Loss of NF-κB1 Causes Gastric Cancer with Aberrant Inflammation and Expression of Immune Checkpoint Regulators in a STAT-1-Dependent Manner.

Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.

The Engineered Lysin, CF-370, is Active Against Antibiotic-Resistant Gram-Negative Pathogens In vitro and Synergizes with Meropenem in Experimental Pseudomonas aeruginosa Pneumonia.

BACKGROUND: Lysins (cell wall hydrolases) targeting Gram-negative organisms require engineering to permeabilize the outer membrane and access subjacent peptidoglycan to facilitate killing. In the current study, the potential clinical utility for engineered lysin, CF-370, was examined in vitro and in vivo against Gram-negative pathogens important in human infections. METHODS: MICs and bactericidal activity were determined using standard methods. An in vivo proof-of-concept efficacy study was conducted using a rabbit acute pneumonia model caused by Pseudomonas aeruginosa. RESULTS: CF-370 exhibited potent antimicrobial activity, with MIC50/90 values (in µg/mL) for: P. aeruginosa, 1/2; Acinetobacter baumannii, 1/1; Escherichia coli, 0.25/1; Klebsiella pneumoniae, 2/4; Enterobacter cloacae 1/4; and Stenotrophomonas maltophilia 2/8. CF-370 furthermore demonstrated: i) bactericidal activity; (ii) activity in serum; iii) a low propensity for resistance; iv) anti-biofilm activity; and v) synergy with antibiotics. In the pneumonia model, CF-370 alone decreased bacterial densities in lungs, kidneys and spleen vs. vehicle control, and demonstrated significantly increased efficacy when combined with meropenem (vs either agent alone). CONCLUSIONS: CF-370 is the first engineered lysin described with potent broad spectrum in vitro activity against multiple clinically-relevant Gram-negative pathogens, as well as potent in vivo efficacy in an animal model of severe invasive multi-system infection.

Convergent CDR3 homology amongst Spike-specific antibody responses in convalescent COVID-19 subjects receiving the BNT162b2 vaccine.

Convalescent coronavirus disease 2019 (COVID-19) subjects who receive BNT162b2 develop robust antibody responses against SARS-CoV-2. However, our understanding of the clonal B cell response pre- and post-vaccination in such individuals is limited. Here we characterized B cell phenotypes and the BCR repertoire after BNT162b2 immunization in two convalescent COVID-19 subjects. BNT162b2 stimulated many B cell clones that were under-represented during SARS-CoV-2 infection. In addition, the vaccine generated B cell clusters with >65% similarity in CDR3 VH and VL region consensus sequences both within and between subjects. This result suggests that the CDR3 region plays a dominant role adjacent to heavy and light chain V/J pairing in the recognition of the SARS-CoV-2 spike protein. Antigen-specific B cell populations with homology to published SARS-CoV-2 antibody sequences from the CoV-AbDab database were observed in both subjects. These results point towards the development of convergent antibody responses against the virus in different individuals.

Oncology clinical trial disruption during the COVID-19 pandemic: a COVID-19 and cancer outcomes study.

BACKGROUND: COVID-19 disproportionately impacted patients with cancer as a result of direct infection, and delays in diagnosis and therapy. Oncological clinical trials are resource-intensive endeavors that could be particularly susceptible to disruption by the pandemic, but few studies have evaluated the impact of the pandemic on clinical trial conduct. PATIENTS AND METHODS: This prospective, multicenter study assesses the impact of the pandemic on therapeutic clinical trials at two large academic centers in the Northeastern United States between December 2019 and June 2021. The primary objective was to assess the enrollment on, accrual to, and activation of oncology therapeutic clinical trials during the pandemic using an institution-wide cohort of (i) new patient accruals to oncological trials, (ii) a manually curated cohort of patients with cancer, and (ii) a dataset of new trial activations. RESULTS: The institution-wide cohort included 4756 new patients enrolled to clinical trials from December 2019 to June 2021. A major decrease in the numbers of new patient accruals (-46%) was seen early in the pandemic, followed by a progressive recovery and return to higher-than-normal levels (+2.6%). A similar pattern (from -23.6% to +30.4%) was observed among 467 newly activated trials from June 2019 to June 2021. A more pronounced decline in new accruals was seen among academically sponsored trials (versus industry sponsored trials) (P < 0.05). In the manually curated cohort, which included 2361 patients with cancer, non-white patients tended to be more likely taken off trial in the early pandemic period (adjusted odds ratio: 2.60; 95% confidence interval 1.00-6.63), and substantial pandemic-related deviations were recorded. CONCLUSIONS: Substantial disruptions in clinical trial activities were observed early during the pandemic, with a gradual recovery during ensuing time periods, both from an enrollment and an activation standpoint. The observed decline was more prominent among academically sponsored trials, and racial disparities were seen among people taken off trial.

Spontaneous mutations in maize pollen are frequent in some lines and arise mainly from retrotranspositions and deletions.

While studying spontaneous mutations at the maize bronze (bz) locus, we made the unexpected discovery that specific low-copy number retrotransposons are mobile in the pollen of some maize lines, but not of others. We conducted large-scale genetic experiments to isolate new bz mutations from several Bz stocks and recovered spontaneous stable mutations only in the pollen parent in reciprocal crosses. Most of the new stable bz mutations resulted from either insertions of low-copy number long terminal repeat (LTR) retrotransposons or deletions, the same two classes of mutations that predominated in a collection of spontaneous wx mutations [Wessler S (1997) The Mutants of Maize, pp 385-386]. Similar mutations were recovered at the closely linked sh locus. These events occurred with a frequency of 2-4 × 10-5 in two lines derived from W22 and in 4Co63, but not at all in B73 or Mo17, two inbreds widely represented in Corn Belt hybrids. Surprisingly, the mutagenic LTR retrotransposons differed in the active lines, suggesting differences in the autonomous element make-up of the lines studied. Some active retrotransposons, like Hopscotch, Magellan, and Bs2, a Bs1 variant, were described previously; others, like Foto and Focou in 4Co63, were not. By high-throughput sequencing of retrotransposon junctions, we established that retrotranposition of Hopscotch, Magellan, and Bs2 occurs genome-wide in the pollen of active lines, but not in the female germline or in somatic tissues. We discuss here the implications of these results, which shed light on the source, frequency, and nature of spontaneous mutations in maize.

Development of a Broth Microdilution Method for Exebacase Susceptibility Testing.

Exebacase (CF-301) belongs to a new class of protein-based antibacterial agents, known as lysins (peptidoglycan hydrolases). Exebacase, a novel lysin with antistaphylococcal activity, is in phase 3 of clinical development. To advance into the clinic, it was necessary to develop an accurate and reproducible method for exebacase MIC determination. The Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method using cation-adjusted Mueller-Hinton broth (CAMHB) produced trailing MIC endpoints, and exebacase activity was diminished when frozen BMD panels were used. A modified BMD method was developed using CAMHB supplemented with 25% horse serum and 0.5 mM dl-dithiothreitol (CAMHB-HSD). Preliminary quality control (QC) ranges for Staphylococcus aureus ATCC 29213 of 0.25 to 1 µg/ml and for Enterococcus faecalis ATCC 29212 of 16 to 64 µg/ml were determined based on the results of a CLSI M23-defined MIC QC tier 1 study. These preliminary QC ranges validated the MIC data generated from a systematic study testing a discrete S. aureus strain collection using CAMHB-HSD to investigate the impact of parameters known to influence susceptibility test results and to evaluate the exebacase MIC distribution against clinical S. aureus isolates. Presentation of these data led to the CLSI Subcommittee on Antimicrobial Susceptibility Testing (AST) approval of the use of CAMHB-HSD to determine exebacase susceptibility and commencement of a multilaboratory (tier 2) QC study. Use of a standard BMD method and concomitant QC testing provides confidence in the assessment of test performance to generate accurate and reproducible susceptibility data during antibacterial drug development.

Loss of NFKB1 Results in Expression of Tumor Necrosis Factor and Activation of Signal Transducer and Activator of Transcription 1 to Promote Gastric Tumorigenesis in Mice.

BACKGROUND & AIMS: Activity of nuclear factor κB transcription factors and signaling via signal transducer and activator of transcription (STAT) are frequently altered in gastric cancer cells. Mice lacking NFKB1 (Nfkb1-/- mice) develop invasive gastric cancer, and their gastric tissues have increased levels of cytokines, such as interleukin (IL) 6, IL22, IL11, and tumor necrosis factor (TNF), as well as increased activation of STAT1. We investigated whether these cytokines were required for STAT1 activation in gastric tissues of mice and critical for gastric tumorigenesis. METHODS: We crossed Nfkb1-/- mice with Il6-/-, Il22-/-, Il11Rα-/-, and Tnf-/- mice. Stomach tissues from compound mutant mice were analyzed by histology, immunoblotting, and RNA sequencing. Lymphoid, myeloid, and epithelial cells were isolated from stomachs, and the levels of cytokines were determined by flow cytometric analysis. RESULTS: Nfkb1-/- mice developed gastritis, oxyntic atrophy, gastric dysplasia, and invasive tumors, whereas Nfkb1-/-Stat1-/- mice did not, even when followed for as long as 2 years. The levels of Il6, Il11, Il22, and Tnf messenger RNA were increased in the body and antrum of the stomachs from Nfkb1-/- mice, from 3-6 months of age. However, Nfkb1-/-Il6-/-, Nfkb1-/-Il22-/-, and Nfkb1-/-Il11Rα-/- mice still developed gastric tumors, although the absence of IL11 receptor (IL11R) significantly reduced development of invasive gastric tumors. Stomachs from Nfkb1-/-Tnf-/- mice exhibited significantly less gastritis and oxyntic atrophy and fewer tumors than Nfkb1-/- mice. This correlated with reduced activation of STAT1 and STAT3 and fewer numbers of T cells and B cells infiltrating the gastric body. Loss of STAT1 or TNF significantly reduced expression of PD-L1 on epithelial and myeloid (CD11b+) cells in the gastric mucosa of Nfkb1-/- mice-indeed, to the levels observed on the corresponding cells from wild-type mice. CONCLUSIONS: In studies of gastric tumor development in knockout mice, we found that loss of NFKB1 causes increased expression of TNF in the stomach and thereby drives activation of STAT1, resulting in an inflammatory immune response and the development of gastric cancer. IL11R appears to be required for the progression of gastric tumors to the invasive stage. These findings suggest that inhibitors of TNF, and possibly also inhibitors of IL11/IL11Rα, might be useful in the treatment of gastric cancer.

Clinical and Electroencephalographic Correlates in Pediatric Cardiac Arrest: Experience at a Tertiary Care Center.

Pediatric cardiac arrest is a significant cause of death and neurologic disability; however, there is a paucity of literature specifically evaluating the utility of prognostic factors in the pediatric population. This retrospective chart review examines clinical, laboratory, and electroencephalographic (EEG) data in children following cardiopulmonary arrest to better characterize findings that may inform prognosis. Pre-arrest clinical characteristics, resuscitation details, and post-arrest hospital course variables were analyzed and neurologic outcome was determined using the Pediatric Cerebral Performance Category scale. Forty-one patients were identified who had cardiac arrest from March, 2011 to January, 2015. Duration of cardiopulmonary resuscitation (p = 0.013), out-of-hospital arrest (p = 0.005), arterial pH (0.014), arterial lactate (0.004), lack of pupil reactivity to light (p < 0.001), absent motor response to noxious stimuli (p < 0.001), and absent brainstem reflexes (p < 0.001) were all predictors of poor neurologic outcome. EEG background suppression (p = 0.005) was associated with poor outcome. Nine patients had electrographically recorded seizures, which began up to 1 week following cardiac arrest. Two patients (4.9%) experienced post-anoxic myoclonic status epilepticus and both had a poor outcome.

HBV/HIV coinfection is associated with poorer outcomes in hospitalized patients with HBV or HIV.

We examined the impact of HBV/HIV coinfection on outcomes in hospitalized patients compared to those with HBV or HIV monoinfection. Using the 2011 US Nationwide Inpatient Sample, we identified patients who had been hospitalized with HBV or HIV monoinfection or HBV/HIV coinfection using ICD-9-CM codes. We compared liver-related admissions between the three groups. Multivariable logistic regression was performed to identify independent predictors of in-hospital mortality, length of stay and total charges. A total of 72 584 discharges with HBV monoinfection, 133 880 discharges with HIV monoinfection and 8156 discharges with HBV/HIV coinfection were included. HBV/HIV coinfection was associated with higher mortality compared to HBV monoinfection (OR 1.67, 95% CI 1.30-2.15) but not when compared to HIV monoinfection (OR 1.22, 95% CI 0.96-1.54). However, the presence of HBV along with cirrhosis or complications of portal hypertension was associated with three times greater in-hospital mortality in patients with HIV compared to those without these complications (OR 3.00, 95% CI 1.80-5.02). Length of stay and total hospitalization charges were greater in the HBV-/HIV-coinfected group compared to the HBV monoinfection group (+1.53 days, P < 0.001; $17595, P < 0.001) and the HIV monoinfection group (+0.62 days, P = 0.034; $8840, P = 0.005). In conclusion, HBV/HIV coinfection is a risk factor for in-hospital mortality, particularly in liver-related admissions, compared to HBV monoinfection. Overall healthcare utilization from HBV/HIV coinfection is also higher than for either infection alone and higher than the national average for all hospitalizations, thus emphasizing the healthcare burden from these illnesses.

Evaluation of microbially enhanced composting of sophora flavescens residues.

The effects of inoculants on the composting of Sophora flavescens residues were evaluated based on several physical, chemical and biological parameters, as well as the infrared spectra. Compared to the control compost without inoculants, the treatment compost with inoculants (Bacillus subtilis strain G-13 and Chaetomium thermophilum strain GF-1) had a significantly longer thermophilic duration, higher cellulase activity and a higher degradation rate of cellulose, hemicellulose and lignin (P < 0.05). Thus, a higher maturity degree of compost with apparently lower C:N ratio (15.88 vs. 17.77) and NH4-N:NO3-N ratio (0.16 vs. 0.20) was obtained with the inoculation comparing with the control (P < 0.05). Besides, the inoculants could markedly accelerate the composting process and increase the maturity degree of compost as indicated by the germination index (GI) in which the treatment reached the highest GI of 133.2% at day 15 while the control achieved the highest GI of 125.7% at day 30 of the composting. Inoculation with B. subtilis and C. thermophilum is a useful method to enhance the S. flavescens residues composting according to this study.

The spectrum and frequency of self-inflicted and host gene mutations produced by the transposon Ac in maize.

The autonomous transposon Activator (Ac) is a powerful mutagen. Ac-induced mutations range from small footprints of host sequences to large rearrangements of transposon or host sequences. These mutations arise by different repair mechanisms of the double-strand break produced by Ac excision: footprints by nonhomologous end joining and rearrangements by various mechanisms, including DNA replication repair. Footprints greatly outnumber other mutations, masking them because they usually share a nonfunctional phenotype. To determine the spectrum and frequencies of host and self-mutations generated by Ac, we used an allele harboring Ac in the 5' untranslated region bronze (bz). In this system, simple excisions produce purple revertants, whereas deletions of host or transposon sequences produce stable bronze (bz-s) mutants. Internal and terminal deletions of Ac predominated among the 72 bz-s derivatives. Most internal deletions (52 of 54) behaved as nonautonomous Dissociation (Ds) elements. All nine terminal deletions or fractured Ac (fAc) elements had rearrangements of adjacent host sequences. Most Ds and fAc deletion junctions displayed microhomologies and contained filler DNA from nearby sequences, suggesting an origin by DNA repair synthesis followed by microhomology-mediated end joining. All mutations occurred more frequently in pollen, where one in 200 grains carried new Ds or fAc elements.

Huntingtin gene CAG repeat numbers in Chinese patients with Huntington's disease and controls.

BACKGROUND AND PURPOSE: Huntington's disease is due to a CAG triplet repeat elongation in the huntingtin gene. Boundaries in CAG numbers have been found between healthy people with and without risk to pass the disorder to the next generation, and between people without, with a mild, or with a fully penetrant phenotype. These data have been generated in western populations and it is not clear whether they are also valid amongst Chinese. METHODS: In order to establish normative data in the huntingtin gene for Chinese people, 966 chromosomes from normal controls were tested. Further, the range of CAG repeats was examined in a cohort from six centres and a total of 368 patients with the disease were included. RESULTS: The CAG triplet repeat range in normal controls was between 9 and 35 (mean 18.9, SD 2.57). Triplets in the range between 26 and 35 were found in 2.5%. In the patient cohort, triplet repeats in the shorter allele were between 8 and 37 (mean 17.7, SD 1.6). In the longer allele, a range between 36 and 120 was found. There was a negative correlation (-0.65, r = 0.42) between age at onset and the number of triplet repeats in the larger allele. The mean age at onset was 38 years, with a range between 2 and 70 years. In 23 patients (6%) a childhood or juvenile onset was noted. CONCLUSION: These data show comparable ranges of huntingtin gene CAG triplet repeats in normal people and in patients with Huntington's disease as in western populations.

Biomechanical analysis of the longitudinal ligament of upper cervical spine in maintaining atlantoaxial stability.

STUDY DESIGN: In vitro human cadaveric biomechanical study. OBJECTIVES: To investigate the roles of transverse atlantal ligament (TAL) and longitudinal ligament (LL) of the upper cervical spine (UCS) in maintaining atlantoaxial stability. SETTING: China. METHODS: Six intact UCS specimens were harvested and embedded in polymethylmethacrylate. Three-dimensional movements including flexion, extension, right and left lateral bending, and axial rotation, as well as the C1-C2 displacement in flexion (atlantodental interval, ADI), were tested on specimens with the following state sequentially: (1) intact (intact group), (2) TAL transected (TAL group) and (3) TAL and LL disrupted (TAL+LL group) using an electromechanical testing machine. RESULTS: Compared with intact group, the flexion/extension motion range and ADI were significantly higher in TAL group when the loading was 10 N or >100 N. However, no significant differences were detected between the two groups within a range of physiological loading (10-100 N). Similarly, significant differences in right-left lateral bending and axial rotation between TAL and intact groups occurred only when the loading was 150 N. However, when both of the TAL and LL were resected, the atlantoaxial joint showed obvious instability compared with TAL or intact group, which were further demonstrated in the analyses of the three-dimensional movements (significant differences at any loading). CONCLUSION: Within physiological loading range, the LLs have sufficient capacities to maintain the stability of atlantoaxial joint even if there are TAL injuries in atlas fractures.

Genetic mapping of three quantitative trait loci for soybean aphid resistance in PI 567324.

Host-plant resistance is an effective method for controlling soybean aphid (Aphis glycines Matsumura), the most damaging insect pest of soybean (Glycine max (L.) Merr.) in North America. Recently, resistant soybean lines have been discovered and at least four aphid resistance genes (Rag1, Rag2, Rag3 and rag4) have been mapped on different soybean chromosomes. However, the evolution of new soybean aphid biotypes capable of defeating host-plant resistance conferred by most single genes demonstrates the need for finding germplasm with multigenic resistance to the aphid. This study was conducted to map quantitative trait loci (QTL) for aphid resistance in PI 567324. We identified two major QTL (QTL_13_1 and QTL_13_2) for aphid resistance on soybean chromosome 13 using 184 recombinant inbred lines from a 'Wyandot' × PI 567324 cross. QTL_13_1 was located close to the previously reported Rag2 gene locus, and QTL_13_2 was close to the rag4 locus. A minor QTL (QTL_6_1) was also detected on chromosome 6, where no gene for soybean aphid resistance has been reported so far. These results indicate that PI 567324 possesses oligogenic resistance to the soybean aphid. The molecular markers closely linked to the QTL reported here will be useful for development of cultivars with oligogenic resistance that are expected to provide broader and more durable resistance against soybean aphids compared with cultivars with monogenic resistance.

Characterization of a novel dual murine model of chemotherapy-induced oral and intestinal mucositis.

Oral and intestinal mucositis are debilitating inflammatory diseases observed in cancer patients undergoing chemo-radiotherapy. These are devastating clinical conditions which often lead to treatment disruption affecting underlying malignancy management. Although alimentary tract mucositis involves the entire gastrointestinal tract, oral and intestinal mucositis are often studied independently utilizing distinct organ-specific pre-clinical models. This approach has however hindered the development of potentially effective whole-patient treatment strategies. We now characterize a murine model of alimentary tract mucositis using 5-Fluorouracil (5-FU). Mice were given 5-FU intravenously (50 mg/kg) or saline every 48 h for 2 weeks. Post initial injection, mice were monitored clinically for weight loss and diarrhea. The incidence and extent of oral mucositis was assessed macroscopically. Microscopical and histomorphometric analyses of the tongue and intestinal tissues were conducted at 3 interim time points during the experimental period. Repeated 5-FU treatment caused severe oral and intestinal atrophy, including morphological damage, accompanied by body weight loss and mild to moderate diarrhea in up to 77.8% of mice. Oral mucositis was clinically evident throughout the observation period in 88.98% of mice. Toluidine blue staining of the tongue revealed that the ulcer size peaked at day-14. In summary, we have developed a model reproducing the clinical and histologic features of both oral and intestinal mucositis, which may represent a useful in vivo pre-clinical model for the study of chemotherapy-induced alimentary tract mucositis and the development of preventative therapies.

High molecular weight hyaluronic acid drastically reduces chemotherapy-induced mucositis and apoptotic cell death.

Oral and intestinal mucositis (OIM) are debilitating inflammatory diseases initiated by oxidative stress, resulting in epithelial cell death and are frequently observed in cancer patients undergoing chemo-radiotherapy. There are currently few preventative strategies for this debilitating condition. Therefore, the development of a safe and effective mucositis mitigating strategy is an unmet medical need. Hyaluronic acid (HA) preparations have been tentatively used in oral mucositis. However, the protective effects of HA in chemotherapy-induced mucositis and their underlying mechanisms remain to be fully elucidated. This study aimed to assess these mechanisms using multiple formulations of enriched HA (Mucosamin®), cross-linked (xl-), and non-crosslinked high molecular weight HA (H-MW-HA) in an oxidative stress-induced model of human oral mucosal injury in vitro and an in vivo murine model of 5-flurouracil (5-FU)-induced oral/intestinal mucositis. All tested HA formulations protected against oxidative stress-induced damage in vitro without inducing cytotoxicity, with H-MW-HA also significantly reducing ROS production. Daily supplementation with H-MW-HA in vivo drastically reduced the severity of 5-FU-induced OIM, prevented apoptotic damage and reduced COX-2 enzyme activity in both the oral and intestinal epithelium. In 5-FU-injected mice, HA supplementation also significantly reduced serum levels of IL-6 and the chemokine CXCL1/KC, while the serum antioxidant activity of superoxide dismutase was elevated. Our data suggest that H-MW-HA attenuates 5-FU-induced OIM, at least partly, by impeding apoptosis, inhibiting of oxidative stress and suppressing inflammatory cytokines. This study supports the development of H-MW-HA preparations for preventing OIM in patients receiving chemotherapy.

Disruption of Nox2 and TNFRp55/p75 eliminates cardioprotection induced by anisomycin.

Transient activation of p38 through anisomycin is demonstrated to precondition the heart against myocardial injury. However, it remains unknown whether specific TNF-α receptor (TNFR) p55/p75 and Nox2, a subunit of NADPH oxidase, are involved in this event. We sought to investigate whether the genetic disruption of TNFRp55/p75 and Nox2 eliminated cardioprotection elicited by anisomycin and whether p38-dependent activation of Nox2 stimulated TNFR to ultimately achieve protective effects. Adult wild-type and TNFR p55/p75(-/-) and Nox2(-/-) mice received intraperitoneal injections of anisomycin (0.1 mg/kg), a potent activator of p38. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after 24 h. Left ventricular function was measured, and infarct size was determined. Myocardial TNF-α protein, Nox2, and superoxides releases were detected. Gel kinase assay was employed to detect the effect of p38 on Nox2 phosphorylation. Activation of p38 through anisomycin produces marked improvements in left ventricular functional recovery, and the reduction of myocardial infarction, which were abrogated by disruption of Nox2 and TNFR p55/p75. Disruption of Nox2 and TNFR p55/p75 abolished the effect of anisomycin-induced reduction of infarct size. Anisomycin induced the production of TNF-α, which was abrogated in Nox2(-/-) mice and by treatment with SB203580, but not by disruption of p55/p75. Anisomycin treatment resulted in an increase in Nox2 protein and the phosphorylation of Nox2, which was blocked by inhibition of p38. Taken together, these results indicate that stimulation of the Nox2 and TNFR p55/p75 pathway is a novel approach to anisomycin-induced cardioprotection.