IL-1 and IL-1ra are key regulators of the inflammatory response to RNA vaccines.

The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here, we show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1ß, which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.

A Stringent Systems Approach Uncovers Gene-Specific Mechanisms Regulating Inflammation.

Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput datasets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif datasets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well-defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks.

Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.

Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.

Defined Sensing Mechanisms and Signaling Pathways Contribute to the Global Inflammatory Gene Expression Output Elicited by Ionizing Radiation.

Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.

Social Risk Factors That Increase Cardiovascular and Breast Cancer Risk.

PURPOSE OF REVIEW: Cardiovascular disease (CVD) and breast cancer (BC) are significant causes of mortality globally, imposing a substantial health burden. This review article aims to examine the shared risk factors and social determinants that contribute to the high prevalence of both diseases, with a focus on social risk factors. RECENT FINDINGS: The common risk factors for CVD and BC, such as hypertension, diabetes, obesity, aging, and physical inactivity, are discussed, emphasizing their modifiability. Adhering to ideal cardiovascular health behaviors has shown a trend toward lower BC incidence. Increased risk of CVD-related mortality is significantly impacted by age and race in BC patients, especially those over 45 years old. Additionally, racial disparities in both diseases highlight the need for targeted interventions. Social determinants of health, including socioeconomic status, education, employment, and neighborhood context, significantly impact outcomes for both CVD and BC. Addressing social factors is vital in reducing the burden of both CVD and BC and improving overall health equity.

Challenges in Cardiovascular Imaging in Women with Breast Cancer.

Cardiovascular imaging in breast cancer patients is paramount for the surveillance of cancer therapy-related cardiac dysfunction (CTRCD); however, it comes with specific limitations. PURPOSE OF REVIEW: This review aims to describe the unique challenges faced in cardiovascular imaging of breast cancer patients, discuss evidence to support the utility of various imaging modalities, and provide solutions for improvement in imaging this unique population. RECENT FINDINGS: Updated clinical society guidelines have introduced more unifying surveillance of CTRCD, although there remains a lack of a universally accepted definition. Traditional and novel multi-modality imaging can be used to detect CTRCD and myocarditis in breast cancer patients. Cardiovascular imaging in breast cancer patients is difficult due to reconstructive surgery. Although echocardiography with myocardial strain is the cornerstone, multi-modality imaging can be used to evaluate for CTRCD and myocarditis. Novel imaging techniques to improve the diagnosis of cardiotoxicities in breast cancer patients are needed.

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression.

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3' end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3'-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

Nucleotide modifications enable rational design of TLR7-selective ligands by blocking RNase cleavage.

Toll-like receptors 7 (TLR7) and 8 (TLR8) each sense single-stranded RNA (ssRNA), but their activation results in different immune activation profiles. Attempts to selectively target either TLR7 or TLR8 have been hindered by their high degree of homology. However, recent studies revealed that TLR7 and TLR8 bind different ligands resulting from the processing of ssRNA by endolysosomal RNases. We demonstrate that by introducing precise 2' sugar-modified bases into oligoribonucleotides (ORNs) containing known TLR7 and TLR8 binding motifs, we could prevent RNase-mediated degradation into the monomeric uridine required for TLR8 activation while preserving TLR7 activation. Furthermore, a novel, optimized protocol for CRISPR-Cas9 knockout in primary human plasmacytoid dendritic cells showed that TLR7 activation is dependent on RNase processing of ORNs and revealed a previously undescribed role for RNase 6 in degrading ORNs into TLR ligands. Finally, 2' sugar-modified ORNs demonstrated robust innate immune activation in mice. Altogether, we identified a strategy for creating tunable TLR7-selective agonists.

Experimental evidence that granulocyte transfusions are efficacious in treatment of neutropenic hosts with pulmonary aspergillosis.

Although polymorphonuclear leukocytes (PMNs) are powerfully anti-Aspergillus, transfusion therapy remains controversial, with conflicting results, and experimental support has been lacking. We devised a pulmonary infection model in neutropenic BALB/c mice, used an antibacterial regimen to prevent confounding sepsis, and optimized PMN induction, purifications, and dose. Mice were given 150 mg/kg cyclophosphamide every 4 days and a gentamicin-vancomycin-clindamycin-imipenem regimen daily beginning 4 days before intranasal challenge with 5 × 10(5) Aspergillus conidia. This regimen produced leukopenia (~10% of normal white blood cell [WBC] count; ≤ 10% PMNs) for 10 days, without bacterial superinfection. PMN donors given 100 µg/kg recombinant murine granulocyte colony-stimulating factor (G-CSF) for 10 days yielded 11 × 10(7) to 13.6 × 10(7) WBC/ml (81 to 87% PMNs). Infected mice were given PMN transfusions intravenously. In 2 experiments with up to 70% mortality of neutropenic controls, transfusion of 10(7) PMNs 1 and 4 days after challenge had negligible effects on peripheral WBC counts but improved survival (P = 0.007, 0.02), decreased lung CFU (P = 0.03, 0.005), and cleared infection in 28 to 50% of survivors. Transfusion of 5 × 10(6) PMNs showed partial protection. Transfusions given every other day did not improve protection. Our present results provide an experimental basis for enthusiasm for PMN transfusions in the therapy of aspergillosis in humans.

Systematic discovery of neoepitope-HLA pairs for neoantigens shared among patients and tumor types.

The broad application of precision cancer immunotherapies is limited by the number of validated neoepitopes that are common among patients or tumor types. To expand the known repertoire of shared neoantigen-human leukocyte antigen (HLA) complexes, we developed a high-throughput platform that coupled an in vitro peptide-HLA binding assay with engineered cellular models expressing individual HLA alleles in combination with a concatenated transgene harboring 47 common cancer neoantigens. From more than 24,000 possible neoepitope-HLA combinations, biochemical and computational assessment yielded 844 unique candidates, of which 86 were verified after immunoprecipitation mass spectrometry analyses of engineered, monoallelic cell lines. To evaluate the potential for immunogenicity, we identified T cell receptors that recognized select neoepitope-HLA pairs and elicited a response after introduction into human T cells. These cellular systems and our data on therapeutically relevant neoepitopes in their HLA contexts will aid researchers studying antigen processing as well as neoepitope targeting therapies.

Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2.

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

Comparative efficacies of lipid-complexed amphotericin B and liposomal amphotericin B against coccidioidal meningitis in rabbits.

In separate previous studies, we have shown that lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious against coccidioidal meningitis in rabbits. Here, we compared ABLC and AmBi directly in a coccidioidal meningitis model. Male New Zealand White rabbits were infected with 5 x 10(4) Coccidioides posadasii arthroconidia by direct cisternal puncture. Therapy with intravenous ABLC or AmBi at 7.5 or 15 mg/kg of body weight or sterile 5% dextrose water (D5W) began 5 days later. Clinical assessments were done daily; cerebrospinal fluid and blood samples were obtained on day 15 and upon euthanasia. Survivors to day 25 were euthanatized, the numbers of CFU in their tissues were determined, and histology analyses of the brains and spinal cords were done. Controls showed progressive disease, whereas animals treated with either dose of either drug showed few clinical signs of infection. All ABLC- or AmBi-treated rabbits survived, whereas eight of nine D5W-treated rabbits were euthanatized before day 25 (P < 0.0001). Numbers of CFU in the brains and spinal cords of ABLC- or AmBi-treated animals were 100- to 10,000-fold lower than those in the corresponding tissues of D5W-treated animals (P < 0.0006 to 0.0001). However, only two or fewer given a regimen of ABLC or AmBi were cured of infection in both tissues. Fewer ABLC-treated rabbits (four of eight treated with 7.5 mg/kg and five of eight treated with 15 mg/kg) than controls (nine of nine) had meningitis at any level of severity (P, 0.015 or 0.043 for animals treated with ABLC at 7.5 or 15 mg/kg, respectively). Although groups of rabbits treated with AmBi regimens did not have significantly fewer animals with meningitis than the control group (P > 0.05), ABLC and AmBi were not significantly different. In this model, intravenous ABLC and AmBi were similarly highly effective, with few clinical signs of infection, 100% survival, and significantly reduced fungal burdens among treated animals. There appeared to be little benefit in using the 15-mg/kg dosage of either formulation. There was no significant advantage of one drug over the other for this indication. Further studies are required to determine the lowest effective doses of these formulations.

Transposable element expression in tumors is associated with immune infiltration and increased antigenicity.

Profound global loss of DNA methylation is a hallmark of many cancers. One potential consequence of this is the reactivation of transposable elements (TEs) which could stimulate the immune system via cell-intrinsic antiviral responses. Here, we develop REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observe increased expression of over 400 TE subfamilies, of which 262 appear to result from a proximal loss of DNA methylation. The most recurrent TEs are among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent results in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing inflammation and the display of potentially immunogenic neoantigens.

Significant differences in drug susceptibility among species in the Candida parapsilosis group.

Candida parapsilosis family has 3 proposed species: C. parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. C. parapsilosis sensu stricto had significantly higher caspofungin (CAS) and anidulafungin MICs than C. orthopsilosis or C. metapsilosis; C. metapsilosis was least susceptible to fluconazole. C. parapsilosis sensu stricto more frequently displayed (37%) paradoxical growth in CAS (P < or = 0.02). These species susceptibility differences could affect therapeutic choices.

A Cross Sectional Study on Knowledge, Attitude and Practice related to Human Papillomavirus Vaccination for Cervical Cancer Prevention between Medical and Non-Medical Students in Hong Kong

Background: One of the most important aetiologies of cervical cancer is Human Papillomavirus (HPV) infection. While vaccination is an effective way in preventing high risk HPV infection, HPV vaccine uptake rate in Hong Kong has been low. Considering the proven effectiveness of HPV vaccination and the low vaccination uptake rate in Hong Kong, this study was conducted to compare the knowledge, attitude and practice towards HPV vaccination for cervical cancer prevention between medical and non-medical students in the University of Hong Kong. Methods: A total of 420 full time undergraduates from the University of Hong Kong were recruited and evaluated. Questionnaires covering demographics, sexual risk profile, knowledge, attitude and practice towards HPV vaccination were applied, with the Chi-square test analysis. Results: Medical students had more comprehensive knowledge than their non-medical counterparts on HPV vaccination, including the carcinogenicity of HPV (P<0.001), available vaccines on the market (P<0.001) and the outcome of vaccination (P<0.001). In particular, senior medical students (Year 3 or above) were shown to be more knowledgeable than their juniors (below Year 3) with statistical significance (P<0.001). Positive attitudes toward HPV vaccination were observed more frequently among medical students when compared to non-medical students, especially regarding the usefulness of HPV vaccination in males (P<0.001). However, there was no significant difference in the vaccination rate between medical and non-medical students (P=0.671), suggesting an importance for factors other than knowledge, such as cost of vaccination and anxiety of side effects. Conclusions: Medical students in Hong Kong, especially those in senior years, had more comprehensive knowledge and positive attitudes towards HPV vaccination than non-medical students. Yet, there was no significant difference in the practice of HPV vaccination between medical and non-medical students. In addition to medical education, other factors such as health beliefs, risk perception and financial considerations, may have a role in determining HPV vaccination for cervical cancer prevention.

Cardiovascular complications of cancer therapy: diagnosis, pathogenesis, and management.

The cardiotoxicity of anticancer agents can lead to significant complications that can affect patients being treated for various malignancies. The severity of such toxicity depends on many factors such as the molecular site of action, the immediate and cumulative dose, the method of administration, the presence of any underlying cardiac condition, and the demographics of the patient. Moreover, toxicity can be affected by current or previous treatment with other antineoplastic agents. Cardiotoxic effects can occur immediately during administration of the drug, or they may not manifest themselves until months or years after the patient has been treated. In this article we review commonly used chemotherapy agents, including several recently approved medications, for their propensity to cause cardiotoxicity. Further research will be required to more accurately predict which patients are at risk for developing cardiotoxicity. In addition, management plans, as well as strategies to reduce cardiotoxicity, need to be developed.

Phase II study of denileukin diftitox for relapsed/refractory B-Cell non-Hodgkin's lymphoma.

PURPOSE: Denileukin diftitox is a fusion protein combining diphtheria toxin and interleukin-2 (IL-2) that targets tumor cells expressing the IL-2 receptor. Its efficacy has been shown in CD25+ cutaneous T-cell lymphoma, but not in B-cell non-Hodgkin's lymphoma (NHL). A phase II study was performed to evaluate the efficacy and tolerability of denileukin diftitox for relapsed or refractory B-cell NHL. PATIENTS AND METHODS: Patients with relapsed or refractory B-cell NHL were eligible. Tumor CD25 expression was determined by immunohistochemistry or flow cytometry. Denileukin diftitox was administered intravenously at a dose of 18 microg/kg once daily for 5 days every 3 weeks, up to eight cycles. RESULTS: Of the 45 patients assessable for response, 32 (71%) were refractory to the last chemotherapy treatment, and all were previously treated with rituximab. Three complete responses (6.7%) and eight partial responses (17.8%) were observed, for an overall response rate of 24.5%. Nine patients (20%) had stable disease. Objective response rates were similar in CD25+ (22%) and CD25- histologies (29%), as were stable disease rates (22% and 18%, respectively). For responding patients, the median time to treatment failure was 7 months, with a median follow-up in survivors of 18 months (range, 9 to 28 months), and the projected progression-free survival at 20 months was 24% (95% CI, 0% to 60%). Most toxicities were low-grade and transient. CONCLUSION: Denileukin diftitox seems to be effective in relapsed or refractory, CD25+ and CD25- B-cell NHL and is well-tolerated at the dosage evaluated. Evaluation of denileukin diftitox in combination with other agents may be warranted.

Transesophageal echocardiography improves risk assessment of thrombolysis of prosthetic valve thrombosis: results of the international PRO-TEE registry.

OBJECTIVES: The goal of this study was to evaluate whether quantitation of thrombus burden with transesophageal echocardiography (TEE) can help risk-stratify patients undergoing thrombolysis of prosthetic valve thrombosis (PVT). BACKGROUND: Thrombolytic therapy of PVT has an unpredictable risk of embolization and complications. METHODS: An international registry of patients with suspected PVT undergoing two-dimensional/Doppler and TEE before thrombolysis was established. All TEE studies were reviewed and quantitated by a single observer blinded to all data. RESULTS: From 1985 to 2001, 107 patients (71 females; age 24 to 86 years) from 14 centers (6 in the U.S.) were identified. The majority of cases involved the mitral valve (79 mitral, 13 aortic, and 15 tricuspid). Hemodynamic success rate was achieved in 85% and was similar across valves. Overall complications were observed in 17.8%, and death in 5.6%. Predictors of complications were: New York Heart Association (NYHA) functional class, presence of shock, sinus tachycardia, hypotension, previous history of stroke, thrombus extension beyond the valve ring, and thrombus area. Multivariate analysis demonstrated that two variables were independent predictors of complications: thrombus area by TEE (odds ratio [OR] 2.41 per 1 cm2 increment, 95% confidence interval [CI] 1.12 to 5.19) and prior history of stroke (OR 4.55, 95% CI 1.35 to 15.38). A thrombus area <0.8 cm2 identified patients at lower risk for complications from thrombolysis, irrespective of NYHA functional class. CONCLUSIONS: In PVT, the thrombus size imaged with TEE is a significant independent predictor of outcome. Transesophageal echocardiography can identify low-risk groups for thrombolysis irrespective of symptom severity and is therefore recommended in the management of prosthetic valve thrombosis.

Toxic epidermal necrolysis associated with denileukin diftitox (DAB389IL-2) administration in a patient with follicular large cell lymphoma.

Denileukin diftitox (DAB(389)IL-2 or Ontak) is a synthetic fusion protein with demonstrated efficacy in a number of lymphoproliferative disorders, including non-Hodgkin's lymphoma. We report the case of a 45-year-old man with progressive follicular large cell lymphoma following an autologous stem cell transplant treated with denileukin diftitox who developed a fatal skin rash associated with extensive erythema, edema and large bullae involving his entire body. The clinical features and pathology were compatible with toxic epidermal necrolysis. This is the first reported case of toxic epidermal necrolysis in the literature associated with denileukin diftitox.