<em>In vitro</em> Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

University of Colorado

Chronic beryllium disease (CBD) is characterized by granulomatous inflammation and the accumulation of CD4(+) T cells in the lung. Patch testing of CBD patients with beryllium sulfate results in granulomatous inflammation in the skin. We investigated whether the T cell clonal populations present in the lung of CBD patients would also be present in the involved skin of a positive beryllium patch test and thus mirror the granulomatous process in the lung. CBD patients with clonal TCR expansions in bronchoalveolar lavage (BAL) were selected for study. All three CBD patients studied had a positive response to beryllium sulfate application and a negative patch test to normal saline. Immunohistochemistry showed extensive infiltration with CD4(+) T cells and few, if any, CD8(+) T cells both at 3 days and at later times when granulomas were apparent. T cell infiltration early after skin testing appeared to be nonspecific with the TCR repertoire of infiltrating T cells being distinct from that present in BAL. At later times when granulomas were present, T cell clones in skin overlapped with those in BAL in all patients tested. Total TCR matches in skin and BAL were as high as 40% in selected Vbeta T cell subsets. Studies of peripheral blood T cells before and after patch testing provided evidence for mobilization of large numbers of pathogenic beryllium-reactive T cells into the circulating pool. These studies using skin patch testing provide new insight into the dynamics of T cell influx and mobilization during granulomatous inflammation.

Beryllium skin patch testing to analyze T cell stimulation and granulomatous inflammation in the lung.

Sulfur mustard (bis-2-(chloroethyl) sulfide; SM) is a highly reactive vesicating and alkylating chemical warfare agent. A SM analog, 2-chloroethyl ethyl sulfide (CEES), has been utilized to elucidate mechanisms of toxicity and as a screen for therapeutics. Previous studies with SM and CEES have demonstrated a role for oxidative stress as well as decreased injury with antioxidant treatment. We tested whether posttreatment with the metalloporphyrin catalytic antioxidant AEOL 10150 would improve outcome in CEES-induced lung injury. Anesthetized rats inhaled 5% CEES for 15 min via a nose-only inhalation system. At 1 and 9 h after CEES exposure, rats were given AEOL 10150 (5 mg/kg, sc). At 18 h post-CEES exposure BALF lactate dehydrogenase activity, protein, IgM, red blood cells, and neutrophils were elevated but were decreased by AEOL 10150 treatment. Lung myeloperoxidase activity was increased after CEES inhalation and was ameliorated by AEOL 10150. The lung oxidative stress markers 8-OHdG and 4-HNE were elevated after CEES exposure and significantly decreased by AEOL 10150 treatment. These findings demonstrate that CEES inhalation increased lung injury, inflammation, and oxidative stress, and AEOL 10150 was an effective rescue agent. Further investigation utilizing catalytic antioxidants as treatment for SM inhalation injury is warranted.

Treatment with the catalytic metalloporphyrin AEOL 10150 reduces inflammation and oxidative stress due to inhalation of the sulfur mustard analog 2-chloroethyl ethyl sulfide.

Sulfur mustard (SM) is a frequently used chemical warfare agent, even in modern history. SM inhalation causes significant respiratory tract injury, with early complications due to airway obstructive bronchial casts, akin to those seen after smoke inhalation and in single-ventricle physiology. This process with SM is poorly understood because animal models are unavailable.

Airway obstruction due to bronchial vascular injury after sulfur mustard analog inhalation.

The inhalation of sulfur mustard (SM) causes substantial deposition in the nasal region. However, specific injury has not been characterized. 2-chloroethyl ethyl sulfide (CEES) is an SM analogue used to model injury and screen potential therapeutics. After the inhalation of CEES, damage to the olfactory epithelium (OE) was extensive. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were present by 4 hours, and maximal at 18-72 hours. Cleaved caspase 3 immunohistochemistry (IHC) was maximal at 18 hours after the inhalation of 5% CEES. Olfactory marker protein (OMP)-positive olfactory neurons were markedly decreased at 18 hours. IHC-positive cells for 3-nitrotyrosine (3-NT) within epithelium were elevated by 8 hours, waning by 18 hours, and absent by 72 hours. AEOL 10150, a catalytic manganoporphyrin antioxidant, administered both subcutaneously (5 mg/kg) and intranasally (50 μM, "combined treatment"), decreased OE injury. CEES-induced increases in markers of cell death were decreased by combined treatment involving AEOL 10150. CEES-induced changes in OMP and 3-NT immunostaining were markedly improved by combined treatment involving AEOL 10150. The selective inducible nitric oxide synthase inhibitor 1400W (5 mg/kg, subcutaneous), administered 1 hour after inhalation and thereafter every 4 hours (five doses), also reduced OE damage with improved OMP and 3-NT staining. Taken together, these data indicate that reactive oxygen and nitrogen species are important mediators in CEES-induced nasal injury.

Role of reactive oxygen and nitrogen species in olfactory epithelial injury by the sulfur mustard analogue 2-chloroethyl ethyl sulfide.

Acute lung injury is a principal cause of morbidity and mortality in response to mustard gas (SM) inhalation. Obstructive, fibrin-containing airway casts have recently been reported in a rat inhalation model employing the SM analog 2-chloroethyl ethyl sulfide (CEES). The present study was designed to identify the mechanism(s) causing activation of the coagulation cascade after CEES-induced airway injury. Here we report that CEES inhalation elevates tissue factor (TF) activity and numbers of detached epithelial cells present in lavage fluid (BALF) from rats after exposure (18 h). In vitro studies using 16HBE cells, or with rat BALF, indicated that detached epithelial cells could convert factor X (FX) to the active form FXa when incubated with factor VII and could elicit rapid clotting of plasma. In addition, immunocytochemical analysis demonstrated elevated cell surface (TF) expression on CEES-exposed 16HBE cells as a function of time. However, total cell TF expression did not increase. Since membrane surfaces bearing TF are important determinants of clot initiation, anticoagulants directed against these entities were tested for ability to limit plasma clotting or FX activation capacity of BALF or culture media. Addition of tifacogin, a TF pathway inhibitor, effectively blocked either activity, demonstrating that the procoagulant actions of CEES were TF pathway dependent. Lactadherin, a protein capable of competing with clotting factors for phospholipid-binding sites, was partially effective in limiting these procoagulant actions. These findings indicate that TF pathway inhibition could be an effective strategy to prevent airway obstruction after SM or CEES inhalation.

Airway tissue factor-dependent coagulation activity in response to sulfur mustard analog 2-chloroethyl ethyl sulfide.

&#x9610;&#x91CA;&#xFF1A; &#x7EC4;&#x7EC7;&#x56E0;&#x5B50; (TF) &#x542F;&#x52A8;&#x5916;&#x6E90;&#x6027;&#x51DD;&#x8840;&#x7EA7;&#x8054;&#x548C;&#x662F;&#x51DD;&#x8840;&#x56E0;&#x5B50;&#x4E03;&#x9AD8;&#x4EB2;&#x548C;&#x529B;&#x53D7;&#x4F53;&#x3002;TF &#x8FD8;&#x53C2;&#x52A0;&#x4E86;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x53D7;&#x4F53;&#x3001; PAR1 &#x548C; PAR2&#xFF0C;&#x6FC0;&#x6D3B;&#x3002;&#x57FA;&#x5E95;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x7EC4;&#x7EC7;&#x56E0;&#x5B50; (TF) &#x8868;&#x8FBE;&#x7684;&#x57FA;&#x7840;&#x4E0A;&#xFF0C;&#x4EE5;&#x524D;&#x7684;&#x51C0;&#x5316;&#x3002;&#x76EE;&#x6807;&#xFF1A; &#x8FD9;&#x9879;&#x7814;&#x7A76;&#x7684;&#x76EE;&#x7684;&#x662F;&#x786E;&#x5B9A;&#x6C14;&#x7BA1;&#x3001; &#x652F;&#x6C14;&#x7BA1;&#x4E0A;&#x76AE;&#x7684;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x5173;&#x8054; TF &#x662F;&#x5426;&#x9A71;&#x52A8;&#x5668;&#x51DD;&#x8840;&#x548C;/&#x6216;&#x6FC0;&#x6D3B; &mdash; &mdash; &#x5E15;&#x5C14;&#x65AF;&#xFF0C;&#x4FC3;&#x8FDB;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x3002;&#x65B9;&#x6CD5;: &#x6211;&#x4EEC;&#x5229;&#x7528;&#x4EBA;&#x4F53;&#x6C14;&#x7BA1;&#x3001; &#x652F;&#x6C14;&#x7BA1;&#x7EC4;&#x7EC7;&#x9694;&#x79BB;&#x547C;&#x5438;&#x9053;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x7684;&#x6D41;&#x5F0F;&#x7EC6;&#x80DE;&#x672F;&#x4F7F;&#x7528;&#x7279;&#x5B9A;&#x7EC6;&#x80DE;&#x7684;&#x8868;&#x9762;&#x6807;&#x5FD7;&#x7269;&#x548C;&#x6807;&#x672C;&#x7814;&#x7A76; TF &#x8868;&#x8FBE;&#x3002;TF &#x4F9D;&#x8D56;&#x7EA4;&#x7EF4;&#x86CB;&#x767D;&#x7F51;&#x7EDC;&#x5F62;&#x6210;&#x7EA2;&#x6F6E;&#x5171;&#x7126;&#x548C;&#x626B;&#x63CF;&#x7535;&#x5B50;&#x663E;&#x5FAE;&#x955C; (SEM)&#x3002;&#x505A; TF &#x7EC4;&#x5408;&#x5F0F;&#x662F;&#x4F7F;&#x7528; shRNA &#x548C; TF mRNA &#x7528;&#x6765;&#x8861;&#x91CF;&#x5B9A;&#x91CF;&#x9006;&#x8F6C;&#x5F55;-&#x805A;&#x5408;&#x9176;&#x94FE;&#x53CD;&#x5E94;&#x3002;&#x7ED3;&#x679C;&#xFF1A; &#x6211;&#x4EEC;&#x53D1;&#x73B0; 97&plusmn;5%&#x7684;&#x7B2C;&#x4E00;&#x901A;&#x9053;&#x4EBA;&#x6C14;&#x7BA1;&#x3001; &#x652F;&#x6C14;&#x7BA1;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x662F;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#xFF0C;100%&#x7684;&#x8FD9;&#x4E9B;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x8868;&#x8FBE; TF&#x3002;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x5173;&#x8054;&#x7684; TF &#x5F88;&#x6D3B;&#x8DC3;&#xFF0C;&#x4F46; TF &#x6D3B;&#x6027;&#x662F;&#x53D6;&#x51B3;&#x4E8E;&#x6DFB;&#x52A0;&#x5916;&#x6E90;&#x6027;&#x51DD;&#x8840;&#x7EA7;&#x8054;&#x56E0;&#x7D20;&#x3002;TF &#x6291;&#x5236;&#x5F15;&#x8D77;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x548C;&#x574F;&#x6B7B;&#x3002;&#x8FD9;&#x662F;&#x56E0;&#x4E3A;&#x4E24;&#x4E2A;&#x5E73;&#x884C;&#x4F46;&#x76F8;&#x4E92;&#x4F9D;&#x5B58;&#x7684; TF &#x8C03;&#x8282;&#x8FC7;&#x7A0B;&#xFF1A; &#x672A;&#x80FD;&#x751F;&#x6210;&#x4E00;&#x4E2A;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x5173;&#x8054;&#x7EA4;&#x7EF4;&#x86CB;&#x767D;&#x7F51;&#x7EDC;&#x548C;&#x6B21;&#x4F18; PAR1/2 &#x6D3B;&#x52A8;&#x3002;&#x7ED3;&#x8BBA;&#xFF1A; &#x6570;&#x636E;&#x8868;&#x660E;&#xFF0C;&#x8BE5;&#x819C;&#x8868;&#x9762; TF &#x4ECB;&#x5BFC;&#x6C14;&#x9053;&#x4E0A;&#x76AE;&#x7684;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x9644;&#x4EF6;&#xFF0C;&#x7EF4;&#x6301;&#x7EC6;&#x80DE;&#x751F;&#x5B58;&#x548C;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x7684;&#x6F5C;&#x529B;&#x3002;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x5173;&#x8054; TF &#x6B63;&#x5E38;&#x548C;&#x53D7;&#x4F24;&#x7684;&#x7EC4;&#x7EC7;&#x548C;&#x6F5C;&#x5728;&#x7684;&#x80BA;&#x75BE;&#x75C5;&#x6C14;&#x9053;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x7684;&#x4FEE;&#x590D;&#x6D3B;&#x52A8;&#x7684;&#x8303;&#x56F4;&#x5185;&#x8BA8;&#x8BBA;&#x8FD9;&#x4E9B;&#x7ED3;&#x8BBA;&#x7684;&#x5F71;&#x54CD;&#x3002;

&#x7EC4;&#x7EC7;&#x56E0;&#x5B50;&#x4FE1;&#x53F7;&#x6C14;&#x9053;&#x4E0A;&#x76AE;&#x7684;&#x57FA;&#x5E95;&#x7EC6;&#x80DE;&#x751F;&#x5B58;&#x901A;&#x8FC7;&#x51DD;&#x8840;&#x548C; PAR1/2&#x3002;

Rationale: Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high affinity receptor for coagulation factor VII. TF also participates in Protease Activated Receptor, PAR1 and PAR2, activation. Human epithelial basal cells were previously purified on the basis of tissue factor (TF) expression. Objective: The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. Methods: We utilized human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy (SEM). TF knockdown was done using shRNA, and TF mRNA was measured using quantitative RT-PCR. Results: We found that 97Â±5% of first passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1/2 activity. Conclusions: The data indicates that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues, and potential for repair of airway epithelium in lung disease.

Tissue Factor Signals Airway Epithelial Basal Cell Survival via Coagulation and PAR1/2.

Justification : Facteur tissulaire (TF) d&eacute;clenche la cascade de coagulation extrins&egrave;que et est un r&eacute;cepteur de haute affinit&eacute; pour le facteur de coagulation VII. TF participe &eacute;galement &agrave; la prot&eacute;ase Activated Receptor, PAR1 et PAR2, activation. Les cellules basales &eacute;pith&eacute;liales humaines ont &eacute;t&eacute; purifi&eacute;es pr&eacute;c&eacute;demment sur la base de l&#39;expression du facteur (TF) tissu. Objectif : Le but de cette &eacute;tude &eacute;tait de d&eacute;terminer si TF d&#39;associ&eacute; aux cellules basales &eacute;pith&eacute;lial trach&eacute;o-bronchique entra&icirc;ne la coagulation et/ou PARs pour promouvoir les fonctions de la cellule basale d&#39;activer. M&eacute;thodes:, Nous avons utilis&eacute; des tissus humains trach&eacute;o-bronchique pour isoler les cellules &eacute;pith&eacute;liales des voies respiratoires humaines &agrave; l&#39;aide de marqueurs de surface sp&eacute;cifiques de cellules par cytom&eacute;trie en flux et &eacute;tudi&eacute; expression TF par immunomarquage. Formation de r&eacute;seau de fibrine TF-d&eacute;pendante a &eacute;t&eacute; observ&eacute;e par microscopie confocale et balayage (SEM). Knockdown TF a &eacute;t&eacute; fait &agrave; l&#39;aide de shARN et ARNm TF a &eacute;t&eacute; mesur&eacute;e par RT-PCR quantitative. R&eacute;sultats : Nous avons trouv&eacute; que 97&plusmn;5 % des cellules &eacute;pith&eacute;liales humaines de premiers passage trach&eacute;o-bronchique &eacute;taient des cellules basales, et 100 % de ces cellules basales part TF. Associ&eacute; aux cellules basales TF &eacute;tait active, mais l&#39;activit&eacute; TF est tributaire de facteurs de coagulation extrins&egrave;que ajout&eacute; cascade. Inhibition de la TF provoqu&eacute; la n&eacute;crose et l&#39;apoptose des cellules basales. Cela est d&ucirc; &agrave; deux processus parall&egrave;les mais interd&eacute;pendants r&eacute;glement&eacute;s TF : &eacute;chec pour g&eacute;n&eacute;rer un r&eacute;seau de fibrine associ&eacute;e aux cellules basales et sous-optimale activit&eacute; PAR1/2. Conclusions : Les donn&eacute;es indiquent que la surface membranaire TF m&eacute;die attachement de cellules basales &eacute;pith&eacute;liales des voies respiratoires, qui maintient la survie cellulaire et potentiel mitotique. Les implications de ces r&eacute;sultats sont discut&eacute;es dans le cadre de la cellule basale associ&eacute;e &agrave; l&#39;activit&eacute; TF dans les tissus normaux et bless&eacute;s et le potentiel pour la r&eacute;paration de l&#39;&eacute;pith&eacute;lium des voies a&eacute;riennes dans les maladies pulmonaires.

Tissu facteur signaux Airway Epithelial baso-cellulaire survie par Coagulation et PAR1/2.

Begr&uuml;ndung: Tissue-Faktor (TF) initiiert die extrinsischen Gerinnungskaskade und ist ein hoher Affinit&auml;t-Rezeptor f&uuml;r Gerinnungsfaktor VII. TF beteiligt sich auch an Protease aktivierter Rezeptor PAR1 und PAR2, Aktivierung. Menschlichen Epithelzellen der basalen wurden auf der Grundlage von Gewebe Faktor (TF) Ausdruck zuvor gereinigt. Ziel: Das Ziel dieser Studie war, um festzustellen, ob tracheobronchial epitheliale Basalzell-assoziierte TF Koagulation f&auml;hrt und/oder aktiviert PARs Basalzell-Funktionen zu f&ouml;rdern. Methoden: Wir tracheobronchial Humangewebe zum Isolieren von menschlichen Atemwege Epithelzellen mit spezifischen Zelle Oberfl&auml;che Markern durch Durchflusszytometrie genutzt und TF Ausdruck von Paraffinschnitten studiert. TF-abh&auml;ngige Fibrinbildung Netzwerk wurde durch konfokale und Scan-Elektronen-Mikroskopie (SEM) beobachtet. TF Niederschlag erfolgte mit ShRNA und TF mRNA mit quantitativen RT-PCR gemessen wurde. Ergebnisse: Wir fanden, dass 97&plusmn;5 % der erste Durchgang menschlichen tracheobronchial Epithelzellen Basalzellen, und 100 % dieser basalen Zellen TF ausgedr&uuml;ckt. Basalzell-assoziierte TF war aktiv, aber TF Aktivit&auml;t war abh&auml;ngig von zus&auml;tzlichen extrinsischen Gerinnungsfaktoren Kaskade. TF-Hemmung verursacht Basale Zellen Apoptose und Nekrose. Dies ist auf zwei parallel, aber voneinander abh&auml;ngigen TF-regulierten Prozesse: Scheitern, ein Basalzell-assoziierte Fibrin-Netz und suboptimale PAR1/2 Aktivit&auml;t zu generieren. Schlussfolgerungen: Die Daten bedeutet, dass die Membranoberfl&auml;che TF Airway basalen Epithelzellen Anlage, vermittelt der Zelle &uuml;berleben und mitotische Potenzial unterh&auml;lt. Die Folgerung aus diesen Erkenntnissen werden im Rahmen des Basalzell-assoziierte TF-Aktivit&auml;t in normalen und verletzten Gewebes und Potenzial f&uuml;r die Reparatur von Airway-Epithel in Lungenerkrankung diskutiert.

Gewebe Faktor Signale Airway basalen Epithelzellen &Uuml;berleben &uuml;ber Koagulation und PAR1/2.

Spiegazione logica: Fattore del tessuto (TF) avvia la cascata della coagulazione estrinseca ed &egrave; un recettore ad alta affinit&agrave; per il fattore di coagulazione VII. TF partecipa anche proteasi recettori attivati, PAR1 e PAR2, attivazione. Le cellule basali epiteliali umane erano precedentemente purificate sulla base dell&#39;espressione di tissue factor (TF). Obiettivo: Lo scopo di questo studio era di determinare se tracheobronchiali epiteliali associate a cellule basali TF azionamenti della coagulazione e/o attiva PARs per promuovere le funzioni delle cellule basali. Metodi: Abbiamo utilizzato tessuti umani tracheo-bronchiale per isolare le cellule epiteliali delle vie aeree umane utilizzando marcatori di superficie specifica delle cellule tramite flusso cytometry e studiato espressione di TF di immunostaining. Formazione di rete di fibrina TF-dipendente &egrave; stata osservata da microscopia confocal e scansione (SEM). Atterramento TF &egrave; stato fatto utilizzando shRNA e TF mRNA &egrave; stato misurato mediante RT-PCR quantitativa. Risultati: Abbiamo scoperto che 97&plusmn;5% di prime passaggio tracheo-bronchiale cellule epiteliali umane erano cellule basali, e 100% di queste cellule basali espresso TF. TF basocellulare associato era attivo, ma attivit&agrave; TF dipendono da fattori cascata della coagulazione estrinseca aggiunto. Inibizione TF ha causato la necrosi e l&#39;apoptosi delle cellule basali. Questo era a causa di due paralleli ma interdipendenti TF-regolato processi: incapacit&agrave; di generare una rete di fibrina associata a cellule basali e attivit&agrave; PAR1/2 non ottimali. Conclusioni: I dati indicano che superficie di membrana TF da intermediario attaccamento delle cellule basali epiteliali delle vie aeree, che mantiene la sopravvivenza delle cellule e potenziale mitotica. Le implicazioni di queste scoperte sono discussi nel contesto delle attivit&agrave; TF associati a cellule basali in tessuti normali e feriti e il potenziale per la riparazione dell&#39;epitelio delle vie aeree in malattia polmonare.

Fattore di tessuto i segnali delle vie aeree sopravvivenza delle cellule basali epiteliali tramite coagulazione e PAR1/2.

&#x53C2;&#x8003;: &#x7D44;&#x7E54;&#x56E0;&#x5B50; (TF) &#x5916;&#x56E0;&#x6027;&#x51DD;&#x56FA;&#x30AB;&#x30B9;&#x30B1;&#x30FC;&#x30C9;&#x3092;&#x958B;&#x59CB;&#x3057;&#x3001;&#x9AD8;&#x89AA;&#x548C;&#x6027;&#x53D7;&#x5BB9;&#x4F53;&#x304C;&#x8840;&#x6DB2;&#x51DD;&#x56FA;&#x7B2C; VII &#x56E0;&#x5B50;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;TF &#x307E;&#x305F;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x5316;&#x53D7;&#x5BB9;&#x4F53;&#x3001;PAR1 &#x3068; PAR2&#x3001;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3067;&#x53C2;&#x52A0;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3072;&#x3068;&#x4E0A;&#x76AE;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x306F;&#x4EE5;&#x524D;&#x7D44;&#x7E54;&#x56E0;&#x5B50; (TF) &#x306E;&#x767A;&#x73FE;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x7CBE;&#x88FD;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x76EE;&#x7684;: &#x672C;&#x7814;&#x7A76;&#x306E;&#x76EE;&#x7684;&#x306F;&#x6C17;&#x7BA1;&#x6C17;&#x7BA1;&#x652F;&#x4E0A;&#x76AE;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F; TF &#x51DD;&#x56FA;&#x30C9;&#x30E9;&#x30A4;&#x30D6;&#x304A;&#x3088;&#x3073;/&#x307E;&#x305F;&#x306F;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x6A5F;&#x80FD;&#x4FC3;&#x9032; PARs &#x304C;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x306A;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x3060;&#x3063;&#x305F;&#x3002;&#x65B9;&#x6CD5;&#xFF1A; &#x6211;&#x3005; &#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30C8;&#x30EA;&#x30FC;&#x306B;&#x3088;&#x3063;&#x3066;&#x7279;&#x5B9A;&#x306E;&#x30BB;&#x30EB;&#x8868;&#x9762;&#x306E;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3072;&#x3068;&#x6C17;&#x9053;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x3092;&#x5206;&#x96E2;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x30D2;&#x30C8;&#x306E;&#x6C17;&#x7BA1;&#x7D44;&#x7E54;&#x3092;&#x6D3B;&#x7528;&#x3001;TF &#x5F0F;&#x514D;&#x75AB;&#x67D3;&#x8272;&#x306B;&#x3088;&#x308B;&#x691C;&#x8A0E;&#x3057;&#x307E;&#x3059;&#x3002;TF &#x4F9D;&#x5B58;&#x578B;&#x30D5;&#x30A3;&#x30D6;&#x30EA;&#x30F3; &#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x5F62;&#x6210;&#x306F;&#x5171;&#x7126;&#x70B9;&#x304A;&#x3088;&#x3073;&#x8D70;&#x67FB;&#x578B;&#x96FB;&#x5B50;&#x9855;&#x5FAE;&#x93E1; (SEM) &#x3067;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;TF &#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3;&#x884C;&#x308F;&#x308C;&#x305F; shRNA &#x3068; TF mRNA &#x3092;&#x7528;&#x3044;&#x305F;&#x5B9A;&#x91CF;&#x7684; RT-PCR &#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x6E2C;&#x5B9A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D50;&#x679C;: 97&plusmn;5% &#x6700;&#x521D;&#x306E;&#x901A;&#x8DEF;&#x3072;&#x3068;&#x6C17;&#x9053;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x3067;&#x3042;&#x3063;&#x305F;&#x3057;&#x3001;TF &#x3053;&#x308C;&#x3089;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x306E; 100 &#xFF05; &#x3092;&#x8868;&#x73FE;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;TF &#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306A;&#x304C; TF &#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x306F;&#x8FFD;&#x52A0;&#x5916;&#x56E0;&#x6027;&#x8840;&#x6DB2;&#x51DD;&#x56FA;&#x30AB;&#x30B9;&#x30B1;&#x30FC;&#x30C9;&#x306E;&#x8981;&#x56E0;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3060;&#x3063;&#x305F;&#x3002;TF &#x963B;&#x5BB3;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3068;&#x58CA;&#x6B7B;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x306F; 2 &#x3064;&#x5E73;&#x884C;&#x304C;&#x76F8;&#x4E92;&#x4F9D;&#x5B58; TF &#x898F;&#x5236;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x304C;&#x539F;&#x56E0;&#x3060;&#x3063;&#x305F;: &#x30D5;&#x30A3;&#x30D6;&#x30EA;&#x30F3;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x3068;&#x6E96;&#x6700;&#x9069; PAR1/2 &#x6D3B;&#x52D5;&#x306E;&#x751F;&#x6210;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D50;&#x8AD6;: &#x30C7;&#x30FC;&#x30BF;&#x306F;&#x305D;&#x306E;&#x819C;&#x8868;&#x9762; TF &#x7D30;&#x80DE;&#x751F;&#x5B58;&#x7387;&#x3068;&#x7D30;&#x80DE;&#x5206;&#x88C2;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x6C17;&#x9053;&#x4E0A;&#x76AE;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x306E;&#x6DFB;&#x4ED8;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x8ABF;&#x67FB;&#x7D50;&#x679C;&#x306E;&#x542B;&#x610F;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F; TF &#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x6B63;&#x5E38;&#x3068;&#x8CA0;&#x50B7;&#x8005;&#x306E;&#x7D44;&#x7E54;&#x3068;&#x6F5C;&#x5728;&#x6027;&#x80BA;&#x75BE;&#x60A3;&#x306B;&#x304A;&#x3051;&#x308B;&#x6C17;&#x9053;&#x4E0A;&#x76AE;&#x306E;&#x4FEE;&#x7406;&#x306E;&#x305F;&#x3081;&#x306E;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x3067;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;

&#x7D44;&#x7E54;&#x56E0;&#x5B50;&#x51DD;&#x56FA;&#x3068; PAR1/2 &#x3092;&#x4ECB;&#x3057;&#x3066;&#x6C17;&#x9053;&#x4E0A;&#x76AE;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x751F;&#x5B58;&#x3092;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC124;&#xBA85;: &#xC870;&#xC9C1; &#xC694;&#xC18C; (TF) &#xC678;&#xBD80; &#xC751;&#xACE0; &#xCE90;&#xC2A4;&#xCF00;&#xC774;&#xB4DC;&#xB97C; &#xC2DC;&#xC791; &#xD558; &#xACE0; &#xC751;&#xACE0; &#xC778;&#xC790; VII&#xC5D0; &#xB300; &#xD55C; &#xB192;&#xC740; &#xC120;&#xD638;&#xB3C4; &#xC218;&#xC6A9; &#xCCB4; &#xC774;&#xB2E4;. TF&#xB294; &#xB610;&#xD55C; &#xB2E8;&#xBC31;&#xC9C8; &#xD65C;&#xC131;&#xD654; &#xC218;&#xC6A9; &#xCCB4;, PAR1 &#xBC0F; PAR2, &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xCC38;&#xC5EC;&#xD569;&#xB2C8;&#xB2E4;. &#xC778;&#xAC04; &#xC0C1;&#xD53C; &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xC870;&#xC9C1; &#xC778;&#xC790; (TF) &#xC2DD;&#xC5D0; &#xADFC;&#xAC70; &#xD558; &#xC5EC; &#xC815;&#xD654; &#xC774;&#xC804;&#xC5D0; &#xC788;&#xC5C8;&#xB2E4;. &#xBAA9;&#xC801;:&#xC774; &#xC5F0;&#xAD6C;&#xC758; &#xBAA9;&#xC801;&#xC740; tracheobronchial &#xC0C1;&#xD53C; &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xAD00;&#xB828; TF &#xC751;&#xACE0; &#xB4DC;&#xB77C;&#xC774;&#xBE0C; &#xB610;&#xB294; &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xAE30;&#xB2A5; &#xCD09;&#xC9C4;&#xC5D0; &#xAC08; &#xAC70; &#xC608;&#xC694;&#xB97C; &#xD65C;&#xC131;&#xD654; &#xD558;&#xB294; &#xACBD;&#xC6B0;&#xB97C; &#xD655;&#xC778; &#xD588;&#xB2E4;. &#xBC29;&#xBC95;: &#xC6B0;&#xB9AC; &#xC778;&#xAC04;&#xC758; &#xAE30;&#xB3C4; &#xC0C1;&#xD53C; &#xC138;&#xD3EC; cytometry &#xC5EC; &#xD2B9;&#xC815; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xACA9;&#xB9AC;&#xB97C; &#xC778;&#xAC04;&#xC758; tracheobronchial &#xC870;&#xC9C1; &#xD65C;&#xC6A9; &#xBC0F; immunostaining&#xC5D0; &#xC758;&#xD574; TF &#xC2DD; &#xACF5;&#xBD80;. TF &#xC885;&#xC18D; &#xC12C;&#xC720; &#xC18C; &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xD615;&#xC131; confocal &#xBC0F; &#xC2A4;&#xCE90;&#xB2DD; &#xC804;&#xC790; &#xD604;&#xBBF8;&#xACBD; (SEM)&#xC73C;&#xB85C; &#xAD00;&#xCC30; &#xB418;&#xC5C8;&#xB2E4;. TF &#xCD5C;&#xC800;&#xC758; &#xC774;&#xB8E8;&#xC5B4;&#xC84C;&#xB2E4; shRNA, TF Mrna&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC591;&#xC801; RT-PCR&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xCE21;&#xC815; &#xD588;&#xB2E4;. &#xACB0;&#xACFC;: &#xCCAB; &#xBC88;&#xC9F8; &#xD1B5;&#xB85C; &#xC778;&#xAC04; tracheobronchial &#xC0C1;&#xD53C; &#xC138;&#xD3EC;&#xC758; 97&plusmn;5% &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xB418;&#xC5C8;&#xACE0; &#xC774;&#xB7EC;&#xD55C; &#xAE30;&#xC800; &#xC138;&#xD3EC;&#xC758; 100% &#xD45C;&#xD604; &#xB41C; TF&#xB97C; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xAD00;&#xB828; TF, &#xC774;&#xC5C8;&#xC9C0;&#xB9CC; TF &#xD65C;&#xB3D9; &#xCD94;&#xAC00; &#xC678;&#xBD80; &#xC751;&#xACE0; &#xCE90;&#xC2A4;&#xCF00;&#xC774;&#xB4DC; &#xC694;&#xC778;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. TF &#xC800;&#xD574; &#xAE30;&#xC800; &#xC138;&#xD3EC; apoptosis&#xC640; &#xAD34; &#xC0AC;&#xB97C; &#xBC1C;&#xC0DD;&#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xAC83;&#xC740; &#xB450; &#xAC1C;&#xC758; &#xBCD1;&#xB82C; &#xD558;&#xC9C0;&#xB9CC; &#xC0C1;&#xD638; &#xC758;&#xC874;&#xC801;&#xC778; TF &#xADDC;&#xC81C; &#xD504;&#xB85C;&#xC138;&#xC2A4; &#xB54C;&#xBB38;: &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xAD00;&#xB828; &#xB41C; &#xC12C;&#xC720; &#xC18C; &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xBC0F; &#xCC28;&#xC120; PAR1/2 &#xD65C;&#xB3D9; &#xC0DD;&#xC131;&#xC744; &#xC2E4;&#xD328; &#xD569;&#xB2C8;&#xB2E4;. &#xACB0;&#xB860;: &#xB370;&#xC774;&#xD130;&#xB294; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xD45C;&#xBA74; TF &#xC911;&#xC7AC; &#xAE30;&#xB3C4; &#xC0C1;&#xD53C; &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xCCA8;&#xBD80; &#xD30C;&#xC77C; &#xC720;&#xC9C0; &#xD558;&#xB294; &#xC138;&#xD3EC; &#xC0DD;&#xC874;&#xACFC; mitotic &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC;&#xC758; &#xC758;&#xBBF8;&#xB294; &#xC815;&#xC0C1;&#xC801;&#xC774; &#xACE0; &#xBD80;&#xC0C1; &#xC870;&#xC9C1;&#xACFC; &#xD3D0; &#xC9C8;&#xD658;&#xC5D0;&#xC11C; &#xAE30;&#xB3C4; &#xC0C1;&#xD53C;&#xC758; &#xC218;&#xB9AC;&#xC5D0; &#xB300; &#xD55C; &#xC7A0;&#xC7AC;&#xB825;&#xC5D0; &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xAD00;&#xB828; TF &#xD65C;&#xB3D9;&#xC758; &#xB9E5;&#xB77D;&#xC5D0;&#xC11C; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;.

&#xC870;&#xC9C1; &#xC694;&#xC18C; &#xC2E0;&#xD638; &#xC751;&#xACE0; &#xBC0F; PAR1/2&#xB97C; &#xD1B5;&#xD574; &#xAE30;&#xB3C4; &#xC0C1;&#xD53C; &#xAE30;&#xC800; &#xC138;&#xD3EC; &#xC0DD;&#xC874;.

Justificaci&oacute;n: Factor tisular (TF) inicia la cascada de coagulaci&oacute;n extr&iacute;nseco y es un receptor de alta afinidad para el factor de coagulaci&oacute;n VII. TF tambi&eacute;n participa en la proteasa activa receptores y PAR1, PAR2, activaci&oacute;n. C&eacute;lulas basales epiteliales humanas fueron purificadas previamente sobre la base de expresi&oacute;n del factor (TF) del tejido. Objetivo: El prop&oacute;sito de este estudio fue determinar si traqueobronquial TF epitelial de c&eacute;lulas basales-asociados unidades de coagulaci&oacute;n y/o activa las igualdades para promover las funciones de la c&eacute;lula b&aacute;sica. M&eacute;todos: Utilizan tejidos humanos traqueobronquiales para aislar c&eacute;lulas epiteliales de las v&iacute;as respiratorias humanas utilizando marcadores de superficie celular espec&iacute;ficos mediante citometr&iacute;a de flujo y estudiado TF expresi&oacute;n immunostaining. Formaci&oacute;n de redes de fibrina TF-dependiente fue observada por microscop&iacute;a confocal y exploraci&oacute;n (SEM). Ca&iacute;da TF se realiz&oacute; utilizando shRNA y TF mRNA se midi&oacute; usando RT-PCR cuantitativa. Resultados: Encontramos que el 97&plusmn;5% de primeras paso humano traqueobronquial c&eacute;lulas epiteliales eran c&eacute;lulas basales, y 100% de estas c&eacute;lulas basales expres&oacute; TF. TF basocelular asociado era activo, pero TF actividad depend&iacute;a de factores de la cascada de coagulaci&oacute;n extr&iacute;nseco a&ntilde;adido. Inhibici&oacute;n de TF causa necrosis y apoptosis de c&eacute;lulas basales. Esto fue debido a dos procesos paralelos pero interdependientes con suelos de TF-regulado: fracaso para generar una red de fibrina asociada a c&eacute;lulas basales y actividad sub&oacute;ptima de PAR1/2. CONCLUSIONES: Los datos indican que la superficie de la membrana TF media accesorio de c&eacute;lula basal epitelial de las v&iacute;as respiratorias, que mantiene la supervivencia celular y potencial mit&oacute;tico. Las implicaciones de estos resultados se discuten en el contexto de la actividad asociada a c&eacute;lulas basales TF en tejidos normales y heridos y el potencial para la reparaci&oacute;n del epitelio de las v&iacute;as respiratorias en enfermedad pulmonar.

Tejido Factor se&ntilde;ales v&iacute;a a&eacute;rea c&eacute;lula Basal epitelial supervivencia mediante coagulaci&oacute;n y PAR1/2.

Sulfur mustard (SM) inhalation causes the rare but life-threatening disorder of plastic bronchitis, characterized by bronchial cast formation, resulting in severe airway obstruction that can lead to respiratory failure and death. Mortality in those requiring intubation is greater than 80%. To date, no antidote exists for SM toxicity. In addition, therapies for plastic bronchitis are solely anecdotal, due to lack of systematic research available to assess drug efficacy in improving mortality and/or morbidity. Adult rats exposed to SM analog were treated with intratracheal tissue plasminogen activator (tPA) (0.15-0.7 mg/kg, 5.5 and 6.5 h), compared with controls (no treatment, isoflurane, and placebo). Respiratory distress and pulse oximetry were assessed (for 12 or 48 h), and arterial blood gases were obtained at study termination (12 h). Microdissection of fixed lungs was done to assess airway obstruction by casts. Optimal intratracheal tPA treatment (0.7 mg/kg) completely eliminated mortality (0% at 48 h), and greatly improved morbidity in this nearly uniformly fatal disease model (90-100% mortality at 48 h). tPA normalized plastic bronchitis-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress (i.e., clinical scores) while decreasing airway fibrin casts. Intratracheal tPA diminished airway-obstructive fibrin-containing casts while improving clinical respiratory distress, pulmonary gas exchange, tissue oxygenation, and oxygen utilization in our model of severe chemically induced plastic bronchitis. Most importantly, mortality, which was associated with hypoxemia and clinical respiratory distress, was eliminated.

Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction.

Hypoxia-inducible transcription factors HIF-1α and HIF-2α can contribute to pulmonary hypertension and vascular remodeling, but their mechanisms remain unknown. This study investigated the role of HIF-1α and HIF-2α in pulmonary artery endothelial and smooth muscle cells. The exposure of human pulmonary artery endothelial cells (HPAECs) to hypoxia (10% O₂ or 5% O₂) increased proliferation over 48 hours, compared with cells during normoxia (21% O₂). The adenovirus-mediated overexpression of HIF-2α that is transcriptionally active during normoxia (mutHIF-2α) increased HPAEC proliferation, whereas the overexpression of HIF-1α, which is transcriptionally active during normoxia (mutHIF-1α), exerted no effect. The knockdown of HIF-2α decreased proliferation during both hypoxia and normoxia. Both HIFs increased migration toward fibrinogen, used as a chemoattractant. In an angiogenesis tube formation assay, mutHIF-2α-transduced cells demonstrated increased tube formation, compared with the mutHIF-1α-transduced cells. In addition, the tubes formed in HIF-2α-transduced cells were more enduring than those in the other groups. In human pulmonary artery smooth muscle cells (HPASMCs), chronic exposure to hypoxia increased proliferation, compared with cells during normoxia. For HPASMCs transduced with adenoviral HIFs, HIF-1α increased proliferation, whereas HIF-2α exerted no such effect. Thus, HIF-1α and HIF-2α exert differential effects in isolated cells of the human pulmonary vasculature. This study demonstrates that HIF-2α plays a predominant role in the endothelial growth pertinent to the remodeling process. In contrast, HIF-1α appears to play a major role in pulmonary smooth muscle growth. The selective targeting of each HIF in specific target cells may more effectively counteract hypoxic pulmonary hypertension and vascular remodeling.

Differential regulation of pulmonary vascular cell growth by hypoxia-inducible transcription factor-1α and hypoxia-inducible transcription factor-2α.

Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence.

Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation.

The superoxide dismutase (SOD)-mimetic effectiveness of [meso-tetrakis (R)porphyrinato]manganese with R = 1,3-di-N-ethylimidazolium-2-yl (Mn-TDEIP), 1,3-di-N-methylimidazolium-2-yl (Mn-TDMIP), 1,3-di-N-propylimidazolium-2-yl (Mn-TDPIP), N-ethyl-2-pyridyl (Mn-T2EPyP), 4-sulphonatophenyl (Mn-TSP), 1-methyl-4-pyridyl (Mn-T4PyP), 4-carboxyphenyl (Mn-TBAP), and β-octabromomeso-tetrakis(4-carboxyphenyl porphyrinato)manganese (MnBr8TBAP) was compared with Cu, Zn SOD. Superoxide generated by reaction of xanthine oxidase with hypoxanthine was trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), forming BMPO-OOH, which was monitored by electron paramagnetic resonance. Manganoporphyrins with redox potentials ranging from -0.190 to 0.346 V relative to the standard hydrogen electrode were selected for this study. With 0.1 µM manganoporphyrins and 20 mM BMPO, the effectiveness of the manganoporphyrins in inhibiting formation of BMPO-OOH increases in the order Mn-TSP < Mn-TBAP < MnBr8TBAP < Mn-T4PyP < Mn-T2EPyP < Mn-TDEIP ~ Mn-TDMIP ~ Mn-TDPIP ~ Cu, Zn SOD. However, at higher concentrations of manganoporphyrin and BMPO, a BMPO-OH signal was observed. The formation of BMPO-OH was not inhibited by catalase or dimethylsulfoxide, which demonstrated that it was not produced from hydroxyl radical. The artifactual formation of BMPO-OH is attributed to oxidation of the water adduct of BMPO by the manganoporphyrins or decomposition of BMPO-OOH. Although spin trapping is an effective method for evaluating SOD-mimetic efficacy, caution must be exercised to ensure that artifact signals are not interpreted improperly.

Impact of SOD-Mimetic Manganoporphyrins on Spin Trapping of Superoxide and Related Artifacts.

Background: Inhalation of sulfur mustard (SM) and SM analog, 2-chloroethyl ethyl sulfide (CEES), cause fibrinous cast formation that occludes the conducting airways, similar to children with Fontan physiology-induced plastic bronchitis. These airway casts cause significant mortality and morbidity, including hypoxemia and respiratory distress. Our hypothesis was that intratracheal heparin, a highly cost effective and easily preserved rescue therapy, could reverse morbidity and mortality induced by bronchial cast formation. Methods: Sprague-Dawley rats were exposed to 7.5% CEES via nose-only aerosol inhalation to produce extensive cast formation and mortality. The rats were distributed into three groups: non-treated, phosphate-buffered saline (PBS)-treated, and heparin-treated groups. Morbidity was assessed with oxygen saturations and clinical distress. Blood and bronchoalveolar lavage fluid (BALF) were obtained for analysis, and lungs were fixed for airway microdissection to quantify the extent of airway cast formation. Results: Heparin, given intratracheally, improved survival (100%) when compared to non-treated (75%) and PBS-treated (90%) controls. Heparin-treated rats also had improved oxygen saturations, clinical distress and airway cast scores. Heparin-treated rats had increased thrombin clotting times, factor Xa inhibition and activated partial thromboplastin times, indicating systemic absorption of heparin. There were also increased red blood cells (RBCs) in the BALF in 2/6 heparin-treated rats compared to PBS-treated control rats. Conclusions: Intratracheal heparin 1 hr after CEES inhalation improved survival, oxygenation, airway obstruction, and clinical distress. There was systemic absorption of heparin in rats treated intratracheally. Some rats had increased RBCs in BALF, suggesting a potential for intrapulmonary bleeding if used chronically after SM inhalation. Pediatr Pulmonol. © 2014 Wiley Periodicals, Inc.

Tara Hendry-Hofer

Pediatric Airway Research Center,
Department of Pediatrics,
Pediatric Airway Research Center, Department of Pediatrics

University of Colorado

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Tara Hendry-Hofer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Pediatric Airway Research Center,Department of Pediatrics,Pediatric Airway Research Center, Department of Pediatrics

University of Colorado

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Pediatric Airway Research Center,
Department of Pediatrics,
Pediatric Airway Research Center, Department of Pediatrics