[The value of CMR high-risk attributes in predicting ventricular remodeling in ST-segment-elevation myocardial infarction patients with mildly reduced or preserved ejection fraction].

Objective: To evaluate the predictive value of a multiparametric cardiac magnetic resonance (CMR) approach for ventricular remodeling in ST-segment-elevation myocardial infarction (STEMI) patients with mildly reduced or preserved left ventricular ejection fraction (LVEF). Methods: This study is a prospective cohort study. STEMI patients with acute LVEF>40% after primary percutaneous coronary intervention (PCI) in Beijing Anzhen Hospital from October 2019 to September 2021 were enrolled. All patients received acute (3-7 days) and follow-up (3 months) CMR post-PCI. According to absence or presence of ventricular remodeling, patients were divided into ventricular remodeling group and non-ventricular remodeling group. Basic clinical characteristics and CMR indicators were analyzed and compared between the two groups. Logistic regression and receiver operating characteristic (ROC) curves were used to explore the predictive performance of CMR high-risk attributes for ventricular remodeling in STEMI patients with mildly reduced or preserved LVEF. The predictive value of combining multiple high-risk characteristics of CMR for ventricular remodeling was analyzed and compared with the traditional clinical risk factor model. Results: A total of 123 STEMI patients were enrolled (aged (57.1±11.1) years, 102 (82.9%) males). There were 97 cases (78.9%) patients in the non-ventricular remodeling group and 26 cases (21.1%) in the ventricular remodeling group. After adjustment for clinical risk factors, stroke volume<51.6 ml, global circumferential strain>-13.7%, infarct size>39.2%, microvascular obstruction>0.5%, and myocardial salvage index<43.9 were independently associated with ventricular remodeling in STEMI patients with mildly reduced or preserved LVEF. The incidence of ventricular remodeling increased with the increasing number of CMR high-risk attributes (P<0.01). The number of CMR high-risk attributes ≥3 was an independent predictor of adverse remodeling (adjusted OR=5.95, 95 CI%: 2.25-15.72, P<0.01) in STEMI patients with mildly reduced or preserved LVEF. Furthermore, the number of CMR high-risk attributes had incremental predictive value over baseline clinical risk factors (area under curve: 0.843 vs. 0.696, P<0.01). Conclusions: In STEMI patients with mild reduced or preserved LVEF, 5 CMR characteristics are associated with ventricular remodeling. The combination of ≥3 CMR high-risk characteristics is an independent predictor of ventricular remodeling, which has incremental predictive value beyond traditional risk factors in this patient cohort.

[Impact of obstructive sleep apnea on the long-term cardiovascular outcomes in patients with acute coronary syndrome].

Objective: To investigate the impact of obstructive sleep apnea (OSA) on long-term cardiovascular outcomes in patients with acute coronary syndrome (ACS). Methods: This is a single-center, prospective cohort study. Between June 2015 to January 2020, consecutive ACS patients hospitalized at Beijing Anzhen Hospital, Capital Medical University were enrolled. All patients underwent portable sleep breathing monitoring, and they were then divided into moderate/severe OSA group (apnea-hypopnea index (AHI)≥15 events/hour) and no/mild OSA group (AHI<15 events/hour). The primary endpoint was major adverse cardiac and cerebrovascular event (MACCE), defined as a composite of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, ischemia-driven revascularization and hospital admission for unstable angina or heart failure. MACCE were compared yearly by the log-rank test. Multivariable Cox regression analyses were performed to determine the independent predictors of MACCE. Results: A total of 1 927 patients with ACS were enrolled, including 1 629 males (84.5%), aged (56.4±10.5) years. Moderate/severe OSA was present in 1 014 (52.6%) patients. Compared with no/mild OSA group, moderate/severe OSA group exhibited a higher body mass index (P<0.05). Hypertension, prior PCI were more prevalent in moderate/severe OSA group (both P<0.05). The difference of ACS category between the two groups was statistically significant (P=0.021). The rate of patients who underwent PCI and the number of stents were higher in the moderate/severe OSA group. During a 5-year follow-up (median 2.9 years (IQR 1.5-3.6 years)), the cumulative incidence of MACCE was significantly higher in the moderate/severe OSA group than in the no/mild OSA group (34.0% vs. 24.0%, HR=1.346, 95%CI 1.100-1.646, log-rank P=0.004). The cumulative incidence of MACCE remained statistically higher at 4 and 5 year in the moderate/severe OSA group as compared to the no/mild OSA group (33.3% vs. 22.9%, HR=1.397, 95%CI 1.141-1.710, log-rank P=0.001; 34.0% vs. 24.0%, HR=1.341, 95%CI 1.096-1.640, log-rank P=0.004, respectively). Multivariate analysis showed that moderate/severe OSA (HR=1.312, 95%CI 1.054-1.631, P=0.015) was an independent predictor of long-term MACCE in ACS patients. Conclusions: Moderate/severe OSA is observed in more than 52% ACS patients. Moderate/severe OSA is an independent predictor of long-term MACCE.

[Prospective cohort study on the impact of moderate/severe obstructive sleep apnea on the prognosis of patients with acute myocardial infarction].

Objective: To investigate the impact of moderate/severe obstructive sleep apnea (OSA) on the prognosis of acute myocardial infarction. Methods: We prospectively selected patients with acute myocardial infarction (AMI) who were hospitalized at the Emergency Critical Care Center of Beijing Anzhen Hospital from June 2015 to May 2017. Patients who met the inclusion criteria were examined with portable sleep respiration monitoring. Patients were divided into moderate/severe OSA group (apnea-hypopnea index (AHI)≥15 beats/hour) and no/mild OSA group (AHI<15 beats/hour) according to sleep AHI. The incidence of major adverse cerebrovascular events (MACCE) after discharge was compared between the two groups, and the independent risk factors of MACCE were analyzed. Results: A total of 432 patients were enrolled in this study, including 211 moderate/severe OSA patients (48.8%). Compared with no/mild OSA group,patients with moderate/severe OSA had higher body mass index ((27.17±3.22) kg/m(2) vs. (25.55±3.44) kg/m(2), t=-5.033,P<0.001), higher proportion of history of percutaneous coronary intervention (PCI) (18.5%(39/211) vs. 8.6%(19/221), χ(2)=9.076,P=0.003), and higher proportion of 3-vessel disease (31.3%(66/211) vs. 24.9%(55/221), χ(2)=10.196,P=0.017). The median follow-up time was 1.0 (0.7, 1.7) years. The incidence of MACCE in the moderate/severe OSA and no/mild group was 19.9%(42/211) and 11.3%(25/221), respectively. Kaplan-Meier analysis showed a higher cumulative risk of MACCE in patients with moderate/severe OSA (log-rank test,χ(2)=5.467, P=0.019). Multivariate Cox regression analysis showed that moderate/severe OSA (HR=1.915, 95%CI 1.016-3.611, P=0.045) and diabetes mellitus (HR=1.819, 95%CI 1.022-3.238, P=0.042) were independent risk factors for MACCE at 1 year post discharge in patients with AMI. Conclusions: Nearly half of AMI patients are complicated with moderate/severe OSA in this patient cohort. Coronary artery disease is more severe in AMI patients complicating with moderate/severe OSA. Moderate/severe OSA is an independent risk factor for MACCE at 1 year after discharge in patients with AMI. Whether the prognosis of AMI can be improved by intervention of OSA remains to be investigated. Trial Registration: Clinical Trial.gov, NCT03362385.

[Association between hypothyroidism and sleep breathing disorders in patients with coronary heart disease].

Objective: To explore the association between hypothyroidism and sleep breathing disorders in patients with coronary heart disease (CHD). Methods: A total of 784 patients with CHD were consecutively enrolled at the Emergency & Critical Care Center of Beijing Anzhen Hospital from June 2015 to May 2017. According to thyroid function test results, patients were divided into hypothyroidism group (79 cases) and non-hypothyroidism group (705 cases). All patients had undergone sleep monitoring. The sleep apnea status was compared between the two groups. Multivariate logistic regression and linear regression models were used to analyze the association between hypothyroidism and sleep breathing disorders in patients with CHD. Results: The proportion of females, mean body weight and body mass index in the hypothyroidism group were higher than those in the non-hypothyroidism group [26.6% vs.16.2%, (78.6±11.6) kg vs. (75.7±12.0) kg, (27.7±3.2) kg/m(2) vs. (26.6±3.5) kg/m(2), all P<0.05]. Patients in hypothyroidism group had a decreased average oxygen saturation (SaO(2)) compared with patients in non-hypothyroidism group [ (93.2±2.9) % vs. (93.9±2.0) %, P=0.030]. In addition, events of hypoventilation in hypothyroidism group were significantly higher than those in non-hypothyroidism group[92.5 (45.8, 758.3) times vs. 68.0 (33.0, 125.0) times, P=0.013]. There were no significant differences in apnea hypopnea index, diagnosis of obstructive sleep apnea and other sleep breathing parameters between the two groups (P>0.05). A multiple linear regression analysis found that in patients with CHD, the correlation between hypothyroidism and average sleep SaO(2) was significant (ß=-0.508, 95%CI -0.989--0.026, P=0.039). Conclusions: CHD patients with hypothyroidism had a lower sleep average SaO(2), and a higher sleep hypopnea events. There is a correlation between hypothyroidism and sleep hypoxia in patients with CHD. Clinical trial registration: clinicalTrials.gov, NCT03362385.

Complement activation promotes colitis-associated carcinogenesis through activating intestinal IL-1ß/IL-17A axis.

Colitis-associated colorectal cancer (CAC) is the most serious complication of inflammatory bowel disease (IBD). Excessive complement activation has been shown to be involved in the pathogenesis of IBD. However, its role in the development of CAC is largely unknown. Here, using a CAC model induced by combined administration of azoxymethane (AOM) and dextran sulfate sodium (DSS), we demonstrated that complement activation was required for CAC pathogenesis. Deficiency in key components of complement (e.g., C3, C5, or C5a receptor) rendered tumor repression in mice subjected to AOM/DSS. Mechanistic investigation revealed that complement ablation dramatically reduced proinflammatory cytokine interleukin (IL)-1ß levels in the colonic tissues that was mainly produced by infiltrating neutrophils. IL-1ß promoted colon carcinogenesis by eliciting IL-17 response in intestinal myeloid cells. Furthermore, complement-activation product C5a represented a potent inducer for IL-1ß in neutrophil, accounting for downregulation of IL-1ß levels in the employed complement-deficient mice. Overall, our study proposes a protumorigenic role of complement in inflammation-related colorectal cancer and that the therapeutic strategies targeting complement may be beneficial for the treatment of CAC in clinic.

Neutrophil infiltration favors colitis-associated tumorigenesis by activating the interleukin-1 (IL-1)/IL-6 axis.

Neutrophil infiltration is a key event in chronic intestinal inflammation and associated colorectal cancer, but how these cells support cancer development is poorly understood. In this study, using a mouse model of colitis-associated cancer (CAC), we have demonstrated that infiltrated neutrophils produce large amounts of interleukin-1 (IL)-1ß that is critical for the development of CAC. Depletion of neutrophil or blockade of IL-1ß activity significantly reduced mucosal damage and tumor formation. This protumorigenic function of IL-1ß was mainly attributed to increased IL-6 secretion by intestine-resident mononuclear phagocytes (MPs). Furthermore, commensal flora-derived lipopolysaccharide (LPS) was identified to trigger IL-1ß expression in neutrophils. Importantly, accumulation of IL-1ß-expressing neutrophils was seen in lesions of patients suffering from ulceratic CAC and these infiltrated neutrophils induced IL-6 production by intestinal MPs in an IL-1ß-dependent manner. Overall, these findings reveal that in CAC milieu, infiltrating neutrophils secrete IL-1ß that promotes tumorigenesis by inducing IL-6 production by intestinal MPs.

Association of methylenetetrahydrofolate reductase and thymidylate synthase promoter polymorphisms with genetic susceptibility to esophageal and cardia cancer in a Chinese high-risk population.

SUMMARY: Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) are key enzymes in folate metabolism, which is essential for normal DNA methylation and synthesis. Common polymorphisms at the MTHFR nucleotides position 677 (C-T) and a 28-bp tandem repeat polymorphism (2R or 3R) in the TS promoter enhancer region (TSER) have been reported to be functional and are supposed to disturb the normal DNA methylation and synthesis leading to carcinogenesis. To investigate the association between these polymorphisms and the risk of esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA), we conducted a case-control study in the Anyang area where the incidence of ESCC is highest in northern China. Subjects consisted of 275 cases with ESCC, 129 cases with GCA and 310 sex- and age-matched cancer-free controls. The risk was evaluated in terms of age-sex adjusted odds ratios (ORs) and 95% confidence intervals (CIs) by unconditional logistic regression model. The ORs for the MTHFR677TT genotype compared with the MTHFR677CC/CT genotype were 1.62 (95% CI = 1.15-2.30) and 1.81 (1.17-2.81) for ESCC and GCA, respectively. The ORs for the TSER 2R/2R genotype relative to the other genotypes were 2.44 (0.89-6.73) and 3.94 (1.29-12.0) for SCC and GCA, respectively. These findings indicated that the folate metabolism plays an important role in carcinogenesis of ESCC and GCA and the common functionally polymorphisms MTHFRC677T and TSER have substantial influence in this metabolic pathway.

Exogenous nitric oxide donor and related compounds protect against lung inflammatory response after hemorrhagic shock and resuscitation.

BACKGROUND: Resuscitation from hemorrhagic shock triggers an inflammatory response characterized by upregulation of cytokine and adhesion molecule expression, increased leukocyte activity, and accumulation of polymorphonuclear neutrophils in a variety of tissues. This study investigated the capability of an exogenous nitric oxide (NO) donor, sodium nitroprusside (NP); a NO substrate, L-arginine; and an inducible NO synthase inhibitor, L-N6-(1-iminoethyl)lysine (L-NIL) to reduce lung injury in an animal model of mixed controlled and uncontrolled hemorrhagic shock. METHODS: For this study, 72 Sprague-Dawley rats weighing 250 to 300 g were subjected to a model of uncontrolled hemorrhagic shock for 150 minutes. Six groups of animals were included in this study (12 per group): sham-saline, sham-NP, shock-saline, shock-NP, shock-L-arginine, and shock-L-N6-(1-iminoethyl)lysine. After the period of hemorrhagic shock, resuscitation of the groups was accomplished using normal saline (groups 1 and 3), NP (0.5 mg/kg) (groups 2 and 4), L-arginine (300 mg/kg) (group 5), or L-NIL (50 mg/kg) (group 6). The following indices were evaluated: fluid requirements for resuscitation, mean arterial pressure (MAP), arterial po2, pco2, and pH, lung wet-to-dry weight ratio, lung histology and cytokine (interleukin [IL]-1 alpha, IL-beta 1, tumor necrosis factor-beta [TNF beta], IL-3, IL-4, IL-5, IL-6, IL-10, TNF alpha, IL-2, interferon-gamma [IFN gamma]), and mRNA expression in the lung by a ribonuclease protection assay (RPA). RESULTS: Sodium nitroprusside significantly increased MAP and reduced fluid requirements during resuscitation after hemorrhage. There also was a significant improvement in lung function, as expressed by improvements in po2, pco2, and pH, and reduction of the wet-to-dry weight ratio. In addition, a significant reduction in acute lung injury was observed in the histologic studies. Furthermore, the expression of cytokines was reduced by NP treatment. The use of L-arginine and L-NIL offered similar protective results for the injured lung. CONCLUSIONS: These data suggest that limiting inducible NO synthase-generated NO availability with the exogenous NO donor, sodium nitroprusside, may reduce lung injury after severe hemorrhage, possibly, among other effects, by downregulating the expression of inflammatory cytokines. L-arginine and L-NIL also had a beneficial effect on lung function and structure.

The attenuation of hemorrhage-induced liver injury by exogenous nitric oxide, L-arginine, and inhibition of inducible nitric oxide synthase.

We investigated the role of nitric oxide (NO) in its ability to reduce liver injury in an animal model of hemorrhagic shock (HS). Ninety-six Sprague-Dawley rats weighing 250 to 300 g were divided in 6 groups (n = 16 per group) that included treatment at the beginning of resuscitation with normal saline (groups 1, 3) sodium nitroprusside (NP) (0.5 mg/kg) (groups 2, 4) L-arginine (300 mg/kg) (group 5), and L-N6-(1-iminoethyl) lysine (L-NIL, 40 mg/kg) (group 6). The experimental model of HS consisted of the withdrawal of 3 mL blood per 100 g in a 15-min period, tail amputation (75%), and drug administration at 30 min. This was followed by fluid resuscitation (FR) with lactated Ringer's (LR) solution to reach a mean arterial pressure (MAP) of 40 mm Hg, then a hospital phase of 60 min with hemostasis and FR with LR solution to reach a MAP of 70 mm Hg with a 3-day observation phase. NP, L-Arginine, and L-NIL significantly reduced fluid requirements for resuscitation (p =.0001) as well as significantly increased MAP after resuscitation from hemorrhage. We also observed an improved statistically significant difference (p =.001) in tests demonstrating less hepatic injury and histology damage. The mRNA expression of cytokines in the liver (interleukin [IL]-1alpha, IL-beta1, tumor necrosis factor [TNF]beta, IL-3, IL-4, IL-5, IL-6, IL-10, TNFalpha, IL-2, interferon [IFN]gamma) was reduced by NP treatment, L-arginine, and L-NIL. These data suggest that excess NO mediates hemorrhage-induced liver injury and that the suppression of inducible nitric oxide synthase (iNOS)-generated NO bioavailability with the NO donor sodium nitroprusside may reduce the pathophysiologic consequences of severe hemorrhage. This effect could be possibly related to the scavenging of to superoxide radicals (O2-) or the blockade of the deleterious effects of TNF and other inflammatory cytokines. The protective action noted with L-arginine cannot be fully explained within the context of this article, although it could be most likely associated with the supplementation of eNOS-generated NO.

Role of IL-18 in acute lung inflammation.

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2-4 h in inflamed lungs and remained elevated at 24 h, and the "mature" protein form of IL-18 (18 kDa) appeared in BAL fluids 1-8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-gamma. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.

Regulatory effects of eotaxin on acute lung inflammatory injury.

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-kappaB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-alpha levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of (125)I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.

Protective effects of anti-C5a peptide antibodies in experimental sepsis.

We evaluated antibodies to different peptide regions of rat C5a in the sepsis model of cecal ligation and puncture (CLP) for their protective effects in rats. Rabbit polyclonal antibodies were developed to the following peptide regions of rat C5a: amino-terminal region (A), residues 1-16; middle region (M), residues 17-36; and the carboxyl-terminal region (C), residues 58-77. With rat neutrophils, the chemotactic activity of rat C5a was significantly inhibited by antibodies with the following rank order: anti-C > anti-M >> anti-A. In vivo, antibodies to the M and C (but not A) regions of C5a were protective in experimental sepsis, as determined by survival over a 10-day period, in a dose-dependent manner. The relative protective efficacies of anti-C5a preparations (in descending order of efficacy) were anti-C > anti-M >> anti-A. In CLP rats, a delay in infusion of antibodies, which were injected at 6 or 12 h after CLP, still resulted in significant improvement in survival rates. These in vivo and in vitro data suggest that there are optimal targets on C5a for blockade during sepsis and that delayed infusion of anti-C5a antibody until after onset of clinical evidence of sepsis still provides protective effects.

Role of C5a in multiorgan failure during sepsis.

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O(2), rising partial pressure of CO(2)). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.

Molecular cloning and characterization of DEFCAP-L and -S, two isoforms of a novel member of the mammalian Ced-4 family of apoptosis proteins.

We report the deduced amino acid sequences of two alternately spliced isoforms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box (Mg(2+)-binding site). Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; 2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; 3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery.

Role of RANTES in experimental cardiac allograft rejection.

The host response to alloantigen results in upregulation of Class II MHC antigens and associated cytokine production (especially IL-2 and interferon-gamma (IFN-gamma)) as well as lymphocytic infiltration and cellular activation which leads to graft damage/destruction. RANTES (Regulated upon Activation, Normal T-cell Expressed and presumably Secreted) is a member of the beta subfamily (CC) of chemokines and has been shown to function as a lymphocyte chemoattractant. We now describe the requirement for RANTES in cardiac heterotopic allograft (brown Norway into Lewis rats) rejection in rats. By Northern blot analysis, mRNA could be detected in allografts at 6 and 8 days post-transplantation but not in isogenic (Lewis) grafts. RANTES protein could be demonstrated by Western blot analysis in homogenates from allografts at day 8 but not at day 0 and could not be identified in isogenic cardiac transplants. By immunostaining, RANTES protein was present in mononuclear cells of allografts at day 6 but was absent in the isogenic transplants. When rats were treated with anti-RANTES serum, there was a significant delay in rejection time (cessation of beating) of the allografts. These data demonstrate that expression of RANTES in rat cardiac allografts is linked to the rejection phenomenon.

Protective effects of anti-C5a in sepsis-induced thymocyte apoptosis.

Multiorgan apoptosis occurs during sepsis. Following cecal ligation and puncture (CLP) in rats, thymocytes underwent apoptosis in a time-dependent manner. C5a blockade dramatically reduced thymocyte apoptosis as measured by thymic weight, binding of annexin V to thymocytes, and laddering of thymocyte DNA. When C5a was generated in vivo by infusion of purified cobra venom factor (CVF), thymocyte apoptosis was significantly increased. Similar results were found when CVF was injected in vivo during the early stages of CLP. In animals 12 hours after induction of CLP, there was an increase in the activities of caspase-3, -6, and -9, but not caspase-1 and -8. Cytosolic cytochrome c levels increased by twofold, whereas mitochondrial levels showed a 50% decrease. Western blot analysis revealed that the content of Bcl-X(L) (but not of Bcl-2, BAX, Bad, and Bim) significantly decreased in thymocytes after CLP. C5a blockade in the sepsis model almost completely inhibited caspase-3, -6, and -9 activation, significantly preserved cytochrome c in the mitochondrial fraction, and restored Bcl-X(L) expression. These data suggest that systemic activation of complement induces C5a-dependent apoptosis of thymocytes and that the blockade of C5a during sepsis rescues thymocytes from apoptosis.

Anti-inflammatory effects of mutant forms of secretory leukocyte protease inhibitor.

The secretory leukocyte protease inhibitor (SLPI) is found in a variety of secreted fluids in mammals and is a known inhibitor of serine proteases. Wild-type (WT) SLPI has recently been shown to block nuclear factor kappaB (NF-kappaB) activation in rat lungs and to interfere with the ensuing inflammatory response and recruitment of neutrophils after an intrapulmonary deposition of IgG immune complexes. In this study, WT SLPI and SLPI mutants with various degrees of protease-inhibitory capacity (for trypsin, chymotrypsin, and elastase) were evaluated for their ability to suppress the lung-vascular leak, neutrophil accumulation, and NF-kappaB activation in the lung inflammatory model. The SLPI mutant with Gly(72) (replacing Leu(72) ) lost its ability to block in vivo activation of NF-kappaB, as well as its ability to suppress the lung vascular leak and neutrophil recruitment. The Phe(72) and Gly(20) mutants were as effective as the WT SLPI in suppressing NF-kappaB activation and neutrophil recruitment. The Lys(72) mutant had the most suppressive effects of the lung vascular leak and for neutrophil recruitment into the lung. The in vivo suppressive effects of SLPI mutants on lung vascular permeability, neutrophil recruitment, and NF-kappaB activation appear to be most closely related to their trypsin-inhibiting activity. These data suggest that the suppressive effects of SLPI on the intrapulmonary activation of NF-kappaB and neutrophil recruitment into the lung may be linked to their antiprotease activity, directed, perhaps, at the intracellular proteases.

Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats.

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1 beta Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-alpha in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1 beta, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1 beta, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1 beta, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.

Eotaxin expression in Sephadex-induced lung injury in rats.

The CC chemokine eotaxin is a potent and specific eosinophil chemoattractant. Eosinophil-dependent tissue injury has been shown to contribute to airway inflammation such as that in asthma. In the present study, We investigated eotaxin expression in a rat model of pulmonary inflammation (featuring accumulation of eosinophils) induced by intratracheal instillation of cross-linked dextran beads (Sephadex G200). Intratracheal instillation of 5 mg/kg Sephadex caused a time-dependent eosinophil infiltration into the lung, reaching a peak at 24 hours. Eotaxin mRNA in the lung paralleled the eosinophil influx. Eotaxin protein in bronchoalveolar (BAL) fluids and lung homogenates was shown by Western blot and immunostaining to be maximally expressed by 24 hours. Sephadex-induced lung injury, as measured by (125)I-labeled albumin leakage from the pulmonary vasculature, developed in a time-dependent manner. Intravenous injection of blocking antibody to eotaxin significantly decreased eosinophil infiltration and lung permeability. These data suggest that, in the Sephadex model of lung inflammation, eotaxin up-regulation mediates intrapulmonary accumulation of eosinophils and the development of lung injury.

Molecular cloning and characterization of a novel human CC chemokine, SCYA26.

By searching the Expressed Sequence Tag database, a full-length cDNA for a novel human CC chemokine was cloned. This cDNA encoded a 94-amino-acid protein with a putative signal peptide of 26 amino acids. The deduced mature protein had the four conserved cysteine residues characteristic of CC chemokines and showed 44% identity with MIP-1beta and 40% identity with MIP-1alpha, RANTES, and MCP-4. mRNA for this chemokine was expressed constitutively in human heart and liver and with lesser but detectable levels in skeletal muscle, kidney, and small intestine. To investigate its biological activity, the protein was expressed in mammalian cells and purified by affinity chromatography. The recombinant protein demonstrated chemotactic activity in vitro for T cells and monocytes but not for neutrophils. The gene was mapped to chromosome 7q11.2 by fluorescence in situ hybridization. Based on its structural identity with other CC chemokines and the chemotactic activity and chromosomal location of this chemokine, we designate this chemokine small inducible cytokine subfamily A, member 26 (SCYA26). This gene symbol has been approved by the HUGO Gene Nomenclature Committee.