to higher body temperatures and remained elevated for at least 3 h. In case of IL-1β the increased temperature thresholds were observed 30 min after i.v. administration, only. I.v. applied IL-6 did not influence the thresholds neither 30 or 180 min after injection. Thus, peripheral IL-6 rather induced hyperthermia than fever.When injected i.h. all cytokines studied induced a long lasting increase in body temperature similar to that after i.v. injection of LPS. Temperature thresholds for induction of all thermoregulatory outputs were increased and remained increased for at least 3 h. No changes in hypothalamic thermosensitivity were observed. Data indicate a nonspecific effect of central cytokines on body temperature control.IL-1β appeared to be the most potent fever inducer. Nanogram doses of IL-1β injected i.v. induced a similar febrile response as microgram doses of other cytokines. On the other hand, TNF-α induced a longer lasting fever than other cytokines.     

Steroidal saponins from Tribulus terrestris

Five new steroidal saponins were isolated from the fruits of Tribulus terrestris. Their structures were fully established by spectroscopic and chemical analysis as (23S,25S)-5α-spirostane-24-one-3β,23-diol-3-O-{α-l-rhamnopyranosyl-(1 → 2)-O-[β-d-glucopyranosyl-(1 → 4)]-β-d-galactopyranoside} (1), (24S,25S)-5α-spirostane-3β,24-diol-3-O-{α-l-rhamnopyranosyl-(1 → 2)-O-[β-d-glucopyranosyl-(1 → 4)]-β-d-galactopyranoside} (2), 26-O-β-d-glucopyranosyl-(25R)-5α-furostan-2α,3β,22α,26-tetraol-3-O-{β-d-glucopyranosyl-(1 → 2)-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside} (3), 26-O-β-d-glucopyranosyl-(25R)-5α-furostan-20(22)-en-2α,3β,26-triol-3-O-{β-d-glucopyranosyl-(1 → 2)-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside} (4), and 26-O-β-d-glucopyranosyl-(25S)-5α-furostan-12-one-22-methoxy-3β,26-diol-3-O-{α-l-rhamnopyranosyl-(1 → 2)-O-[β-d-glucopyranosyl-(1 → 4)]-β-d-galactopyranoside} (5). The isolated compounds were evaluated for cytostatic activity against HL-60 cells.     

Saponins in aerial parts of Helleborus viridis L.

In the search of natural compounds inhibiting methane production in ruminants three novel steroidal saponins have been isolated from the aerial parts of Helleborus viridis L. Their structures have been established based on spectral analyses as: (25R)-26-O-β-d-glucopyranosyl-5β-furostan-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1 → 6)-O-β-d-glucopyranoside, (25R)-26-O-β-d-glucopyranosyl-5α-furostan-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1 → 6)-O-β-d-glucopyranoside and (25R)-26-O-β-d-glucopyranosyl-furost-5-ene-1β,3β,22α,26-tetraol 1-O-{α-l-rhamnopyranosyl-(1 → 2)-O-[β-d-glucopyranosyl-(1 → 3)]-6-O-acetoxy-β-d-glucopyranoside}.     

Spatial and temporal expression of CCR3 and the common beta chain of the IL-3, IL-5 and GM-CSF receptor in the nasal epithelium and lymphoid tissues in a rat model of allergic rhinitis

BackgroundAllergic rhinitis (AR) and asthma are closely related conditions that often co-exist, and are characterized by a Th2 inflammatory response where eosinophils occupy a predominant role. Strategies aimed at blocking signaling through the CC chemokine receptor 3 (CCR3) and/or the common beta chain of the IL-3, IL-5 and GM-CSF receptor (βc) efficiently reduced eosinophilic inflammation in both animal models and in asthmatic patients. This study was therefore aimed at characterizing the spatio-temporal expression pattern of βc and CCR3 using a rat model of AR.MethodsSensitized rats were challenged with ovalbumin and sacrificed at 2 h, 8 h, 16 h or 24 h post-challenge. Nasal tissues were microdissected and used for mRNA quantification by QPCR, while histological evaluation determined the presence of eosinophils and mucosubstances.ResultsAllergen-induced recruitment of eosinophils in the distal septum and turbinates was maximal at 8 h post-challenge, and was correlated with 2–4-fold increase in CCR3 and βc mRNA. Recruitment of eosinophils was also accompanied by upregulated IL-5, IL-4Rα, TNF-α and IFN-γ mRNA at early time-points. In contrast, IL-13 and MUC5AC mRNA, as well as production of mucosubstances were maximal at 24 h.Conclusionsβc and CCR3 could play important roles in the modulation of the allergic response, and their inhibition may represent a promising therapeutic approach for AR.     

Serum cytokine profiles of Khorasan veterans 23 years after sulfur mustard exposure

Sulfur mustard (SM) is an incapacitating chemical warfare agent that was used against Iranian soldiers during the period from 1983 to 1988. We have investigated serum cytokines profiles of Khorasan veterans who were exposed to SM >23 years earlier. Forty-four male Iranian veterans who had >40% disabilities due to delayed complications of SM poisoning and had disabilities were investigated. A total of 30 healthy male volunteers (relatives of the veterans) were selected as the control group. Cytokine levels were measured in the serum of case and control subjects using commercial ELISA kits. Hematologic parameters (white/red blood cell counts, hemoglobin levels, immune cell differentials) were also performed on blood samples from the study subjects. The results indicated that serum levels of ICAM-1 were significantly higher in the samples from SM-exposed veterans (772.8 [±15.1] ng/ml [p = 0.014] vs. control values of 710.2 [±20.0] ng/ml). On the other hand, serum IL-1β, IL-8 levels and TNFα, were significantly lower for the veterans than the controls (IL-1β: 3.8 [±0.1] vs. 4.3 [±0.2] pg/ml, p = 0.037; IL-8: 21.0 [±6.1] vs. 84.6 [±20.3] pg/ml, p = 0.002; TNFα: 4.5 [±0.1] vs. 5.5 [± 0.1] pg/ml, p = 0.027). Levels of other assayed cytokines, e.g., IL-2, -4, -5, -6, -10, and -12, IFNγ, TNFβ, and sVCAM-1 were not significantly different between the study populations. None of the assayed hematologic parameters appeared to differ as well. It seems possible that dysfunctions could have been induced in the innate immune functions of the SM-exposed veterans as a result of these changes in cytokine expression and that these, in turn, may have contributed to the increased incidence of a myriad of diseases that have been documented in these veterans, including cancers. Future studies must focus on examining the significance of these changes in circulating cytokines and their potential contribution to the development of different diseases in veterans exposed to SM.     

Simultaneous quantification of GABAergic 3α,5α/3α,5β neuroactive steroids in human and rat serum

The 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography–mass spectrometry to simultaneously identify serum levels of the eight 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3α,5α- and 3α,5β-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3α,5α-THP (+1488%, p < 0.001), (3α,5α)-3,21-dihydroxypregnan-20-one (3α,5α-THDOC, +205%, p < 0.01), (3α,5α)-3-hydroxyandrostan-17-one (3α,5α-A, +216%, p < 0.001), (3α,5α,17β)-androstane-3,17-diol (3α,5α-A-diol, +190%, p < 0.01). (3α,5β)-3-hydroxypregnan-20-one (3α,5β-THP) and (3α,5β)-3-hydroxyandrostan-17-one (3α,5β-A) were not altered, while (3α,5β)-3,21-dihydroxypregnan-20-one (3α,5β-THDOC) and (3α,5β,17β)-androstane-3,17-diol (3α,5β-A-diol) were increased from undetectable levels to 271 ± 100 and 2.4 ± 0.9 pg ± SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3α,5α-THP (+1806%, p < 0.0001), 3α,5β-THP (+575%, p < 0.001), 3α,5α-THDOC (+309%, p < 0.001). 3α,5β-THDOC levels were increased by 307%, although this increase was not significant because this steroid was detected only in 3/16 control subjects. Levels of 3α,5α-A, 3α,5β-A and pregnenolone were not altered. This method can be used to investigate the physiological and pathological role of neuroactive steroids and to develop biomarkers and new therapeutics for neurological and psychiatric disorders.     

Cytokine expression profile over time in burned mice

The persistent inflammatory response induced by a severe burn increases patient susceptibility to infections and sepsis, potentially leading to multi-organ failure and death. In order to use murine models to develop interventions that modulate the post-burn inflammatory response, the response in mice and the similarities to the human response must first be determined. Here, we present the temporal serum cytokine expression profiles in burned mice in comparison to sham mice and human burn patients. Male C57BL/6 mice were randomized to control (n = 47) or subjected to a 35% TBSA scald burn (n = 89). Mice were sacrificed 3, 6, 9, 12, 24, and 48 h and 7, 10, and 14 days post-burn; cytokines were measured by multi-plex array. Following the burn injury, IL-6, IL-1β, KC, G-CSF, TNF, IL-17, MIP-1α, RANTES, and GM-CSF were increased, p < 0.05. IL-2, IL-3, and IL-5 were decreased, p < 0.05. IL-10, IFN-γ, and IL-12p70 were expressed in a biphasic manner, p < 0.05. This temporal cytokine expression pattern elucidates the pathogenesis of the inflammatory response in burned mice. Expression of 11 cytokines were similar in mice and children, returning to lowest levels by post-burn day 14, confirming the utility of the burned mouse model for development of therapeutic interventions to attenuate the post-burn inflammatory response.     

Vocal fold fibroblasts immunoregulate activated macrophage phenotype

Recent evidence suggests that fibroblasts play a critical role in regulating inflammation during wound healing because they express several inflammatory mediators in response to bacteria. The objective of this study was to analyze the effects of lipopolysaccharide (LPS) on the immunomodulatory properties of vocal fold fibroblasts (VFFs) derived from polyps, scar and normal tissue co-cultured with macrophages, to provide insight into their interactions during the inflammatory process. Fibroblasts were co-cultured with CD14+ monocytes and after 7 days, wells were treated with LPS for 24 and 72 h. Culture supernatants were collected and concentrations of TNF-α, IL-6, IL-8, IL-10, IL-12, IL-1β and MCP-1 were quantified by ELISA. Normal VFF and CD14+ monocultures were used as controls. Twenty-four hours after LPS activation, macrophages co-cultured with polyp VFF had significantly increased expression of TNF-α, IL-1β, IL-12 and IL-10 compared to controls (p < 0.0001). In contrast, macrophages co-cultured with scar VFF had significantly lower expression of TNF-α, IL-1β and IL-12 with significantly higher IL-10 compared to control (p < 0.0001). After 72 h, macrophages co-cultured with polyp VFF increased expression of TNF-α, IL-1β, IL-10, IL-6, IL-8, MCP-1 and TGF-β (p < 0.01) and macrophages co-cultured with scar VFF significantly decreased their expression of IL-1β and IL-12 compared to control (p < 0.0001). Scar VFF at both time points produced significantly lower levels of IL-8, MCP-1, IL-6 and TGF-β compared to controls (p < 0.05). Based on our findings, VFF and macrophages secrete several inflammatory mediators that modify their diverse functions. Polyp and scar VFF may play a role in regulating abnormal inflammatory responses, which could result in excessive ECM deposition that disrupts the function of the vocal folds.