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.

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.

High-throughput identification of conditional MHCI ligands and scaled-up production of conditional MHCI complexes.

Despite the need to monitor the impact of Cancer Immunotherapy (CI)/Immuno-Oncology (IO) therapeutics on neoantigen-specific T-cell responses, very few clinical programs incorporate this aspect of immune monitoring due to the challenges in high-throughput (HTP) generation of Major Histocompatibility Complex Class I (MHCI) tetramers across a wide range of HLA alleles. This limitation was recently addressed through the development of MHCI complexes with peptides containing a nonnatural UV cleavable amino acid (conditional MHCI ligands) that enabled HTP peptide exchange upon UV exposure. Despite this advancement, the number of alleles with known conditional MHCI ligands is limited. We developed a novel workflow to enable identification and validation of conditional MHCI ligands across a range of HLA alleles. First, known peptide binders were screened via an enzyme-linked immunosorbent assay (ELISA) assay. Conditional MHCI ligands were designed using the highest-performing peptides and evaluated in the same ELISA assay. The top performers were then selected for scale-up production. Next-generation analytical techniques (LC/MS, SEC-MALS, and 2D LC/MS) were used to characterize the complex after refolding with the conditional MHCI ligands. Finally, we used 2D LC/MS to evaluate peptide exchange with these scaled-up conditional MHCI complexes after UV exposure with validated peptide binders. Successful peptide exchange was observed for all conditional MHCI ligands upon UV exposure, validating our screening approach. This approach has the potential to be broadly applied and enable HTP generation of MHCI monomers and tetramers across a wider range of HLA alleles, which could be critical to enabling the use of MHCI tetramers to monitor neoantigen-specific T-cells in the clinic.

Sex disparities in cancer incidence by period and age.

BACKGROUND: Cancer epidemiology articles often point out that cancer rates tend to be higher among males than females yet rarely is this theme the subject of investigation. METHODS: We used the Surveillance, Epidemiology and End Results program data to compute age-adjusted (2000 U.S. standard population) sex-specific incidence rates and male-to-female incidence rate ratios (IRR) for specific cancer sites and histologies for the period 1975 to 2004. RESULTS: The 10 cancers with the largest male-to-female IRR were Kaposi sarcoma (28.73), lip (7.16), larynx (5.17), mesothelioma (4.88), hypopharynx (4.13), urinary bladder (3.92), esophagus (3.49), tonsil (3.07), oropharynx (3.06), and other urinary organs (2.92). Only 5 cancers had a higher incidence in females compared with males: breast (0.01), peritoneum, omentum, and mesentery (0.18), thyroid (0.39), gallbladder (0.57), and anus, anal canal, and anorectum (0.81). Between 1975 and 2004, the largest consistent increases in male-to-female IRR were for cancers of the tonsil, oropharynx, skin excluding basal and squamous, and esophagus, whereas the largest consistent decreases in IRR were for cancers of the lip and lung and bronchus. Male-to-female IRRs varied considerably by age, the largest increases of which were for ages 40 to 59 years for tonsil cancer and hepatocellular carcinoma. The largest decreases in male-to-female IRR by age, meanwhile, were for ages 30 to 49 years for thyroid cancer, ages >70 years for esophageal squamous cell carcinoma, and ages >30 years for lung and bronchus cancer. CONCLUSION: These observations emphasize the importance of sex in cancer etiopathogenesis and may suggest novel avenues of investigation.

Multicolor Tracking of Molecular Motors at Nanometer Resolution.

Molecular motors move processively along cytoskeletal filaments through stepping of their catalytic head domains. Observation of the stepping movement of the heads reveals the mechanism of motor processivity and how they coordinate the cycles of the catalytic heads during processive motility. This chapter will discuss recent developments in simultaneous observation of the stepping motions of the two heads using multicolor single particle tracking microscopy.

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots.

Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.

DNA repair gene polymorphisms and risk of adult meningioma, glioma, and acoustic neuroma.

Although the etiology of primary brain tumors is largely unknown, prior studies suggest that DNA repair polymorphisms may influence risk of glioma. Altered DNA repair is also likely to affect the risk of meningioma and acoustic neuroma, but these tumors have not been well studied. We estimated the risk of glioma (n = 362), meningioma (n = 134), and acoustic neuroma (n = 69) in non-Hispanic whites with respect to 36 single nucleotide polymorphisms from 26 genes involved in DNA repair in a hospital-based, case-control study conducted by the National Cancer Institute. We observed significantly increased risk of meningioma with the T variant of GLTSCR1 rs1035938 (OR(CT/TT) = 3.5; 95% confidence interval: 1.8-6.9; P(trend) .0006), which persisted after controlling for multiple comparisons (P = .019). Significantly increased meningioma risk was also observed for the minor allele variants of ERCC4 rs1800067 (P(trend) .01); MUTYH rs3219466 (P(trend) .02), and PCNA rs25406 (P(trend) .03). The NBN rs1805794 minor allele variant was associated with decreased meningioma risk (P(trend) .006). Risk of acoustic neuroma was increased for the ERCC2 rs1799793 (P(trend) .03) and ERCC5 rs17655 (P(trend) .05) variants and decreased for the PARP1 rs1136410 (P(trend) .03). Decreased glioma risk was observed with the XRCC1 rs1799782 variant (P(trend) .04). Our results suggest that common DNA repair variants may affect the risk of adult brain tumors, especially meningioma.