Airways exposure of bacterial superantigen SEB enhances bone marrow eosinophil population and facilitates its egress to blood and lung tissue.

BACKGROUND: Differentiation of bone marrow eosinophils (BM-EO) and its trafficking to peripheral blood and respiratory mucosa are a hallmark of inflammatory diseases. Staphylococcal enterotoxin B (SEB) has been shown to aggravate airways eosinophilic inflammation. This study aimed to investigate the effects of mouse airways SEB exposure on BM-EO population, as well as its adhesive properties and release of cytokines/chemokines that orchestrate BM-EO trafficking to lungs. METHODS: Male BALB/c mice were intranasally exposed to SEB (1 µg), and at 4, 16, 24 and 48 h thereafter, bone marrow (BM), circulating blood and bronchoalveolar lavage (BAL) fluid were collected. Levels of cytokines/chemokines and expressions of VLA-4 and CCR3 in BM were evaluated. Adhesion of BM to ICAM-1 and VCAM-1 were also evaluated. RESULTS: SEB exposure promoted a marked eosinophil influx to BAL at 16 and 24 h after exposure, which was accompanied by significant increases in counts of immature (16 h) and mature (4 to 48 h) forms of eosinophil in BM, along with blood eosinophilia (16 h). In BM, higher levels of eotaxin, IL-5, IL-4, IL-3 and IL-7 were detected at 16 to 48 h. SEB also significantly increased CCR3 expression and calcium levels in BM-EO, and enhanced the cell adhesion to ICAM-1 (24 h) and ICAM-1 (48 h). CONCLUSION: Airways SEB exposure increases the number of eosinophils in BM by mechanisms involving a network of cytokine and chemokine release, facilitating the BM-EO adhesion to ICAM-1 and VCAM-1 to gain access to the peripheral blood and lung tissues.

Airway exposure to Staphylococcal enterotoxin type B (SEB) enhances the number and activity of bone marrow neutrophils via the release of multiple cytokines.

BACKGROUND: The lung infections by Staphylococcus aureus are strongly associated with its ability to produce enterotoxins. However, little is known about the mechanisms underlying trafficking of bone marrow (BM) neutrophils during airway inflammation induced by Staphylococcal enterotoxin B (SEB). We therefore aimed to investigate the effects of mouse airways SEB exposure on BM neutrophil counts and its adhesive properties as well as on the release of cytokines/chemokines that orchestrate BM neutrophils trafficking to lung tissue. METHODS: Male BALB/c mice were intranasally exposed to SEB (1 µg), and at 4, 16 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. BM neutrophils adhesion, MAC-1 and LFA1-α expressions (by flow cytometry) as well as measurement of cytokine and/or chemokines levels were assayed after SEB-airway exposure. RESULTS: Prior exposure to SEB promoted a marked influx of neutrophils to BAL and lung tissue, which was accompanied by increased counts of BM immature neutrophils and blood neutrophilia. BM neutrophil expressions of LFA1-α and MAC-1 were unchanged by SEB exposure whereas a significant enhancement of adhesion properties to VCAM-1 was observed. The early phase of airway SEB exposure was accompanied by high levels of GM-CSF, G-CSF, IFN-γ, TNF-α and KC/CXCL1, while the latter phase by the equilibrated actions of SDF1-α and MIP-2. CONCLUSION: Mouse airways exposure to SEB induces BM cytokines/chemokines release and their integrated actions enhance the adhesion of BM neutrophils leading to acute lung injury.

Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice.

Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1µg), and at 4, 12 and 24h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression.

Heterodimeric structure of superoxide dismutase in complex with its metallochaperone.

The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 A resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.

Copper stabilizes a heterodimer of the yCCS metallochaperone and its target superoxide dismutase.

The copper chaperone for superoxide dismutase (CCS) activates the antioxidant enzyme Cu,Zn-SOD (SOD1) by directly inserting the copper cofactor into the apo form of SOD1. Neither the mechanism of protein-protein recognition nor of metal transfer is clear. The metal transfer step has been proposed to occur within a transient copper donor/acceptor complex that is either a heterodimer or heterotetramer (i.e. a dimer of dimers). To determine the nature of this intermediate, we generated a mutant form of SOD1 by replacing a copper binding residue His-48 with phenylalanine. This protein cannot accept copper from CCS but does form a stable complex with apo- and Cu-CCS, as observed by immunoprecipitation and native gel electrophoresis. Fluorescence anisotropy measurements corroborate the formation of this species and further indicate that copper enhances the stability of the dimer by an order of magnitude. The copper form of the heterodimer was isolated by gel filtration chromatography and contains one copper and one zinc atom per heterodimer. These results support a mechanism for copper transfer in which CCS and SOD1 dock via their highly conserved dimer interfaces in a manner that precisely orients the Cys-rich copper donor sites of CCS and the His-rich acceptor sites of SOD1 to form a copper-bridged intermediate.

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS.

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.

Heterodimer formation between superoxide dismutase and its copper chaperone.

Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS). The molecular mechanisms by which CCS recognizes and docks with SOD1 for metal ion insertion are not well understood. Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one monomer of SOD1 and a dimer of dimers involving interactions between the two homodimers. We have investigated protein-protein complex formation between copper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replaced with phenylalanine (H48F-SOD1). According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical cross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer. No higher order oligomers were detected. Heterodimer formation is facilitated by the presence of zinc but does not depend on copper loading of yCCS. The complex formed with H48F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species. Notably, heterodimer formation between copper-loaded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc. These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.

[Thyrotoxic periodic paralysis. Report of a case]. / Paralisia periódica tireotóxica. Registro de um caso.

A case of thyrotoxic periodic paralysis based on clinical grounds, laboratory data and therapeutic response as well is reported. The authors comment on the differential diagnosis with the most frequent types of periodic paralysis. The importance of a correct diagnosis and treatment as early as possible is stressed as to prevent further development of permanent paralysis due to irreversible degenerative myofibril changes as stated in literature.