Minimization of the E. coli ribosome, aided and optimized by community science.

The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome.

Intranasal Delivery of Recombinant S100A8 Protein Delays Lung Cancer Growth by Remodeling the Lung Immune Microenvironment.

Lung cancer is the leading cause of cancer-related death worldwide. Increasing evidence indicates a critical role for chronic inflammation in lung carcinogenesis. S100A8 is a protein with reported pro- and anti-inflammatory functions. It is highly expressed in myeloid-derived suppressor cells (MDSC) that accumulate in the tumor microenvironment and abrogate effective anti-cancer immune responses. Mechanisms of MDSC-mediated immunosuppression include production of reactive oxygen species and nitric oxide, and depletion of L-arginine required for T cell function. Although S100A8 is expressed in MDSC, its role in the lung tumor microenvironment is largely unknown. To address this, mouse recombinant S100A8 was repeatedly administered intranasally to mice bearing orthotopic lung cancers. S100A8 treatment prolonged survival from 19 days to 28 days (p < 0.001). At midpoint of survival, whole lungs and bronchoalveolar lavage fluid (BALF) were collected and relevant genes/proteins measured. We found that S100A8 significantly lowered expression of cytokine genes and proteins that promote expansion and activation of MDSC in lungs and BALF from cancer-bearing mice. Moreover, S100A8 enhanced activities of antioxidant enzymes and suppressed production of nitrite to create a lung microenvironment conducive to cytotoxic lymphocyte expansion and function. In support of this, we found decreased MDSC numbers, and increased numbers of CD4+ T cells and natural killer T (NK-T) cells in lungs from cancer-bearing mice treated with S100A8. Ex-vivo treatment of splenocytes with S100A8 protein activated NK cells. Our results indicate that treatment with S100A8 may favourably modify the lung microenvironment to promote an effective immune response in lungs, thereby representing a new strategy that could complement current immunotherapies in lung cancer.

Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone tumours.

Chimeric Antigen Receptor (CAR) T-cell therapy, as an approved treatment option for patients with B cell malignancies, demonstrates that genetic modification of autologous immune cells is an effective anti-cancer regimen. Erythropoietin-producing Hepatocellular receptor tyrosine kinase class A2 (EphA2) is a tumour associated antigen expressed on a range of sarcomas, including paediatric osteosarcoma (OS) and Ewing sarcoma (ES). We tested human EphA2 directed CAR T cells for their capacity to target and kill human OS and ES tumour cells using in vitro and in vivo assays, demonstrating that EphA2 CAR T cells have potent anti-tumour efficacy in vitro and can eliminate established OS and ES tumours in vivo in a dose and delivery route dependent manner. Next, in an aggressive metastatic OS model we demonstrated that systemically infused EphA2 CAR T cells can traffic to and eradicate tumour deposits in murine livers and lungs. These results support further pre-clinical evaluation of EphA2 CAR T cells to inform the design of early phase clinical trial protocols to test the feasibility and safety of this immune cell therapy in paediatric bone sarcoma patients.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

High-Throughput In Vitro, Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction.

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

S100A8/A9 and S100A9 reduce acute lung injury.

S100A8 and S100A9 are myeloid cell-derived proteins that are elevated in several types of inflammatory lung disorders. Pro- and anti-inflammatory properties are reported and these proteins are proposed to activate TLR4. S100A8 and S100A9 can function separately, likely through distinct receptors but a systematic comparison of their effects in vivo are limited. Here we assess inflammation in murine lung following S100A9 and S100A8/A9 inhalation. Unlike S100A8, S100A9 promoted mild neutrophil and lymphocyte influx, possibly mediated in part, by increased mast cell degranulation and selective upregulation of some chemokine genes, particularly CXCL-10. S100 proteins did not significantly induce proinflammatory mediators including TNF-α, interleukin-1ß (IL-1ß), IL-6 or serum amyloid A3 (SAA3). In contrast to S100A8, neither preparation induced S100A8 or IL-10 mRNA/protein in airway epithelial cells, or in tracheal epithelial cells in vitro. Like S100A8, S100A9 and S100A8/A9 reduced neutrophil influx in acute lung injury provoked by lipopolysaccharide (LPS) challenge but were somewhat less inhibitory, possibly because of differential effects on expression of some chemokines, IL-1ß, SAA3 and IL-10. Novel common pathways including increased induction of an NAD+-dependent protein deacetylase sirtuin-1 that may reduce NF-κB signalling, and increased STAT3 activation may reduce LPS activation. Results suggest a role for these proteins in normal homeostasis and protective mechanisms in the lung.

Biomechanics of a Posterior Lumbar Motion Stabilizing Device: In Vitro Comparison to Intact and Fused Conditions.

STUDY DESIGN: Nondestructive flexibility tests were performed in vitro, comparing multiple conditions of fixation in a single group of specimens. OBJECTIVE: To compare the biomechanical behavior of the lumbar spine in the intact condition, after implanting a novel motion stabilizer, and after implanting a rigid fixator. SUMMARY OF BACKGROUND DATA: Two specific scenarios that may benefit from dynamic lumbar stabilization are single-level moderate instability, where the stabilizing tissues are relatively incompetent, and juxta-level to fusion, where the last instrumented level requires intermediate stiffness ("topping off") to prevent transfer of high stresses from the stiffer fusion construct to the intact adjacent levels. Both scenarios were evaluated in vitro. METHODS: Seven human cadaveric L2-S1 segments were tested (1) intact, (2) after moderate destabilization, (3) after 2-level hybrid posterior fixation, consisting of bilateral dynamic pedicle screws at L4 interconnected with rigid rods to standard pedicle screws at L5 and S1, (4) after 2-level rigid fixation, (5) after 1-level (L4-L5) dynamic fixation, and (6) after 1-level rigid fixation. In each condition, angular range of motion (ROM) and sagittal instantaneous axis of rotation (IAR) were assessed. RESULTS: In 1-level constructs, dynamic hardware allowed 104% of intact ROM, whereas rigid hardware allowed 49% of intact ROM. Relative to the intact, the IAR was shifted significantly farther posterior by rigid 1-level instrumentation than by dynamic 1-level instrumentation. In 2-level constructs, the dynamic level allowed significantly greater ROM than the rigid level in all directions but allowed significantly less ROM than the intact level in all directions except axial rotation. CONCLUSION: Dynamic instrumentation shifted the IAR less than rigid instrumentation, providing more favorable kinematics. This dynamic stabilizer provided 1-level ROM that was close to intact ROM during all loading modes in vitro. In the topping-off construct, the dynamic segment allowed intermediate ROM to give balanced transitional flexibility. LEVEL OF EVIDENCE: N/A.

T-bet and interleukin-27: possible TH1 immunomodulators of sarcoidosis.

BACKGROUND: Sarcoidosis has often been termed an "immune paradox" as there is peripheral anergy to common recall antigens despite pronounced TH1-dominant inflammation at disease sites, such as the lung, with up-regulation of interferon γ, IL-27 and transcription factors. Peripheral blood may reflect the anergic state, while exhaled breath condensate (EBC) analysis may offer insights into the lung disease. METHODS: A cross-sectional study was conducted to investigate the expression of TH1 cytokines and transcription factors (IFNγ, IL-27 and T-bet) in the peripheral blood and/or EBC of sarcoidosis patients and healthy controls. Whole blood and EBC were collected from sarcoidosis patients and healthy controls. TH1 cytokine expression levels were then measured in peripheral blood mononuclear cells (PBMCs) and/or plasma and EBC using quantitative real-time PCR, ELISA and via Western blotting. RESULTS: Compared to healthy controls, PBMC IL-27 mRNA was higher in patients (p = 0.0019). There were no significant differences in plasma IL-27 protein between patients and controls (p = 0.20). T-bet mRNA and protein were lower (p = 0.010 and p = 0.0043, respectively) in patients compared to controls. There were no significant differences in PBMC IFNγ mRNA and protein expression (p = 0.68 and p = 0.74, respectively) nor in EBC IL-27 levels. CONCLUSIONS: Our data indicate that depressed T-bet mRNA and protein expression could contribute to the peripheral anergy in sarcoidosis and that IL-27 mRNA levels are elevated in the PBMC from those with sarcoidosis.

ISU201 enhances the resolution of airway inflammation in a mouse model of an acute exacerbation of asthma.

Glucocorticoids are commonly used for treating asthma and its exacerbations but have well-recognised adverse effects and are not always effective. Few alternative treatments exist. Using a murine model of an acute exacerbation of asthma, we assessed the ability of ISU201, a novel protein drug, to suppress the inflammatory response when administered after induction of an exacerbation. Sensitised mice were chronically challenged with a low mass concentration of aerosolised ovalbumin, and then received a single moderate-level challenge to simulate an allergen-induced exacerbation. ISU201 was administered to mice 2 and 8 hours later, while pulmonary inflammation and expression of mRNA for chemokines and proinflammatory cytokines were assessed after 4, 12, and 24 hours. Relative to vehicle-treated controls, ISU201 suppressed accumulation of pulmonary neutrophils and eosinophils, while accelerating the decline in CXCL1, TNF-α, and IL-6 in lavage fluid and lung tissue. ISU201 significantly reduced peak expression of mRNA for the chemokines Cxcl9 and Cxcl10, the adhesion molecules Icam1 and Vcam1, and the proinflammatory cytokines Il1b, Il12p40, and Csf1. The ability of ISU201 to promote resolution of inflammation suggests that it may have potential as an alternative to glucocorticoids in the management of asthma, including when administered after the onset of an acute exacerbation.

TLR9 ligands induce S100A8 in macrophages via a STAT3-dependent pathway which requires IL-10 and PGE2.

S100A8 and S100A9 are highly-expressed calcium-binding proteins in neutrophils and monocytes, and in subsets of macrophages in inflammatory lesions. Unmethylated CpG motifs found in bacterial and viral DNA are potent activators of innate immunity via Toll-like receptor 9 (TLR9). S100A8, but not S100A9, mRNA and protein was directly induced by CpG-DNA in murine and human macrophages. Induction in murine macrophages peaked at 16 h. CpG-DNA-induced S100A8 required de novo protein synthesis; IL-10 and Prostaglandin E2 (PGE2) synergistically enhanced expression and promoted earlier gene induction. Inhibitors of endogenous IL-10, PGE2, and the E prostanoid (EP) 4 receptor strongly suppressed S100A8 expression, particularly when combined. Thus, S100A8 induction by E. coli DNA required both IL-10 and PGE2/EP4 signaling. The MAPKs, PI3K and JAK pathways were essential, whereas ERK1/2 appeared to play a direct role. S100A8 induction by CpG-DNA was controlled at the transcriptional level. The promoter region responsible for activation, either directly, or indirectly via IL-10 and PGE2, was located within a -178 to -34-bp region and required STAT3 binding. Because of the robust links connecting IL-10 and PGE2 with an anti-inflammatory macrophage phenotype, the induction profile of S100A8 strongly indicates a role for this protein in resolution of inflammation.

Rare events in earth history include the LB1 human skeleton from Flores, Indonesia, as a developmental singularity, not a unique taxon.

The original centrally defining features of "Homo floresiensis" are based on bones represented only in the single specimen LB1. Initial published values of 380-mL endocranial volume and 1.06-m stature are markedly lower than later attempts to confirm them, and facial asymmetry originally unreported, then denied, has been established by our group and later confirmed independently. Of nearly 200 syndromes in which microcephaly is one sign, more than half include asymmetry as another sign and more than one-fourth also explicitly include short stature. The original diagnosis of the putative new species noted and dismissed just three developmental abnormalities. Subsequent independent attempts at diagnosis (Laron Syndrome, Majewski osteodysplastic primordial dwarfism type II, cretinism) have been hampered a priori by selectively restricted access to specimens, and disparaged a posteriori using data previously unpublished, without acknowledging that all of the independent diagnoses corroborate the patent abnormal singularity of LB1. In this report we establish in detail that even in the absence of a particular syndromic diagnosis, the originally defining features of LB1 do not establish either the uniqueness or normality necessary to meet the formal criteria for a type specimen of a new species. In a companion paper we present a new syndromic diagnosis for LB1.

Evolved developmental homeostasis disturbed in LB1 from Flores, Indonesia, denotes Down syndrome and not diagnostic traits of the invalid species Homo floresiensis.

Human skeletons from Liang Bua Cave, Flores, Indonesia, are coeval with only Homo sapiens populations worldwide and no other previously known hominins. We report here for the first time to our knowledge the occipitofrontal circumference of specimen LB1. This datum makes it possible to link the 430-mL endocranial volume of LB1 reported by us previously, later confirmed independently by other investigators, not only with other human skeletal samples past and present but also with a large body of clinical data routinely collected on patients with developmental disorders. Our analyses show that the brain size of LB1 is in the range predicted for an individual with Down syndrome (DS) in a normal small-bodied population from the geographic region that includes Flores. Among additional diagnostic signs of DS and other skeletal dysplasiae are abnormally short femora combined with disproportionate flat feet. Liang Bua Cave femora, known only for LB1, match interlimb proportions for DS. Predictions based on corrected LB1 femur lengths show a stature normal for other H. sapiens populations in the region.

S100A8 induces IL-10 and protects against acute lung injury.

S100A8 is considered proinflammatory by activating TLR4 and/or the receptor for advanced glycation end products. The aim was to investigate inflammatory effects of S100A8 in murine lung. S100A8 was administered to BALB/c mice by nasal inhalation and genes induced over a time-course assessed. LPS was introduced intranasally either alone or 2 h after pretreatment of mice with intranasal application of S100A8 or dexamethasone. A Cys(42)-Ala(42) mutant S100A8 mutant was used to assess whether S100A8's effects were via pathways that were dependent on reactive oxygen species. S100A8 induced IL-10 mRNA, and expression was apparent only in airway epithelial cells. Importantly, it suppressed acute lung injury provoked by LPS inhalation by suppressing mast-cell activation and induction of mediators orchestrating leukocyte recruitment, possibly by reducing NF-κB activation via an IκBα/Akt pathway and by downmodulating pathways generating oxidative stress. The Cys(42)-Ala(42) S100A8 mutant did not induce IL-10 and was less immunosuppressive, indicating modulation by scavenging oxidants. S100A8 inhibition of LPS-mediated injury was as potent, and outcomes were remarkably similar to immunosuppression by dexamethasone. We challenge the notion that S100A8 is an agonist for TLR4 or the receptor for advanced glycation end products. S100A8 induced IL-10 in vivo and initiates a feedback loop that attenuates acute lung injury.

S100A12 suppresses pro-inflammatory, but not pro-thrombotic functions of serum amyloid A.

S100A12 is elevated in the circulation in patients with chronic inflammatory diseases and recent studies indicate pleiotropic functions. Serum amyloid A induces monocyte cytokines and tissue factor. S100A12 did not stimulate IL-6, IL-8, IL-1ß or TNF-α production by human peripheral blood mononuclear cells but low amounts consistently reduced cytokine mRNA and protein levels induced by serum amyloid A, by ∼49% and ∼46%, respectively. However, S100A12 did not affect serum amyloid A-induced monocyte tissue factor. In marked contrast, LPS-induced cytokines or tissue factor were not suppressed by S100A12. S100A12 did not alter cytokine mRNA stability or the cytokine secretory pathway. S100A12 and serum amyloid A did not appear to form complexes and although they may have common receptors, suppression was unlikely via receptor competition. Serum amyloid A induces cytokines via activation of NF-κB and the MAPK pathways. S100A12 reduced serum amyloid A-, but not LPS-induced ERK1/2 phosphorylation to baseline. It did not affect JNK or p38 phosphorylation or the NF-κB pathway. Reduction in ERK1/2 phosphorylation by S100A12 was unlikely due to changes in intracellular reactive oxygen species, Ca(2+) flux or to recruitment of phosphatases. We suggest that S100A12 may modulate sterile inflammation by blunting pro-inflammatory properties of lipid-poor serum amyloid A deposited in chronic lesions where both proteins are elevated as a consequence of macrophage activation.

Mitochondrial DNA mutations in exhaled breath condensate of patients with lung cancer.

INTRODUCTION: Lung cancer is a leading cause of cancer mortality worldwide. Non-invasively collected biofluids such as exhaled breath condensate (EBC) present a potential sampling medium to detect and study pathological changes implicated in tumourigenesis. Mitochondrial DNA changes have been implicated in the carcinogenesis process. Consequently, the detection of mitochondrial changes in EBC could expand our understanding of lung carcinogenesis as well as identifying specific markers for future studies. METHODS: EBC and saliva was collected from newly diagnosed subjects with lung cancer and control subjects in a cross-sectional study. The EBC and saliva was analysed for mitochondrial DNA D-loop changes using a PCR sequencing approach. The sequences obtained were compared to paired salivary DNA and the revised Cambridge Reference Sequence (rCRS) to identify somatic mutations, and quantitative and qualitative differences in mutations were analysed between groups. RESULTS: A total of 25 subjects (9 NSCLC patients, 10 smokers/ex-smokers and 6 non-smokers) were recruited. A significantly elevated D-loop mutation rate in the lung cancer group compared to the control groups was present (7 vs 3.5 for smokers/ex-smokers, and 7 vs. 4 for non-smokers, p = 0.034). The recognised mutation T16217C showed specificity for lung cancer. CONCLUSIONS: Mitochondrial DNA mutations are more common in the EBC of patients with lung cancer. This suggests that these processes are associated with the carcinogenesis of lung cancer and may be a marker of the disease.

The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein.

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25µg/ml) decreased nitric oxide ((•)NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10µg/ml) stimulated a Ca(2+) influx linked to apocynin-sensitive superoxide radical anion (O(2)(•-)) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24h. Therefore, in addition to modulating (•)NO bioavailability by stimulating O(2)(•-) production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, µg/ml) before (not after) SAA treatment ameliorated the Ca(2+) influx and O(2)(•-) production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating (•)NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.

S100A8 modulates mast cell function and suppresses eosinophil migration in acute asthma.

S100A8 is implicated in the pathogenesis of inflammatory diseases. S100A8 is upregulated in macrophages by Toll-like receptors (TLR)-3, 4, and 9 agonists in an IL-10-dependent manner, and by corticosteroids in vitro and in vivo, and scavenges oxidants generated by activated phagocytes. Because if its elevated expression in various lung disorders, we asked whether S100A8 was protective in allergic inflammation. S100A8, but not Cys(41)-Ala S100A8, in which the single reactive Cys residue was replaced by Ala, reduced mast cell (MC) degranulation and production of particular cytokines (IL-6, IL-4, and granulocyte macrophage colony-stimulating factor) in response to IgE-crosslinking in vitro, likely by inhibiting intracellular reactive oxygen species production, thereby reducing downstream linker for activation of T cells and extracellular signal regulated kinase/mitogen-activated protein kinase phosphorylation. In lungs of mice with acute asthma, S100A8, but not Cys(41)-Ala S100A8, reduced MC degranulation, production of eosinophil chemoattractants (IL-5, eotaxin, and monocyte chemoattractant protein-1), and eosinophil infiltration. Suppression of IL-6 and IL-13 could have contributed to reduced mucus production seen in lungs of S100A8-treated mice. IgE production was unaffected. In asthma, there is an imbalance of anti-oxidant systems that are generally protective. Our results strongly support a protective role for S100A8 in allergic inflammation by modulating MC activation and eosinophil recruitment, and by scavenging oxidants generated by activated leukocytes, in processes reliant on its thiol-scavenging capacity.

Alveolar macrophages stimulate enhanced cytokine production by pulmonary CD4+ T-lymphocytes in an exacerbation of murine chronic asthma.

The mechanisms underlying the exaggerated distal airway inflammation and hyperresponsiveness that characterize acute exacerbations of asthma are largely unknown. Using BALB/c mouse experimental models, we demonstrated a potentially important role for alveolar macrophages (AM) in the development of an allergen-induced exacerbation of asthma. To induce features of airway inflammation and remodeling characteristic of mild chronic asthma, animals were systemically sensitized and exposed to low mass concentrations (≈3 mg/m(3)) of aerosolized ovalbumin for 30 minutes per day, 3 days per week, for 4 weeks. A subsequent single moderate-level challenge (≈30 mg/m(3)) was used to trigger an acute exacerbation. In chronically challenged animals, cytokine expression by AM was not increased, whereas after an acute exacerbation, AM exhibited significantly enhanced expression of proinflammatory cytokines, including interleukin (IL) 1ß, IL-6, CXCL-1, and tumor necrosis factor α. In parallel, there was a marked increase in the expression of several cytokines by CD4(+) T-lymphocytes, notably the Th2 cytokines IL-4 and IL-13. Importantly, AM from an acute exacerbation stimulated the expression of Th2 cytokines when cocultured with CD4(+) cells from chronically challenged animals, and their ability to do so was significantly greater than AM from either chronically challenged or naïve controls. Stimulation was partly dependent on interactions involving CD80/86. We conclude that in an acute exacerbation of asthma, enhanced cytokine expression by AM may play a critical role in triggering increased expression of cytokines by pulmonary CD4(+) T-lymphocytes.