Significant Cytokine Release Research Articles

Abstract B249: Prolonged cytotoxic activity induced by anti-PSMA x anti-CD3 ADAPTIR™ molecule in the absence of significant cytokine release.

Abstract Background: Treatment of metastatic, castrate-resistant prostate cancer (CRPC) remains a highly unmet medical need. We have developed a humanized bispecific ADAPTIR™ (modular protein technology) molecule, ES414, which redirects T-cell cytotoxicity against cells expressing PSMA (Prostate Specific Membrane Antigen), a prostate cancer antigen. In previous preclinical studies, ES414 has been shown to induce cytotoxicity against prostate cancer cells in vitro when measured by target cell lysis and in vivo in multiple mouse xenograft models. ES414 was also previously shown to be well-tolerated in humanized mice and non-human primates. Here, we examine the effects of exposure to ES414 over several days on T-cell function in the presence of PSMA+ target cells.Materials and Methods: Long term cytotoxic activity induced by ES414 against PSMA(+) cell lines in the presence of purified human T cells was followed in vitro by use of high content microscopy. Cytotoxic activity was determined by incorporation of a label such as propidium iodide or 7-aminoactinomycin D. Binding to subsets of peripheral blood mononuclear cells (PBMC) and Fc(gamma) receptor-expressing cell lines was assessed by multi-color flow cytometry. T cells were assessed for activation and proliferation in a similar setting using multi-color flow cytometry. Cytokine release was measured at multiple timepoints using multiplex immunoassays. Results: ES414 selectively bound to T cells, and did not bind to other PBMC subsets nor to Fc (gamma) receptor expressing cell lines. T cells activated by ES414 in the presence of target cells rapidly induced target cell lysis within 4 hours and continued serial lysis for more than 48 hours. Target cell lysis occurred at low effector-to-target cell ratios. In the presence of target cells, ES414 also induced T-cell activation, as measured by upregulation of activation markers, and proliferation, but induced limited levels of cytokine secretion in comparison to control molecules. Conclusions: These preclinical in vitro studies show that ES414 selectively engages T cells, targets T cells towards tumor cells, and induces T-cell activation, with limited cytokine release. Limited cytokine release in the presence of redirected T-cell cytotoxicity is a desirable feature that warrants further investigation of ES414 as a potential therapeutic for CRPC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B249. Citation Format: Gabriela Hernandez-Hoyos, Toddy Sewell, Padma Ravikumar, Megan Aguilar, John Kumer, David Bienvenue, Catherine J. McMahan, Jane Gross, John W. Blankenship. Prolonged cytotoxic activity induced by anti-PSMA x anti-CD3 ADAPTIR™ molecule in the absence of significant cytokine release. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B249.

Anti‐CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model

We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local. A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3(+) human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3(+) T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of pNICC. Humanized mice were transplanted with either control-transduced pNICC or diliximab-transduced pNICC and human T cells within grafts and spleens were enumerated by flow cytometry. Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3(+) T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced pNICC afforded depletion of CD3(+) T cells at the graft site leaving the peripheral immune system intact. Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression.

Abstract P6-11-06: A phase Ib study of LCL161, an oral inhibitor of apoptosis (IAP) antagonist, in combination with weekly paclitaxel in patients with advanced solid tumors

Abstract Background: Impaired apoptosis is a common feature of cancer cells and may contribute to chemoresistance. LCL161 is an oral small molecule antagonist of Inhibitor of Apoptosis Proteins (IAPs) that sensitizes a subset of tumors from diverse lineages to treatment with cytotoxic therapies, including paclitaxel. Multiple breast cancer models are sensitive to LCL161 as a single agent and LCL161 acts synergistically with paclitaxel in these models. A phase I study established an LCL161 dose of 1800 mg once weekly as well tolerated, with strong evidence of pharmacodynamic activity at doses ≥320 mg. This ongoing phase Ib study defines the dose limiting toxicities (DLTs), maximum tolerated dose (MTD), safety, and pharmacokinetics (PK) of LCL161 in combination with weekly paclitaxel. Methods: Patients with advanced/metastatic solid tumors were treated with paclitaxel 80 mg/m2 each week followed by escalating doses of LCL161 administered once weekly immediately following paclitaxel. PK and biomarker sampling was performed. Results: Thirty-two patients have received LCL161 doses of 600 mg (n = 3), 1200 mg (n = 5), 1500 mg (n = 4), and 1800 mg (n = 20). The most frequent adverse events considered LCL161-related included diarrhea (n = 11; 1 Grade 3), nausea (n = 8), fatigue (n = 7; 2 Grade 3), peripheral neuropathy (n = 6; 1 Grade 3), vomiting (n = 6), decreased appetite (n = 5), alopecia (n = 4), and anemia (n = 4). The principal DLTs were neutropenia, fatigue, and neuropathy. Significant cytokine release syndrome, the DLT of single-agent LCL161, has not been observed likely due to the use of dexamethasone as a premedication. No PK interaction between LCL161 and paclitaxel was observed. RECIST partial responses have been observed in 4 patients with diverse tumor types, including breast cancer. Preliminary antitumor activity in the expansion cohort with breast cancer patients will be presented. Discussion: LCL161 and paclitaxel combination therapy is well tolerated, with manageable toxicities and no evidence of a PK interaction that might interfere with the activity of either agent. Enrollment of additional patients with breast and ovarian cancer into an expansion cohort is ongoing, utilizing an approach to identify those more likely to respond to treatment with IAP antagonists. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-06.

Cytokine milieu in renal cavities of immunocompetent mice in response to intravenous challenge of Aspergillus flavus leading to aspergillosis

Investigations in mice have demonstrated that Aspergillus flavus is more virulent than all other Aspergillus species except A. tamari. However, there is a complete lack of information on the immune responses elicited by A. flavus in systemic model. This communication reports the progression of infection and cytokine profile in BALB/c mice in response to intravenous challenge of A. flavus. The pathogenesis of infection was evaluated morphologically and by the analysis of Colony Forming Units (CFUs) in kidney homogenates. The kinetics of regulated cytokines was determined in kidneys by cytokine-specific murine ELISA. During the initial phase of infection the rate of clearance of A. flavus was high, most likely through recruited neutrophils and the resident renal macrophages with concurrent significant release of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-12/IL-23p40, IL-6) indicating antifungal innate immune response to be active at the site. However, at 24h PI there was a significant rise of IL-17 and IL-23 suggesting the activation of IL-17/IL-23 axis of inflammation resulting in rise of CFU. The lack of significant induction in the anti-inflammatory cytokines like IL-4 and IL-10 confirmed the absence of Th2 type of response. In the late phase, after 3days post-infection, there was a rise in the number of pathogen in the kidneys as determined by histopathology and CFU counts. The A. flavus hyphae were evident in the renal pelvis and ureter and we propose the production of blastoconidia by metamorphosed hyphae.

A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans

The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats.We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation.We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ – a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine responses to the individual mAbs, in the concentration-response relationships and the prominent cytokine signatures for individual mAbs in the two formats reflect diverging mechanisms of cytokine release and different levels of dependency on high density coating even for two anti-CD28 super-agonistic antibodies. These results clearly show that one generic approach to assessment of cytokine release using in vitro assays is not sufficient, but rather the choice of the method, i.e. applying the whole blood assay or the PBMC assay needs to be well considered depending on the target characteristics and the mechanistic features of the therapeutic mAbs being evaluated.

Abstract 4625: IMGN529, a CD37-directed antibody-drug conjugate for the treatment of B-cell malignancies, has a favorable in vitro safety profile

Abstract The B-cell surface antigen CD37 is an attractive target for antibody-drug conjugate (ADC) mediated therapies, as it is widely expressed on malignant B cells in NHL and CLL. In normal human tissues, high CD37 expression is mainly restricted to B cells, with substantially lower expression seen in T cells, NK cells, monocytes and granulocytes. The CD37-targeting K7153A antibody was selected from a panel of antibodies for its potent pro-apoptotic, CDC and ADCC activity against B-cell lymphoma cell lines in vitro. The K7153A antibody was conjugated to the maytansinoid DM1 via the SMCC linker to yield the IMGN529 conjugate, which retained the intrinsic functions of the antibody and demonstrated enhanced in vitro and in vivo activity through targeted delivery of its payload. To complement preclinical in vivo toxicity studies with IMGN529 in animal models, we conducted extensive in vitro safety studies, with the aim of identifying any potential negative effects on normal human blood cells. Depletion of B cells, T cells and monocytes from peripheral blood cells by K7153A, IMGN529, the anti-CD20 antibody rituximab or the anti-CD52 antibody alemtuzumab was determined by flow cytometry. Proliferation of peripheral blood mononuclear cells (PBMCs) in response to K7153A or IMGN529 was evaluated by CFDA staining. Cytokine release of TNF-α, IFN-γ, IL-2, IL-4, IL-6 and IL-10 by normal human PBMCs was measured by ELISpot and cytometric bead array following exposure to compounds in both soluble and immobilized form. K7153A and IMGN529 specifically depleted B cells in a dose-dependent manner, but had no effect on T cells or monocytes, consistent with the much lower CD37 expression levels in these cells. K7153A and IMGN529 were more potent than rituximab and more specific than alemtuzumab, which depleted T cells and monocytes in addition to B cells. Incubation with IMGN529 did not increase the percentage of proliferating cells, while the anti-CD3 control antibody was able to strongly stimulate T cell proliferation. Treatment with K7153A or IMGN529 did not specifically induce cytokine release as compared to non-binding antibody controls, while treatment with the anti-CD3 control antibody resulted in significant cytokine release for all donors. Rituximab treatment caused release of low levels of IFN-γ and TNF in some donors. In contrast, treatment with alemtuzumab resulted in release of IFN-γ, TNF-α and IL-6 for most donors consistent with its potential for cytokine release in patients. In conclusion, the CD37-directed IMGN529 maytansinoid conjugate demonstrated potent in vitro and in vivo efficacy combined with a favorable in vitro safety profile with respect to cytokine release. Our analysis confirmed that the primary target cells of IMGN529 are B cells. Together, these data support the clinical development of this ADC for the treatment of B-cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4625. doi:1538-7445.AM2012-4625

The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells

Carbon nanotubes (CNT) are high aspect ratio nanoparticles with diameters in the nanometre range but lengths extending up to hundreds of microns. The structural similarities between CNT and asbestos have raised concern that they may pose a similar inhalation hazard. Recently CNT have been shown to elicit a length-dependent, asbestos-like inflammatory response in the pleural cavity of mice, where long fibres caused inflammation but short fibres did not. However the cellular mechanisms governing this response have yet to be elucidated. This study examined the in vitro effects of a range of CNT for their ability to stimulate the release of the acute phase cytokines; IL-1β, TNFα, IL-6 and the chemokine, IL-8 from both Met5a mesothelial cells and THP-1 macrophages. Results showed that direct exposure to CNT resulted in significant cytokine release from the macrophages but not mesothelial cells. This pro-inflammatory response was length dependent but modest and was shown to be a result of frustrated phagocytosis. Furthermore the indirect actions of the CNT were examined by treating the mesothelial cells with conditioned media from CNT-treated macrophages. This resulted in a dramatic amplification of the cytokine release from the mesothelial cells, a response which could be attenuated by inhibition of phagocytosis during the initial macrophage CNT treatments. We therefore hypothesise that long fibres elicit an inflammatory response in the pleural cavity via frustrated phagocytosis in pleural macrophages. The activated macrophages then stimulate an amplified pro-inflammatory cytokine response from the adjacent pleural mesothelial cells. This mechanism for producing a pro-inflammatory environment in the pleural space exposed to long CNT has implications for the general understanding of fibre-related pleural disease and design of safe nanofibres.

Glutaraldehyde treatment elicits toxic response compared to decellularization in bovine pericardium

Glutaraldehyde-stabilized bovine pericardium is used for clinical application since 1970s because of its desirable features such as less immunogenicity and acceptable durability. However, a propensity for calcification is reported on account of glutaraldehyde treatment. In this study, commercially available glutaraldehyde cross-linked bovine pericardium was evaluated for its in vitro cytotoxic effect, macrophage activation, and in vivo toxic response in comparison to decellularized bovine pericardium. Glutaraldehyde-treated bovine pericardium and its extract were observed to be cytotoxic and it also caused significant inflammatory cytokine release from activated macrophages. Significant antibody response, calcification response, necrotic, and inflammatory response were noticed in glutaraldehyde-treated bovine pericardium in comparison to decellularized bovine pericardium in a rat subcutaneous implantation model. Glutaraldehyde-treated bovine pericardium also failed in acute systemic toxicity testing and intracutaneous irritation testing as per ISO 10993. With respect to healing and implant remodeling, total lack of host tissue incorporation and angiogenesis was noticed in glutaraldehyde-treated bovine pericardium compared to excellent host fibroblast incorporation and angiogenesis within the implant in decellularized bovine pericardium. In conclusion, using in vitro and in vivo techniques, this study has demonstrated that glutaraldehyde-treated bovine pericardium elicits toxic response compared to decellularized bovine pericardium which is not congenial for long-term implant performance.

Nonclinical Evaluation of GMA161—An Antihuman CD16 (FcγRIII) Monoclonal Antibody for Treatment of Autoimmune Disorders in CD16 Transgenic Mice

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16). As CD16 expression and sequence have high species specificity, preclinical assessments were conducted in mice transgenic for both isoforms of human CD16, CD16A, and CD16B. This transgenic mouse model was useful in transitioning into phase I clinical trials, as it generated positive efficacy data in a relevant disease model and an acceptable single-dose safety profile. However, when GMA161 or its murine parent 3G8 were dosed repeatedly in transgenic mice having both human CD16 isoforms, severe reactions were observed that were not associated with significant cytokine release, nor were they alleviated by antihistamine administration. Prophylactic dosing with an inhibitor of platelet-activating factor (PAF), however, completely eliminated all signs of hypersensitivity. These findings suggest that (1) GMA161 elicits a reaction that is target dependent, (2) immunogenicity and similar adverse reactions were observed with a murine version of the antibody, and (3) the reaction is driven by the atypical hypersensitivity pathway mediated by PAF.

Cross-regulation of innate response in human PBMC by an enveloped RNA virus and LPS (67.11)

Abstract The innate response to polymicrobial infections and their pathways are poorly understood. In this work, we used Sindbis virus (SIN), an enveloped RNA virus and LPS as inducers of innate response. PBMC from healthy volunteers were exposed to SIN Toto 1101, in the presence and absence of E.coli LPS. We observed absence of new virus release, SIN/GFP reporter expression and viral RNA synthesis suggesting poor replication of SIN in human PBMC. However, significant lymphocyte activation, cytokine and chemokine release were seen upon exposure of PBMC to SIN. Analysis of lymphocyte activation markers showed that, LPS was a better inducer of CD69 expression in CD4, CD8, and CD19 lymphocytes than SIN. Increased secretion of IL-12p40, IL-1β, TNF-α, IL-6, IL-18, IL-10, IL-8, RANTES, MCP-1, MIP1-α and MIP1-β was induced by LPS whereas, SIN induced IL-6, IFN-α, MCP-1, MIP1-α, MIP1-β, I-TAC and IP-10 secretion. SIN downregulated LPS induced IL-12p40 secretion, whereas LPS inhibited virus induced IFN-α and IP-10. SIN also suppressed both the spontaneous and LPS induced secretion of IFN-α, IL-17, IL-13 and SLC from PBMC. To analyze involvement of specific PBMC subsets and associated signaling events, ELISPOT and phosphoflow are used. The absence and presence of antagonism between SIN and LPS for lymphocyte activation and secretion of select cytokines and chemokines may help in understanding pathogenesis of mixed infections, and find applications in vaccine development and virotherapy.

Calcitonin Gene-Related Peptide-Mediated Enhancement of Purinergic Neuron/Glia Communication by the Algogenic Factor Bradykinin in Mouse Trigeminal Ganglia from Wild-Type and R192Q Cav2.1 Knock-In Mice: Implications for Basic Mechanisms of Migraine Pain

Within the trigeminal ganglion, crosstalk between neurons and satellite glial cells (SGCs) contributes to neuronal sensitization and transduction of painful stimuli, including migraine pain, at least partly through activation of purinergic receptor mechanisms. We previously showed that the algogenic mediator bradykinin (BK) potentiates purinergic P2Y receptors on SGCs in primary trigeminal cultures. Our present study investigated the molecular basis of this effect in wild-type (WT) mice and Ca(V)2.1 α1 R192Q mutant knock-in (KI) mice expressing a human mutation causing familial hemiplegic migraine type 1. Single-cell calcium imaging of WT cultures revealed functional BK receptors in neurons only, suggesting a paracrine action by BK to release a soluble mediator responsible for its effects on SGCs. We identified this mediator as the neuropeptide calcitonin gene-related peptide (CGRP), whose levels were markedly increased by BK, while the CGRP antagonist CGRP(8-37) and the anti-migraine drug sumatriptan inhibited BK actions. Unlike CGRP, BK was ineffective in neuron-free SGC cultures, confirming the CGRP neuronal source. P2Y receptor potentiation induced by CGRP in SGCs was mediated via activation of the extracellular signal-regulated kinase 1/2 pathways, and after exposure to CGRP, a significant release of several cytokines was detected. Interestingly, both basal and BK-stimulated CGRP release was higher in KI mouse cultures, where BK significantly upregulated the number of SGCs showing functional UTP-sensitive P2Y receptors. Our findings suggest that P2Y receptors on glial cells might be considered as novel players in the cellular processes underlying migraine pathophysiology and might represent new targets for the development of innovative therapeutic agents against migraine pain.

S47 RAGE-mediated cytokine release from leukocytes: implications for systemic inflammation

IntroductionSystemic inflammatory response syndrome (SIRS) is the physiological and biochemical changes that result from an inflammation of the body, which commonly occurs in patients after cardiac surgery necessitating cardiopulmonary bypass...

Paracrine effect of canine allogenic umbilical cord blood-derived mesenchymal stromal cells mixed with beta-tricalcium phosphate on bone regeneration in ectopic implantations

The aim of this study was to evaluate the paracrine effects of canine umbilical cord blood (cUCB) mesenchymal stromal cells (MSC) mixed with beta-tricalcium phosphate (beta-TCP) on bone regeneration in ectopic implantation. beta-TCP mixed with cUCB MSC (UCB-MSC group), cell lysates (cell lysate group) or a control (control group) were respectively implanted in a subcutaneous pouches in the back of beagle dogs . The implants were harvested 1, 4, 7, 14, 28, 56, 84 days after implantation. Histological findings and stain analyzes of tartrate-resistant acid phosphatase (TRACP) and assays of alkaline phosphatase (ALP) and TRACP were evaluated. The mRNA expression levels of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) were analyzed using semi-quantitative reverse transcription - polymerase chain reactions (RT-PCR). An enzyme-linked immunosorbent assay (ELISA) was used to confirm the protein expression levels of IL-6, COX-2, VEGF and TGF-beta. TRACP-positive cells were observed in all groups 7 days after implantation. ALP and TRACP activities in the UCB-MSC group 84 days after implantation were significantly higher than those of the control (P>0.05). Histologic findings after 84 days showed that the osteoid matrix area in the UCB-MSC group was significantly larger than that of the control (P<0.05). The mRNAs levels of IL-1, IL-6 and VEGF in UCB-MSC and cell lysate groups on day 1 were up-regulated compared with the control. The protein levels of IL-6 and VEGF in the UCB-MSC group at day 1 were significantly higher than that of the other groups (P<0.05). It is suggested that a significant release of cytokines by cUCB MSC, 1 day following implantation, could enhance bone regeneration.

Skeletal Muscle as an Endocrine organ; the effect of TNF‐alpha

During chronic inflammation muscle is exposed to inflammatory cytokines; a key player in this is TNF‐α. Muscle has protective mechanisms to adapt to stress, via further expression of cytokines and Heat shock proteins (HSPs), which can be released into the extracellular environment and then involved in apocrine or paracrine signalling pathways. C2C12 myotubes in vitro treated with TNF‐α (25ng/ml) showed significant up‐regulation of 7 pro‐inflammatory genes by qPCR array analysis. Significant release of cytokines (IL‐6, MCP‐1, KC, IP‐10 &amp; RANTES) was seen after 3 &amp; 6 hours. In addition, TNF‐α up‐regulated intracellular levels of HSP60 &amp; 70, with specific release of HSP60 at 3 hours. Treatment of C2C12 myotubes with recombinant HSP60 and 70 induced significant release of MCP‐1 and RANTES at 3 hours. Adult and old C57Bl6 mice treated with TNF‐α (7.5μg/kg) i.v. were subject to microdialysis perfusion of the gastrocnemius muscle to measure direct cytokine release from whole muscle. Adult mice showed a significant release of IL‐1β &amp; MCP‐1, 1 hour post TNF‐α; old mice showed elevated basal levels of IL‐1β &amp; MCP‐1, with a blunted response to TNF‐α. Data show TNF‐α induces an immune and stress response in muscle in vitro and HSPs have a paracrine signalling effect inducing release of cytokines. Whole muscle in vivo releases cytokines in response to TNF‐α treatment which is blunted in old mice. Supported by University of Liverpool.

Hypercapnic acidosis in ventilator-induced lung injury

Permissive hypercapnia is established in lung injury management. Therapeutic hypercapnia causes benefit or harm, depending on the context. Ventilator-associated lung injury has a wide spectrum of candidate mechanisms, affording multiple opportunities for intervention such as hypercapnia to exert benefit or harm. To confirm (1) that hypercapnia attenuates in vivo ventilator-induced lung injury (VILI); (2) biological plausibility of such protection (e.g., dose-response, time series, inflammatory profile); and (3) that the associated biochemical events are consistently beneficial. A mouse model of VILI was established in vivo. Injurious ventilation was established, hypercapnia applied and markers of inflammation measured. Lung injury was quantified by gas exchange, elastance, microvascular leak, histology and levels of cytokines and eicosanoids, cyclooxygenase and tissue nitrotyrosine. Injurious ventilation caused significant lung injury (mechanics, microvascular leak, histology) and release of inflammatory cytokines, chemokines and eicosanoids. Hypercapnia attenuated these responses, with dose-response and time-dependent effects. No adverse effects of hypercapnia were observed in controls. Hypercapnia suppressed the transcription (mRNA) and translation (protein) of the major inducible prostanoid-generating enzyme (COX-2), but the effects on the downstream eicosanoids were modest. However, hypercapnia significantly increased lung tissue nitrotyrosine-at PaCO(2) levels that were protective. Hypercapnia provided consistent and biologically plausible in vivo protection against VILI, but elevated lung tissue levels of nitro-tyrosine as previously described in sepsis. Clinicians and those designing clinical trials need to be aware of the potential for detrimental effects when using hypercapnia in order to balance benefits versus harm with this approach.

Cytokines are produced locally by myocytes in rat skeletal muscle during endotoxemia

Cytokines act as chemical mediators during the inflammatory process. Measurements of cytokine levels in tissue have previously been performed in homogenized tissue, but the true concentrations in native interstitial fluid (ISF), i.e., the compartment where cytokines exert their biologically active role, have remained unknown. The role of skeletal muscle myocytes as a source for cytokines during endotoxemia was explored by collecting muscle ISF using a wick method, and the levels of 14 cytokines in ISF and plasma were related to the corresponding changes in mRNA levels to reveal any potential discrepancies between gene expression and protein release of cytokines to ISF. The majority of investigated cytokines were elevated in muscle ISF during endotoxemia, and an analysis of cytokine mRNA levels revealed consistency between gene expression and protein release. The elevated cytokine level in ISF, in addition to elevated gene expression in muscle, indicated a significant local production and release of several proinflammatory cytokines and chemokines within skeletal muscle tissue during endotoxemia. Immunohistochemistry revealed that myocytes constituted a significant source of IL-1beta and TNF-alpha production during endotoxemia, whereas the contribution from inflammatory cells i.e., leukocytes, was found to be less significant. Muscle cells apparently constitute an important source of several different cytokines during endotoxemia, governing the level in the muscle microenvironment, and are likely to contribute significantly to cytokine levels in plasma.

Oral viral infections of adults

The field of virology has advanced greatly over the past two decades, mainly because of the introduction of sophisticated molecular tools, such as monoclonal antibodies, polymerase chain reaction (PCR)-based amplification, DNA sequencing, DNA and protein microarray chip assays, and rapid diagnostic tests. These technologies have been the driving force in the identification of viral bodies, proteins and nucleic acids in body fluids and tissue samples, and in determining the host response to viral infections. The 5th (2007) edition of Fields Virology provides an indepth description of viral methodology and medical virology, and is an important source of reference for this review (118). Viruses replicate only when present within eukaryotic (animals, plants, protists and fungi) or prokaryotic (bacteria and archaea) cells, not on their own. The extracellular virion particle ranges in size from 20 to 300 nm and consists of either DNA or RNA contained within a protective protein capsid. Some viruses have an additional envelope comprising a lipid bilayer derived from the outer cellular membrane, the internal nuclear membrane, or the endoplasmic reticulum membrane of the infected cell. Taxonomically, viruses are classified according to the presence of DNA or RNA, single-stranded or double-stranded nucleic acid, and an enveloped or nonenveloped nucleocapsid. Additional taxonomical criteria include mode of replication, type of host, capsid shape, immunological properties and disease association. The host recognizes and reacts to the infecting virus by innate and adaptive immune responses. Important cells of the innate immune system include macrophages, dendritic cells and natural killer cells. Viruses activate inflammatory cell types to release antiviral cytokines and cytotoxic agents, and to induce lymphocyte-mediated adaptive immunity. A significant cytokine release is stimulated through activation of the tumor necrosis factor-a receptor ⁄ nuclear factor-jB ⁄ extracellular signal-regulated kinase pathway (49). Virally derived proteins, which are presented by major histocompatibility complex molecules on the surface of infected cells, serve as epitopes for specific host immune cells. Nonenveloped viruses are mainly controlled by the humoral adaptive immunity. Enveloped viruses are controlled by the cellular immunity through the action of natural killer cells and cytotoxic CD8 T lymphocytes. After recognizing viral surface antigens on infected cells, cytotoxic T lymphocytes inhibit virus replication by cytolytic killing and by releasing interferons, chemokines, tumor necrosis factor-a or other pro-inflammatory mediators. Viral disease may be a direct result of cell destruction or a secondary consequence of host immune reactions against viral proteins. Proinflammatory cytokines play important roles in the antiviral immune response, but interleukin-1b, interleukin-6 and tumor necrosis factor-a cytokines can also contribute to disease manifestation. The host usually performs a delicate balancing act between promoting antiviral cytokine responses and limiting the amount of tissue damage. To counteract the immune attack, viruses employ sophisticated immunoevasive strategies to suppress antiviral host responses. For example, some viruses produce proteins that alter the major histocompatibility complex and hence the exposure of viral proteins on the surface of infected cells. Viruses may encode viral homologs of host cytokines and decoy receptors capable of binding and neutralizing host-derived cytokines. A rapid rate of mutation in critical viral genes can help viruses avoid the adaptive host defense. Other viral gene products inhibit apoptosis, which facilitates a prolonged state of replication of infected cells and spread of the virus. Viral diseases of the oral mucosa and the perioral region are often encountered in dental practice, but have received only limited research interest. Viruses are important ulcerogenic and tumorigenic agents of the human mouth. The finding of an abundance of mammalian viruses in periodontitis lesions may suggest a role for viruses in more oral diseases than

Dose-Dependent Effects of Ethanol and E. Coli on Gut Permeability and Cytokine Production

The gut may prime inflammatory responses following shock/trauma insults. Ethanol (EtOH) use is common in trauma patients and may impair intestinal barrier function. We compared varying concentrations of EtOH on proinflammatory cytokine production of Caco2 cell monolayers and the resultant changes in barrier function. We hypothesized that even low concentrations of EtOH would cause significant cytokine release and barrier dysfunction in vitro. Confluent Caco2 cell monolayers were grown in a two-chamber culture system and exposed to varying concentrations of EtOH (0.1%, 0.5%, 1.0%, 1.5%, and 2.0%) with/without Escherichia coli C-25 (EC). Supernatants were collected and TNF and IL6 quantified by ELISA (pg/mL). Monolayer integrity was assessed by apoptosis and permeability measurements. Caco2 production of TNF-alpha increased in a dose-dependent manner when incubated with increasing concentrations of EtoH. A synergistic effect was seen when E. coli was added to the apical chamber. A similar result was seen with the production of IL-6. A dose-dependent effect was also noted with EtOH with or without E. coli on apoptosis and permeability measurements. In addition to alterations in gut permeability, increasing concentrations of ethanol have a synergistic effect with E. coli on Caco2 production of proinflammatory cytokines TNF and IL-6. The creation of a proinflammatory cytokine milieu with an altered barrier integrity may be a mechanism by which ethanol may increase septic complications in the injured patient.

Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells

BackgroundCigarette smoke mediated oxidative stress and inflammatory events in the airway and alveolar epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Previously, individual cell lines were used to assess the oxidative and proinflammatory effects of cigarette smoke with confounding results. In this study, a panel of human and rodent transformed epithelial cell lines were used to determine the effects of cigarette smoke extract (CSE) on oxidative stress markers, cell toxicity and proinflammatory cytokine release and compared the effects with that of primary human small airway epithelial cells (SAEC).MethodsPrimary human SAEC, transformed human (A549, H1299, H441), and rodent (murine MLE-15, rat L2) alveolar epithelial cells were treated with different concentrations of CSE (0.2–10%) ranging from 20 min to 24 hr. Cytotoxicity was assessed by lactate dehydrogenase release assay, trypan blue exclusion method and double staining with acridine orange and ethidium bromide. Glutathione concentration was measured by enzymatic recycling assay and 4-hydroxy-2-nonenal levels by using lipid peroxidation assay kit. The levels of proinflammatory cytokines (e.g. IL-8 and IL-6) were measured by ELISA. Nuclear translocation of the transcription factor, NF-κB was assessed by immunocytochemistry and immunoblotting.ResultsCigarette smoke extract dose-dependently depleted glutathione concentration, increased 4-hydroxy-2-nonenal (4-HNE) levels, and caused necrosis in the transformed cell lines as well as in SAEC. None of the transformed cell lines showed any significant release of cytokines in response to CSE. CSE, however, induced IL-8 and IL-6 release in primary cell lines in a dose-dependent manner, which was associated with the nuclear translocation of NF-κB in SAEC.ConclusionThis study suggests that primary, but not transformed, lung epithelial cells are an appropriate model to study the inflammatory mechanisms in response to cigarette smoke.

Human Tumor-Released Microvesicles Promote the Differentiation of Myeloid Cells with Transforming Growth Factor-β–Mediated Suppressive Activity on T Lymphocytes

Human tumors constitutively release endosome-derived microvesicles, transporting a broad array of biologically active molecules with potential modulatory effects on different immune cells. Here, we report the first evidence that tumor-released microvesicles alter myeloid cell function by impairing monocyte differentiation into dendritic cells and promoting the generation of a myeloid immunosuppressive cell subset. CD14+ monocytes isolated from healthy donors and differentiated with interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in the presence of tumor-derived microvesicles turned into HLA-DR(-/low) cells, retaining CD14 expression and failing to up-regulate costimulatory molecules, such as CD80 and CD86. These phenotypic changes were paralleled by a significant release of different cytokines, including IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and a dose-dependent suppressive activity on activated T-cell-proliferation and cytolytic functions, which could be reversed by anti-TGF-beta-neutralizing antibodies. Microvesicles isolated from plasma of advanced melanoma patients, but not from healthy donors, mediated comparable effects on CD14+ monocytes, skewing their differentiation toward CD14+HLA-DR-/low cells with TGF-beta-mediated suppressive activity on T-cell-functions. Interestingly, a subset of TGF-beta-secreting CD14+HLA-DR- cells mediating suppressive activity on T lymphocytes was found to be significantly expanded in peripheral blood of melanoma patients compared with healthy donors. These data suggest the development in cancer patients of an immunosuppressive circuit by which tumors promote the generation of suppressive myeloid cells through the release of circulating microvesicles and without the need for cell-to-cell contact. Therapeutic interventions on the crucial steps of this pathway may contribute to restore tumor/immune system interactions favoring T-cell-mediated control of tumor growth in cancer patients.