Increased Cytokine Release Research Articles

Enhanced immune response to MMP3 stimulation in microglia expressing mutant huntingtin

Huntington’s Disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. The YAC128 mouse model of HD expresses the full-length human huntingtin protein with 128 CAG repeats and replicates the phenotype and neurodegeneration that occur in HD. Several studies have implicated a role for neuroinflammation in HD pathogenesis. Studies on presymptomatic HD patients have illustrated microgliosis (activated microglia) in brain regions affected in HD. Mutant huntingtin expressing isolated primary monocytes (human HD patients) and primary macrophages (YAC128) are overactive in response to lipopolysaccharide (LPS) stimulation. In this study we demonstrate that cultured primary microglia (the resident immune cells of the brain cells) from YAC128 mice differentially express a wide number of cytokines compared to wildtype microglia cultures in response to LPS. Furthermore, this study outlines a direct interaction between mutant huntingtin and cytokine secretion in HD microglia. Increased cytokine release in YAC128 microglia can be blocked by cannabinoid activation or by mutant huntingtin knockdown with anti-sense oligonucleotide treatment. Matrix metalloprotease 3 (MMP3), an endogenous neuronal activator of microglia, also induces increased cytokine release from YAC128 microglia compared to wildtype microglia. We found elevated MMP levels in HD CSF, and MMP levels correlate with disease severity in HD. These data support a novel role for MMPs and microglial activation in HD pathogenesis. With an improved understanding of the specific cellular processes involved in HD neuroinflammation, novel therapeutic agents targeting these processes can be developed and hold great promise in the treatment of HD.

Kinin Peptides Enhance Inflammatory and Oxidative Responses Promoting Apoptosis in a Parkinson's Disease Cellular Model.

Kinin peptides ubiquitously occur in nervous tissue and participate in inflammatory processes associated with distinct neurological disorders. These substances have also been demonstrated to promote the oxidative stress. On the other hand, the importance of oxidative stress and inflammation has been emphasized in disorders that involve the neurodegenerative processes such as Parkinson's disease (PD). A growing number of reports have demonstrated the increased expression of kinin receptors in neurodegenerative diseases. In this study, the effect of bradykinin and des-Arg10-kallidin, two representative kinin peptides, was analyzed with respect to inflammatory response and induction of oxidative stress in a PD cellular model, obtained after stimulation of differentiated SK-N-SH cells with a neurotoxin, 1-methyl-4-phenylpyridinium. Kinin peptides caused an increased cytokine release and enhanced production of reactive oxygen species and NO by cells. These changes were accompanied by a loss of cell viability and a greater activation of caspases involved in apoptosis progression. Moreover, the neurotoxin and kinin peptides altered the dopamine receptor 2 expression. Kinin receptor expression was also changed by the neurotoxin. These results suggest a mediatory role of kinin peptides in the development of neurodegeneration and may offer new possibilities for its regulation by using specific antagonists of kinin receptors.

The nephroprotective effects of ginkgo biloba extract (EGb761) against l-NG-nitroarginine methyl ester-induced hypertension in rats: role of oxidative stress and inflammatory markers

BackgroundGinkgo biloba extract 761 (EGb761) was studied for its nephroprotective effects in experimentally induced hypertension. Hypertension is increasingly a cause of end-stage renal diseases. Increased cytokine release and oxidative stress are mechanisms that appear to be involved in the pathogenesis of hypertensive renal damage. EGb has antioxidant and anti-inflammatory effects and can attenuate hypertensive renal damage.Methods and resultsMale adult Wistar rats were used in this study. Hypertension was induced in these rats by administering l-NG-nitroarginine methyl ester (l-NAME) (10 mg/kg/day, intraperitoneal) for 12 weeks. Another group of rats received l-NAME and EGb761 (100 mg/kg/day, orally) starting from the ninth week to the end of treatment. It was found that the blood pressure was reduced at the end of 12th week in rats treated with EGb761 compared with l-NAME-treated (hypertensive) rats. EGb761-treated rats showed lower renal tissue malondialdehyde level and renal tissue tumor necrosis factor-α level when compared with l-NAME-treated rats (hypertensive).ConclusionEGb761 has antihypertensive effect; it can protect the kidney from hypertension through the reduction of renal inflammation and oxidative stress.

Multitargeting of selected prostanoid receptors provides agents with enhanced anti-inflammatory activity in macrophages.

A polypharmacologic approach to prostanoid based anti-inflammatory therapeutics was undertaken in order to exploit both the anti- and proinflammatory properties attributed to the various prostanoid receptors. Multitargeting of selected prostanoid receptors yielded a prototype compound, compound 1 (AGN 211377), that antagonizes prostaglandin D2 receptors (DPs) DP1 (49) and DP2 (558), prostaglandin E2 receptors (EPs) EP1 (266) and EP4 (117), prostaglandin F2α receptor (FP) (61), and thromboxane A2 receptor (TP) (11) while sparing EP2, EP3, and prostaglandin I2 receptors (IPs); Kb values (in nanomoles) are given in parentheses. Compound 1 evoked a pronounced inhibition of cytokine/chemokine secretion from lipopolysaccharide or TNF-α stimulated primary human macrophages. These cytokine/chemokines included cluster of designation 40 receptor (CD40), epithelial-derived neutrophil-activating protein 78 (ENA-78), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, IL-18, monocyte chemotactic protein-1 (CCL2) (MCP-1), tissue plasminogen activator inhibitor (PAI-1), and regulated on activation, normal T cell expressed and secreted (RANTES). In contrast, the inhibitory effects of most antagonists selective for a single receptor were modest or absent, and selective EP2 receptor blockade increased cytokine release in some instances. Compound 1 also showed clear superiority to the cyclooxygenase inhibitors diclofenac and rofecoxib. These findings reveal that blockade of multiple prostanoid receptors, with absent antagonism of EP2 and IP, may provide more effective anti-inflammatory activity than global suppression of prostanoid synthesis or highly selective prostanoid receptor blockade. These investigations demonstrate the first working example of prostanoid receptor polypharmacology for potentially safer and more effective anti-inflammatory therapeutics by blocking multiple proinflammatory receptors while sparing those with anti-inflammatory activity.

Sex differences in neuroimmune response to neonatal infection in brain regions important for learning and memory: Implications for developmental disorders

Many developmental and psychiatric disorders exhibit strong sex differences with males being biased towards diagnosis of developmental disorders. Interestingly, many of these disorders have been linked to early-life immune activation. One theory regarding increased male susceptibility involves the activation of immune factors in early life; however factors such as sex and microglia function affecting immune activation are not well understood. Microglia are the resident immune cells of the brain and respond to infections with rapid increases in cytokine release. We recently discovered that males have significantly more microglia in the hippocampus and amygdala compared to females on postnatal (P) day 4. Subsequently, we examined proinflammatory cytokine responses to neonatal infection at P4 in males and females 8 and 24 hours following a low dose of Escherichia coli (E.coli). Contrary to our predictions, males and females showed similar neuroimmune responses at P4 in the hippocampus, cerebellum, amygdala, and prefrontal cortex with neonatally infected animals showing exaggerated cytokine levels compared to saline controls ( p ′ s .05); however; in the periphery males showed significantly greater levels of IL-1Beta and TNF-alpha 8 hours following neonatal infection compared to females ( p ′ s .05). We are currently examining microglia response to an ex vivo immune challenge to address whether microglia may be inherently more reactive in a sex-dependent manner outside of cell number or peripheral immune signals.

Blockade of the N-Methyl-D-Aspartate Glutamate Receptor Ameliorates Lipopolysaccharide-Induced Renal Insufficiency.

N-methyl-D-aspartate (NMDA) receptor activation in rat kidney reduces renal perfusion and ultrafiltration. Hypoperfusion-induced ischemia is the most frequent cause of functional insufficiency in the endotoxemic kidney. Here, we used non-hypotensive rat model of lipopolysaccharide-induced endotoxemia to examine whether NMDA receptor hyperfunction contributes to acute kidney injury. Lipopolysaccharide-induced renal damage via increased enzymuria and hemodynamic impairments were ameliorated by co-treatment with the NMDA receptor blocker, MK-801. The NMDA receptor NR1 subunit in the rat kidney mainly co-localized with serine racemase, an enzyme responsible for synthesizing the NMDA receptor co-agonist, D-serine. The NMDA receptor hyperfunction in lipopolysaccharide-treated kidneys was demonstrated by NR1 and serine racemase upregulation, particularly in renal tubules, and by increased D-serine levels. Lipopolysaccharide also induced cell damage in cultured tubular cell lines and primary rat proximal tubular cells. This damage was mitigated by MK-801 and by small interfering RNA targeting NR1. Lipopolysaccharide increased cytokine release in tubular cell lines via toll-like receptor 4. The release of interleukin-1β from these cells are the most abundant. An interleukin-1 receptor antagonist not only attenuated cell death but also abolished lipopolysaccharide-induced NR1 and serine racemase upregulation and increases in D-serine secretion, suggesting that interleukin-1β-mediated NMDA receptor hyperfunction participates in lipopolysaccharide-induced tubular damage. The results of this study indicate NMDA receptor hyperfunction via cytokine effect participates in lipopolysaccharide-induced renal insufficiency. Blockade of NMDA receptors may represent a promising therapeutic strategy for the treatment of sepsis-associated renal failure.

Platelet and endothelial cell P-selectin are required for host defense against Klebsiella pneumoniae-induced pneumosepsis

Sepsis is associated with activation of platelets and endothelial cells accompanied by enhanced P-selectin surface expression. Both platelet- and endothelial P-selectin have been associated with leukocyte recruitment and induction of inflammatory alterations. Klebsiella (K.) pneumoniae is a common human sepsis pathogen, particularly in the context of pneumonia. Wild-type (WT) and P-selectin-deficient (Selp(-/-) ) mice or bone marrow chimeric mice were infected with K. pneumoniae via the airways to induce pneumosepsis. Mice were sacrificed during early (12 h after infection) or late-stage (44 h) sepsis for analyses, or followed in a survival study. Selp(-/-) mice displayed 10-1000-fold higher bacterial burdens in the lungs, blood and distant organs during late-stage sepsis. P-selectin deficiency did not influence leukocyte recruitment to the lungs, but was associated with decreased platelet-monocyte complexes and increased cytokine release. Bone marrow transfer studies revealed a role for both platelet and endothelial cell P-selectin as mice deficient in platelet or endothelial cell P-selectin displayed an intermediate phenotype in bacterial loads and survival compared with full wild-type or full knockout control mice. Both platelet and endothelial cell P-selectin contribute to host defense during Klebsiella pneumosepsis.

Lipoic acid and N-acetylcysteine prevent ammonia-induced inflammatory response in C6 astroglial cells: The putative role of ERK and HO1 signaling pathways

Hyperammonemia induces significant changes in the central nervous system (CNS) in direct association with astroglial functions, such as oxidative damage, glutamatergic excitotoxicity, and impaired glutamine synthetase (GS) activity and pro-inflammatory cytokine release. Classically, lipoic acid (LA) and N-acetylcysteine (NAC) exhibit antioxidant and anti-inflammatory activities by increasing glutathione (GSH) biosynthesis and decreasing pro-inflammatory mediator levels in glial cells. Thus, we evaluated the protective effects of LA and NAC against ammonia cytotoxicity in C6 astroglial cells. Ammonia decreased GSH levels and increased cytokine release and NFκB transcriptional activation. LA and NAC prevented these effects by the modulation of ERK and HO1 pathways. Taken together, these observations show that LA and NAC prevent the ammonia-induced inflammatory response.

Protective effect of ixerisoside A against UVB-induced pro-inflammatory cytokine production in human keratinocytes.

Human skin is the first line of defense for the protection of the internal organs of the body from different stimuli. Ultraviolet B (UVB), one of the harmful radiations for skin, is widely known to induce abnormally increased cytokine release from keratinocytes leading to inflammatory skin disorders. IL-6 and IL-8 induce an acute-phase response and stimulate leukocyte infiltration in the skin. Previous studies have shown that chronic exposure to UVB radiation increases cyclooxygenase-2 (COX‑2) expression through various cell signaling pathways, resulting in skin cancer. Recent studies have shown that the activation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK is strongly correlated with acute inflammation and development of skin cancer caused by an increased expression of COX-2. Ixerisoside A (IXA) is an active constituent of Ixeris dentata of the Compositae (Asteraceae) family. The effect of IXA on skin inflammation has yet to be elucidated. To determine the anti-inflammatory effects of IXA, we examined its effect on UVB-induced pro-inflammatory cytokine production in human keratinocytes (HaCaT cells) by observing these cells in the presence or absence of IXA. In this study, pro-inflammatory cytokine production was determined by enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (rt-pcr), and western blot analysis to evaluate the activation of mitogen-activated protein kinases (MAPKs). IXA inhibited UVB-induced production of the pro-inflammatory cytokines IL-6 and IL-8 in a dose-dependent manner. Moreover, IXA inhibited the expression of COX-2, ERK, JNK, and p38 MAPKs, indicating that the secretion of the pro-inflammatory cytokines IL-6 and IL-8, and COX-2 expression was inhibited by blocking MAPK phosphorylation. These results indicated that IXA potentially protects against UVB-induced skin inflammation.

Dectin-1 Is Expressed in Human Lung and Mediates the Proinflammatory Immune Response to Nontypeable Haemophilus influenzae

The C-type lectin receptor Dectin-1 is expressed mainly on myeloid cells mediating the immune response targeting respiratory pathogens such as Aspergillus fumigatus and Mycobacterium tuberculosis. The pulmonary epithelium serves as an important interface for interactions between these pathogens and the respiratory tract. Therefore, we analyzed the expression pattern of Dectin-1 in the human lung. Immunohistochemically stained human lung sections from 17 out of 19 individuals were positive for Dectin-1, which was expressed mainly apically on bronchial and alveolar epithelium. Our results showed no correlation with chronic obstructive pulmonary disease (COPD) or the smoking habits of the patients. Nontypeable Haemophilus influenzae (NTHI), an important bacterial pathogen of the respiratory tract with significant importance in COPD, has also been proposed to be recognized by Dectin-1, suggesting a possible impact on the NTHI-dependent immune response in human airways. Therefore, the involvement of Dectin-1 in NTHI-triggered cytokine responses was investigated in primary normal human bronchial epithelial (NHBE) cells and in the A549 cell line stably transfected with Dectin-1. The presence of Dectin-1 significantly increased cytokine release in response to NTHI in NHBE and A549 cells. In addition, phosphorylation of the Dectin-1 hem-immunoreceptor tyrosine-based activation motif (hemITAM) was essential for the Dectin-1-triggered response to NTHI in A549 cells. In conclusion, in human airways, epithelium-expressed Dectin-1 may play a significant role in generating an NTHI-mediated, proinflammatory immune response.

Brain toxicity and inflammation induced in vivo in mice by the amyloid-β forty-two inducer aftin-4, a roscovitine derivative.

Aftins (amyloid forty-two inducers) represent a novel class of tri-substituted purines derived from roscovitine, able to promote the generation of amyloid-β (Aβ)1-42 from amyloid-β protein precursor through γ-secretase activation in cell cultures. We here examined whether aftin-4 could provoke an amyloid-like toxicity in vivo in mice. The intracerebroventricular administration of aftin-4 (3-20 nmol) increased Aβ1-42, but not Aβ1-40, content in the mouse hippocampus, between 5 and 14 days after injection. Aftin-4 injection increased lipid peroxidation levels in the hippocampus, an index of oxidative stress. It increased brain contents in pro-inflammatory cytokines, IL-1β, IL-6, and TNFα, and GFAP immunolabeling, showing astrocytic reaction. Expression of the synaptic marker synaptophysin was decreased by aftin-4. Finally, the treatment provoked marked learning deficits, observed using different memory procedures: Spontaneous alternation in the Y-maze, place learning in the water-maze, and passive avoidance response. The systemic intraperitoneal injection of aftin-4 in the 3-30 mg/kg dose range also induced oxidative stress and learning deficits. All these alterations could be blocked by pre-treatment with the γ-secretase inhibitor BMS-299,897, confirming that the mechanism of action of aftin-4 involves secretase activity. Furthermore, we examined if the cholinesterase inhibitor donepezil and the non-steroidal anti-inflammatory drug ibuprofen could prevent aftin-4-induced memory impairments, cytokine release, and lipid peroxidation. Donepezil prevented all alterations, whereas ibuprofen prevented the increases in cytokine release and lipid peroxidation, but only marginally the memory impairments. As a whole, this study showed that in vivo injection of aftin-4 results in a rapid, acute Alzheimer's disease-like toxicity in the rodent brain.

Mechanical Ventilation Induces an Inflammatory Response in Preinjured Lungs in Late Phase of Sepsis

Mechanical ventilation (MV) may amplify the lung-specific inflammatory response in preinjured lungs by elevating cytokine release and augmenting damage to the alveolar integrity. In this study, we test the hypothesis that MV exerts different negative impacts on inflammatory response at different time points of postlung injury. Basic lung injury was induced by cecal ligation and puncture (CLP) surgery in rats. Physiological indexes including blood gases were monitored during MV and samples were assessed following each experiment. Low V T (tidal volume) MV caused a slight increase in cytokine release and tissue damage at day 1 and day 4 after sepsis induced lung injury, while cytokine release from the lungs in the two moderately ventilated V T groups was amplified. Interestingly, in the two groups where rats received low V T MV, we found that infiltration of inflammatory cells was only profound at day 4 after CLP. Marked elevation of protein leakage indicated a compromise in alveolar integrity in rats that received moderate V T MV at day 4 following CLP, correlating with architectural damage to the alveoli. Our study indicates that preinjured lungs are more sensitive to mechanical MV at later phases of sepsis, and this situation may be a result of differing immune status.

Humoral and cellular immune responses in mice after airway administration of Bacillus thuringiensis Cry1Ab and MON810 cry1Ab-transgenic maize

Genetically modified (GM) crops may bring new proteins with immunogenic and allergenic properties into the food and feed chains. The most commonly grown GM maize, MON810, expresses a modified version of the insecticidal Cry1Ab protein originating in the soil bacterium Bacillus thuringiensis (Bt). Immune reactions following inhalation of pollen and debris from such plants have been scarcely studied. We exposed BALB/c mice to purified Cry1Ab proteins and Cry1Ab-containing MON810 plant materials by intranasal installation. No anti-Cry1Ab antibodies were detected following exposure to the plant materials. Exposure to purified Cry1Ab resulted in specific anti-Cry1Ab IgG1 and IgE production, indicating inherent immunogenicity and allergenicity. Mice exposed to leaf extracts from both MON810 and unmodified maize demonstrated influx of lymphocytes and eosinophils in the broncho-alveolar lavage, and increased cytokine release in mediastinal lymph node cells. The results indicate that the airway exposure to Cry1Ab proteins may be a route of practical relevance.

A functional (pro)renin receptor is expressed in human lymphocytes and monocytes.

The renin-angiotensin system (RAS) is involved in inflammation. The signaling via the ANG II type 1 receptor in human lymphocytes and monocytes, which play key roles in pathophysiology of glomerulonephritis (GN), can enhance inflammation. However, the role of the (pro)renin receptor [(P)RR], a component of the RAS, in inflammatory reactions is unknown. We assessed whether (P)RR is expressed in human lymphocytes and monocytes by RT-PCR, Western blotting, flow cytometry, and immunohistochemistry, and whether (P)RR functions in inflammation. (P)RR mRNA and protein were expressed in human peripheral blood mononuclear cells (PBMCs). Flow cytometric analysis revealed high expression of (P)RR on monocytes. (P)RR was present on PBMCs, infiltrating lymphocytes, and macrophages around glomeruli with a crescent in anti-neutrophil cytoplasmic antibody (ANCA)-associated GN. Renin stimulation of PBMCs from healthy subjects in the presence of the ANG II type 1 receptor and ANG II type 2 receptor blockers induced ERK1/2 phosphorylation and release of IL-6 and expression of cyclooxygenase-2 (COX-2). The increases in cytokine release and COX-2 expression were inhibited in the presence of an ERK1/2 inhibitor. (P)RR knockdown by small interfering RNA in U937 cells, a human leukemic monocyte lymphoma cell line, significantly decreased ERK1/2 phosphorylation after renin stimulation. Thus (P)RR expressed in human inflammatory cells might contribute to inflammation in ANCA-associated GN.

Prognostic Role of Serum Albumin Level in DLBCL before and during the Rituximab Era. Retrospective GISL Study over 738 Cases

Introduction.Serum albumin (SA) has been shown to be a prognostic marker in many hematological malignancies, and in diffuse large B-cell lymphoma (DLBCL) before the introduction of rituximab. SA may be a surrogate for age, comorbid status, and disease severity. Here, we aimed to assess the role of SA as prognostic marker for overall survival (OS) in patients with newly diagnosed DLBCL treated with and without rituximab (R) containing regimens (CHOP and CHOP-like).Patients and methodWe collected 738 patients complete for IPI factors [age, LDH, extra nodal sites, stage and performance status (PS)] and SA recorded in the GISL database from 1998 to 2012. OS was estimated using the Kaplan-Meier method, with Cox proportional hazard model used to identify potential risk factors for time-to-event data. Optimal cut-off for SA was calculated by means of ROC curve at 5 years of follow-up. Stability of the cut-point was analyzed using bootstrap techniques. The role of SA was explored interacting SA with R and adjusting by IPI. The strength of SA as prognostic factor was evaluated counting the bootstrap frequency of inclusion (BIF, 1000 re-samples) of SA, adjusted by the IPI factors, in Cox PH regression.ResultsOf the 738 patients included in the study 322 (44%) were treated with R-CHOP and 416 (56%) were treated without regimens containing R. The median age at diagnosis was 60 years (range 21-89) and 45%, 25% and 30% were categorized in IPI 0-1, 2 and 3/5 respectively; and the median SA was 3.8 g/dL (range 1.0-6.4). Patients treated with R showed a greater % of high IPI 3/5 (43%) compared to those not treated with R (20%, P<0.001) and a lower SA level (3.7 vs 3.9 g/dL, P=0.001). SA showed correlation with IPI (4.0, 3.8 and 3.5 g/dL in IPI 0-1, 2 and 3/5, respectively, P=0.0001).Overall, the median follow-up was 64 months (range 1-157) with 241 dead. The 5-ys OS was 66% (95 CI 62-70) and the best cut-off for SA was 3.7 g/dL with ROC=68.5%. OS at 5-yrs in patients not treated with R was 74% and 52% for SA ≥3.7 and <3.7 g/dL, respectively (P<0.001). In patients treated with R the 5-yrs OS was 78% and 50% for SA ≥3.7 and <3.7 g/dL (P<0.001). The SA <3.7 g/dL, interacted by R and adjusted by IPI, had HR = 1.71 (95 CI 1.22-2.40, P=0.002) and HR = 1.99 (95 CI 1.31-3.01, P=0.001) in cases not treated and treated with R, and the HRs did not vary between treatments (P=0.582). The BIF of SA<3.7 g/dL adjusted by IPI factors was 88% and 79% in patients without and with R chemotherapy.ConclusionThe SA is a simple variable collected from the clinical-chemistry, that showed a good prognostic property in DLBCL. Although correlated with the IPI, SA has maintained a strong prognostic value both before or during the rituximab era. The biologic role of SA over the survival is unclear. It has been hypothesized that low level of SA may be associated with 1) increased inflammatory response to the tumor, 2) increased cytokine release and 3) poor nutritional status, and due to this condition the patients would not be able to receive the full cycles of chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Dectin-1 is expressed in human lung and mediates the proinflammatory immune response to nontypeable Haemophilus influenzae.

ABSTRACTThe C-type lectin receptor Dectin-1 is expressed mainly on myeloid cells mediating the immune response targeting respiratory pathogens such as Aspergillus fumigatus and Mycobacterium tuberculosis. The pulmonary epithelium serves as an important interface for interactions between these pathogens and the respiratory tract. Therefore, we analyzed the expression pattern of Dectin-1 in the human lung. Immunohistochemically stained human lung sections from 17 out of 19 individuals were positive for Dectin-1, which was expressed mainly apically on bronchial and alveolar epithelium. Our results showed no correlation with chronic obstructive pulmonary disease (COPD) or the smoking habits of the patients. Nontypeable Haemophilus influenzae (NTHI), an important bacterial pathogen of the respiratory tract with significant importance in COPD, has also been proposed to be recognized by Dectin-1, suggesting a possible impact on the NTHI-dependent immune response in human airways. Therefore, the involvement of Dectin-1 in NTHI-triggered cytokine responses was investigated in primary normal human bronchial epithelial (NHBE) cells and in the A549 cell line stably transfected with Dectin-1. The presence of Dectin-1 significantly increased cytokine release in response to NTHI in NHBE and A549 cells. In addition, phosphorylation of the Dectin-1 hem-immunoreceptor tyrosine-based activation motif (hemITAM) was essential for the Dectin-1-triggered response to NTHI in A549 cells. In conclusion, in human airways, epithelium-expressed Dectin-1 may play a significant role in generating an NTHI-mediated, proinflammatory immune response.

Antimicrobial Properties of Honey

Honey has been widely accepted as food and medicine by all generations, traditions, and civilizations, both ancient and modern. For at least 2700 years, honey has been used by humans to treat a variety of ailments through topical application, but only recently have the antiseptic and antimicrobial properties of honey been discovered. Honey has been reported to be effective in a number of human pathologies. Clinical studies have demonstrated that application of honey to severely infected cutaneous wounds rapidly clears infection from the wound and improves tissue healing. A large number of in vitro and limited clinical studies have confirmed the broad-spectrum antimicrobial (antibacterial, antifungal, antiviral, and antimycobacterial) properties of honey, which may be attributed to the acidity (low pH), osmotic effect, high sugar concentration, presence of bacteriostatic and bactericidal factors (hydrogen peroxide, antioxidants, lysozyme, polyphenols, phenolic acids, flavonoids, methylglyoxal, and bee peptides), and increase in cytokine release, and to immune modulating and anti-inflammatory properties of honey; the antimicrobial action involves several mechanisms. Despite a large amount of data confirming the antimicrobial activity of honey, there are no studies that support the systemic use of honey as an antibacterial agent.

Evidence of mononuclear cell preactivation in the fasting state in polycystic ovary syndrome

We evaluated mononuclear cell (MNC) preactivation in women with polycystic ovary syndrome (PCOS) by examining the effect of in vitro lipopolysaccharide (LPS) exposure on cytokine release in the fasting state. Twenty women with PCOS (10 lean, 10 obese) and 20 weight-matched controls (10 lean, 10 obese) volunteered for study participation. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release was measured from mononuclear cells isolated from fasting blood samples and cultured in the presence and absence of LPS. Plasma IL-6 was measured from the same fasting blood samples. Insulin sensitivity was derived from an oral glucose tolerance test using the Matsuda index, and truncal fat was measured by dual-energy x-ray absorptiometry. The percent change from baseline in TNF-α and IL-6 release from MNC following LPS exposure was increased (P < .04) in lean and obese women with PCOS and obese controls compared with lean controls. Plasma IL-6 was increased (P < .02) in obese women with PCOS compared with lean women with PCOS, which in turn was increased (P < .02) compared with lean controls. The MNC-derived TNF-α and IL-6 responses from MNCs were negatively correlated with insulin sensitivity (P < .03) and positively correlated with testosterone (P < .03) and androstenedione (P < .006) for the combined groups. Plasma IL-6 was positively correlated with percentage truncal fat (P < .008). In PCOS, increased cytokine release from MNCs following LPS exposure in the fasting state reveals the presence of MNC preactivation. Importantly, this phenomenon is independent of obesity and may contribute to the development of insulin resistance and hyperandrogenism in PCOS. In contrast, the source of plasma IL-6 elevations in PCOS may be excess adiposity.

Novel CD3‐specific antibody induces immunosuppression via impaired phosphorylation of LAT and PLCγ1 following T‐cell stimulation

The activation of T cells is known to be accompanied by the temporary downmodulation of the TCR/CD3 complex on the cell surface. Here, we established a novel monoclonal antibody, Dow2, that temporarily induces downmodulation of the TCR/CD3 complex in mouse CD4(+) T cells without activating T cells. Dow2 recognized the determinant on CD3ε; however, differences were observed in the binding mode between Dow2 and the agonistic anti-CD3ε Ab, 145-2C11. An injection of Dow2 in vivo resulted in T-cell anergy, and prolonged the survival of cardiac allografts without a marked increase in cytokine release. The phosphorylated forms of the signaling proteins PLC-γ1 and LAT in Dow2-induced anergic T cells were markedly decreased upon stimulation. However, the levels of phosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells could be rescued in the presence of the proteasome inhibitor MG-132. These results suggest that proteasome-mediated degradation is involved in hypophosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells. The novel CD3-specific Ab, Dow2, may provide us with a unique tool for inducing immunosuppression.

Prior TLR5 induction in human T cells results in a transient potentiation of subsequent TCR-induced cytokine production

Activation of TLRs by components required for pathogen viability results in increased inflammation and an enhanced immune response to infection. Unlike their effects on other immune cells, TLR activation in the absence of T cell antigen receptor (TCR) induction has little effect on T cell activity. Instead, the simultaneous induction of TLR and TCR results in increased cytokine release compared to TCR treatment alone. Thus, the current model states that TLRs alter T cell function only if activated at the same time as the TCR. In this study, we tested the novel hypothesis that prior TLR induction can also alter TCR-mediated functions. We found that human T cells responded to ligands for TLR2 and TLR5. However, only prior TLR5 induction potentiated subsequent TCR-mediated cytokine production in human T cells. This response required at least 24h of TLR5 induction and lasted for approximately 24–36h after removal of a TLR5 ligand. Interestingly, prior TLR5 induction enhanced TCR-mediated activation of Akt without increasing Lck, LAT or ERK kinase phosphorylation. Together, our studies show that TLR5 induction leads to a transient increase in the sensitivity of T cells to TCR stimulation by selectively enhancing TCR-mediated Akt function, highlighting that timeframe when TLR5 can potentiate TCR-induced downstream functions are significantly longer that previously appreciated.