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.

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.

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.

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.

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.

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.

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.

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.

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.

Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults.

A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development.

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.

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.

Toward an understanding of the gene-specific and global logic of inducible gene transcription.

Virtually all living organisms have evolved mechanisms to adapt to their environment by sensing environmental stresses and inducing the transcription of appropriate sets of response genes in a coordinated fashion. In the vertebrate immune system, the highly selective response to an environmental stimulus, often an invading microorganism, plays an especially important role in regulating the activities of, and interactions among, the many cell types involved in innate and adaptive immunity. It is now widely appreciated that the selective response to a stimulus requires the concerted action of signal transduction pathways, transcription factors, and chromatin structure. Many proteins and pathways that help to regulate a response have been characterized. However, our understanding of the gene-specific and global logic through which a highly selective response is elicited has only recently begun to emerge.

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.

Resistance of MBL gene-knockout mice to experimental systemic aspergillosis.

Mannose binding lectin (MBL) is a protein of the collectin family that appears important in resistance to invasive pulmonary aspergillosis. We assessed the role of MBL in experimental systemic aspergillosis. MBL-sufficient C57BL/6 (WT) mice and B6.129S4--Mb11(tm1Kata) Mb12(tm1Kata)/J MBL A and C gene-knockout (KO) mice were infected intravenously with different inocula of Aspergillus fumigatus conidia. WT and KO mice were dose-responsively susceptible. In no instance were the KO mice more susceptible than WT. At the highest inoculum, all WT and 90% of KO mice died on day 4 (P>0.05). Reduction of the inoculum to 5.5 x 10(6) conidia was lethal, but comparison showed KO mice less susceptible to lethal infection (P<0.015). At the lowest inoculum used, deaths of KO mice were delayed, but survival was not significantly different than WT (P>0.05). These results suggest MBL may play a deleterious role in systemic aspergillosis.

Myocardial perfusion imaging and cardiovascular outcomes in a cancer population.

Myocardial perfusion imaging can predict outcomes in cardiac patients. However, limited data exist regarding its prediction of cardiovascular outcomes in cancer patients. We sought to determine whether myocardial perfusion imaging predicts long-term cardiovascular outcomes in cancer patients.We performed a retrospective review of 787 consecutive patients at our institution who underwent myocardial perfusion imaging from January 2001 through March 2003. The Cox proportional hazard model was applied, and total cardiac events, cardiac death, and all-cause death were determined for 3 years. We considered P <0.05 to be statistically significant.Patients with abnormal myocardial perfusion imaging results were more likely to be male and older, with heart disease, more vascular risk factors, and lower left ventricular ejection fraction (0.52 +/- 0.14 vs 0.63 +/- 0.11; P <0.001) than patients with normal myocardial perfusion imaging results. Multivariate predictors of total cardiac events included age (P = 0.023), hyperlipidemia (P = 0.0021), pharmacologic myocardial perfusion imaging (P <0.01), left ventricular ejection fraction (P <0.001), and abnormal myocardial perfusion imaging (P = 0.012). Multivariate predictors of cardiac death included age (P = 0.026) and left ventricular ejection fraction (P = 0.0001). Multivariate predictors of all-cause death were age (P = 0.0001), atrial fibrillation (P = 0.0012), and smoking (P <0.001). Overall survival was improved when patients took aspirin (P = 0.0002) and upon each unit increase in left ventricular ejection fraction (P <0.001).Myocardial perfusion imaging in cancer patients can predict 3-year cardiac outcomes. Increasing age, atrial fibrillation, and smoking were associated with worse outcomes, whereas higher left ventricular ejection fraction and the taking of aspirin were protective.

The role of nesiritide in heart failure.

BACKGROUND: Heart failure afflicts > 5 million patients in the US, with about 550,000 new diagnoses a year. Side effects and increased mortality limit heart failure treatment. Nesiritide is the newest addition to therapeutic options for treatment of acute decompensated heart failure (ADHF). It has been used as single in-patient infusions, multiple out-patient infusions, and perioperatively to improve hemodynamics and promote diuresis. Initially well-received, meta-analyses suggesting increased mortality and renal dysfunction after nesiritide use were published 4 - 5 years after its introduction in the US. These reports prompted new recommendations on nesiritide use by an expert panel. OBJECTIVE: Critical review of the studies leading to approval of nesiritide and the current recommendations for its use. METHODS: Medline search and Pubmed search using nesiritide or natrecor for text word searches. CONCLUSION: Nesiritide represents the first drug in a new class designed for treatment of ADHF. In patients with ADHF, nesiritide improves hemodynamic parameters and reduces dyspnea. Questions remain about possible increased mortality and side effects. A continuing study expected to enroll 7000 patients by 2010 has been designed to clarify whether nesiritide reduces mortality, hospital length-of-stay and renal parameters in patients with ADHF.

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.

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.