Mechanistic Characterization of Cancer Associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

Cancer-associated fibroblasts (CAFs) are a prominent cell type within the tumor microenvironment where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease Fibroblast Activation Protein (FAP) is expressed on the surface of highly pro-tumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating pro-tumorigenic CAFs will disrupt the crosstalk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate (ADC) using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell co-culture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the pro-inflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of pro-inflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies are through effects on the immune microenvironment and anti-tumor immune response.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase 1 clinical trial investigating a ferritin nanoparticle vaccine displaying H2 hemagglutinin (HA) in H2-naive and H2-exposed adults enabled us to perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited. We temporally map the epitopes targeted by serum antibodies after vaccine prime and boost, revealing that previous H2 exposure results in higher responses to the variable HA head domain. In contrast, initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryoelectron microscopy and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the HA head, including the receptor-binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses post-vaccination.

Liquid Thyroxine Improves Outcomes in Hypothyroid Patients With Small Intestinal Bacterial Overgrowth and Irritable Bowel Syndrome.

OBJECTIVE: Malabsorption of levothyroxine (LT4) is often seen in patients with hypothyroidism and gastrointestinal (GI) conditions. Our study was designed to establish the prevalence of small intestinal bacterial overgrowth (SIBO) in patients with hypothyroidism and irritable bowel syndrome (IBS), and to demonstrate that liquid LT4 is more consistently absorbed vs tablet, leading to improvement in thyroid and GI symptoms. METHODS: This was a single-center, open label, prospective cohort study of liquid LT4 in 75 adult patients with hypothyroidism and IBS. Patients were transitioned from LT4 tablets to solution at equivalent dosing. Patients returned at 6 and 12 weeks for repeat thyroid levels and completion of validated questionnaires. A standard 2-hour SIBO breath test was administered at Week 6. Patients recorded daily stool appearance and frequency. RESULTS: Prevalence of SIBO was 65.3%. Liquid LT4 normalized thyroid stimulating hormone (TSH) in a higher percentage of patients vs tablet (77.55% vs 57.14%); significantly decreased TSH in subjects with SIBO; improved hypothyroid symptoms, IBS symptoms, stool appearance in all groups, and significantly altered bowel frequency among those with SIBO. CONCLUSION: Small intestinal bacterial overgrowth (SIBO) is common in patients with hypothyroidism and IBS. Among SIBO patients, LT4 tablets were inefficiently absorbed, leading to suboptimal thyroid control; however, transitioning from LT4 tablets to solution normalized TSH and improved hypothyroid symptoms. Liquid LT4 also significantly improved GI symptoms in all patients with hypothyroidism and IBS, regardless of SIBO status. Additionally, 1 in 5 patients had complete resolution of IBS symptoms after switching from LT4 tablets to solution, independent of changes in TSH.

Age related trends in the utilization of neoadjuvant chemotherapy for muscle invasive bladder cancer.

PURPOSE: The current standard of care for muscle invasive bladder cancer (MIBC) is neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Previous research has shown under-utilization of NAC for treatment of MIBC, especially among the elderly. Our aim was to stratify NAC use by decade of life and analyze trends in use over time along with recording pathologic downstaging and perioperative outcomes. MATERIALS AND METHODS: The National Cancer Database was queried for patients with cT2-4NanyM0 MIBC treated with RC from 2010 to 2016 with urothelial carcinoma. Nineteen thousand nine hundred fifty seven patients met criteria for analysis. We retrospectively analyzed trends in use of NAC, readmission rate, mortality rate, and pathologic downstaging with NAC all stratified by decade of life. RESULTS: Of the 19,957 patients treated with RC for MIBC, only 30.9% underwent NAC. There was a statistically significant increase in NAC use across all age groups from 2010 to 2016. Receipt of NAC was associated with decreased age on univariate analysis (P < 0.001) and on logistic regression (OR: 0.617 for age 70-79, OR: 0.221 for age ≥80 vs. age <60; P < 0.001). Patients receiving NAC were more likely to exhibit pathologic downstaging at time of RC (OR: 3.907; P < 0.001), and this trend held for each age group examined. Among patients receiving NAC, the risk of 30 and 90-day mortality was associated with increasing age; however, age was not associated with 30-day readmission for those receiving NAC. CONCLUSION: Rates of NAC use prior to RC have increased among all age groups with the lowest utilization rate among the elderly. NAC use was associated with greater pathologic downstaging in all age groups. These data show a promising trend in the uptake of the gold standard for treatment of MIBC; however, the underlying etiology of differing rates of NAC utilization remains to be determined.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase I clinical trial investigating a ferritin nanoparticle displaying H2 hemagglutinin in H2-naïve and H2-exposed adults. Therefore, we could perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited after H2 vaccination. We temporally map the epitopes targeted by serum antibodies after first and second vaccinations and show previous H2 exposure results in higher responses to the variable head domain of hemagglutinin while initial responses in H2-naïve participants are dominated by antibodies targeting conserved epitopes. We use cryo-EM and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the hemagglutinin head including the receptor binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Increasing sensitivity of antibody-antigen interactions using photo-cross-linking.

Understanding antibody-antigen interactions in a polyclonal immune response in humans and animal models is critical for rational vaccine design. Current approaches typically characterize antibodies that are functionally relevant or highly abundant. Here, we use photo-cross-linking and single-particle electron microscopy to increase antibody detection and unveil epitopes of low-affinity and low-abundance antibodies, leading to a broader structural characterization of polyclonal immune responses. We employed this approach across three different viral glycoproteins and showed increased sensitivity of detection relative to currently used methods. Results were most noticeable in early and late time points of a polyclonal immune response. Additionally, the use of photo-cross-linking revealed intermediate antibody binding states and demonstrated a distinctive way to study antibody binding mechanisms. This technique can be used to structurally characterize the landscape of a polyclonal immune response of patients in vaccination or post-infection studies at early time points, allowing for rapid iterative design of vaccine immunogens.

Protocol for analyzing antibody responses to glycoprotein antigens using electron-microscopy-based polyclonal epitope mapping.

Electron microscopy-based polyclonal epitope mapping (EMPEM) can delineate epitope specificities of serum antibodies to a given antigen following vaccination or infection. Here, we present a protocol for the EMPEM method for rapid high-throughput assessment of antibody responses to glycoprotein antigens in vaccination and infection studies. We describe steps for antibody isolation and digestion, antigen complex and purification, and electron microscope imaging. We then detail procedures for processing and analysis of EMPEM data. For complete details on the use and execution of this protocol, please refer to Bianchi et al. (2018).1.

Melanocortin receptor 3 and 4 mRNA expression in the adult female Syrian hamster brain.

Melanocortin 3 receptors (MC3R) and melanocortin 4 receptors (MC4R) are vital in regulating a variety of functions across many species. For example, the dysregulation of these receptors results in obesity and dysfunction in sexual behaviors. Only a handful of studies have mapped the expression of MC3R and MC4R mRNA across the central nervous system, with the primary focus on mice and rats. Because Syrian hamsters are valuable models for functions regulated by melanocortin receptors, our current study maps the distribution of MC3R and MC4R mRNA in the Syrian hamster telencephalon, diencephalon, and midbrain using RNAscope. We found that the expression of MC3R mRNA was lowest in the telencephalon and greatest in the diencephalon, whereas the expression of MC4R mRNA was greatest in the midbrain. A comparison of these findings to previous studies found that MC3R and MC4R expression is similar in some brain regions across species and divergent in others. In addition, our study identifies novel brain regions for the expression of MC3Rs and MC4Rs, and identifies cells that co-express bothMC3 and MC4 receptors within certain brain regions.

Aggression Results in the Phosphorylation of ERK1/2 in the Nucleus Accumbens and the Dephosphorylation of mTOR in the Medial Prefrontal Cortex in Female Syrian Hamsters.

Like many social behaviors, aggression can be rewarding, leading to behavioral plasticity. One outcome of reward-induced aggression is the long-term increase in the speed in which future aggression-based encounters is initiated. This form of aggression impacts dendritic structure and excitatory synaptic neurotransmission in the nucleus accumbens, a brain region well known to regulate motivated behaviors. Yet, little is known about the intracellular signaling mechanisms that drive these structural/functional changes and long-term changes in aggressive behavior. This study set out to further elucidate the intracellular signaling mechanisms regulating the plasticity in neurophysiology and behavior that underlie the rewarding consequences of aggressive interactions. Female Syrian hamsters experienced zero, two or five aggressive interactions and the phosphorylation of proteins in reward-associated regions was analyzed. We report that aggressive interactions result in a transient increase in the phosphorylation of extracellular-signal related kinase 1/2 (ERK1/2) in the nucleus accumbens. We also report that aggressive interactions result in a transient decrease in the phosphorylation of mammalian target of rapamycin (mTOR) in the medial prefrontal cortex, a major input structure to the nucleus accumbens. Thus, this study identifies ERK1/2 and mTOR as potential signaling pathways for regulating the long-term rewarding consequences of aggressive interactions. Furthermore, the recruitment profile of the ERK1/2 and the mTOR pathways are distinct in different brain regions.

Effects of electronic cigarette e-liquid flavouring on cigarette craving.

BACKGROUND: E-liquid flavour restrictions may discourage electronic cigarette (e-cigarette) uptake among youth. However, possible unintended consequences may include reduced appeal and effectiveness of e-cigarettes for smoking cessation. Non-tobacco flavours appear to be important for smoking cessation, but how and why are currently unclear. METHODS: We conducted an experimental study in a UK sample of adult daily smokers using an independent groups design (N=84). Participants were randomised to use an e-cigarette with nicotine-containing fruit/sweet-flavoured e-liquid (blackcurrant, strawberry, vanilla, caramel) or unflavoured e-liquid for 1 week. The primary outcomes were average, peak and cue-elicited cigarette craving (the latter was assessed using a cue exposure task). The secondary outcomes were smoking lapse occurrence, enjoyment of the e-cigarette, ease of transitioning from smoking to using an e-cigarette, intentions to continue using an e-cigarette, intentions and motivation to quit smoking, return to smoking, and continuation of e-cigarette use. RESULTS: E-liquid flavouring did not appear to have an effect on average cigarette craving (b 0.18, 95% CI -0.44 to 0.79, p=0.57), peak cigarette craving (b -0.12, 95% CI -0.59 to 0.35, p=0.62) or cue-elicited cigarette craving (b -0.21, 95% CI -3.86 to 3.43, p=0.91). We did not find evidence of a difference in secondary outcomes. CONCLUSIONS: We did not find evidence to suggest that nicotine-containing fruit/sweet-flavoured and unflavoured e-liquids have different effects on cigarette cravings after 1 week of use. Further research is needed to establish if differences emerge over longer periods of exposure and extend to smoking cessation outcomes.

An insulin hypersecretion phenotype precedes pancreatic ß cell failure in MODY3 patient-specific cells.

MODY3 is a monogenic hereditary form of diabetes caused by mutations in the transcription factor HNF1A. The patients progressively develop hyperglycemia due to perturbed insulin secretion, but the pathogenesis is unknown. Using patient-specific hiPSCs, we recapitulate the insulin secretion sensitivity to the membrane depolarizing agent sulfonylurea commonly observed in MODY3 patients. Unexpectedly, MODY3 patient-specific HNF1A+/R272C ß cells hypersecrete insulin both in vitro and in vivo after transplantation into mice. Consistently, we identified a trend of increased birth weight in human HNF1A mutation carriers compared with healthy siblings. Reduced expression of potassium channels, specifically the KATP channel, in MODY3 ß cells, increased calcium signaling, and rescue of the insulin hypersecretion phenotype by pharmacological targeting ATP-sensitive potassium channels or low-voltage-activated calcium channels suggest that more efficient membrane depolarization underlies the hypersecretion of insulin in MODY3 ß cells. Our findings identify a pathogenic mechanism leading to ß cell failure in MODY3.

Solitary Intracranial Plasmacytoma of the Brain Treated With Primary Radiation Therapy.

We present a rare case of a solitary intracranial plasmacytoma of the brain parenchyma in a 49-year-old female who presented with neck pain/headache, paresthesias, and auditory hallucinations. A workup revealed a solitary left parietal lobe brain lesion and a biopsy demonstrated a plasma cell infiltrate consistent with an extramedullary plasmacytoma. A complete workup for multiple myeloma was negative. As opposed to surgical resection and adjuvant radiation therapy (RT), as described in prior case reports in the literature, this patient was managed with definitive local RT alone to 50 Gy in 25 fractions. Six months following primary RT completion, the patient's presenting symptoms completely resolved and follow-up imaging revealed regression of the primary tumor. To our knowledge, this is the first reported case of a solitary extramedullary plasmacytoma of the brain treated with localized definitive RT alone.

Glacial Legacies: Microbial Communities of Antarctic Refugia.

In the cold deserts of the McMurdo Dry Valleys (MDV) the suitability of soil for microbial life is determined by both contemporary processes and legacy effects. Climatic changes and accompanying glacial activity have caused local extinctions and lasting geochemical changes to parts of these soil ecosystems over several million years, while areas of refugia may have escaped these disturbances and existed under relatively stable conditions. This study describes the impact of historical glacial and lacustrine disturbance events on microbial communities across the MDV to investigate how this divergent disturbance history influenced the structuring of microbial communities across this otherwise very stable ecosystem. Soil bacterial communities from 17 sites representing either putative refugia or sites disturbed during the Last Glacial Maximum (LGM) (22-17 kya) were characterized using 16 S metabarcoding. Regardless of geographic distance, several putative refugia sites at elevations above 600 m displayed highly similar microbial communities. At a regional scale, community composition was found to be influenced by elevation and geographic proximity more so than soil geochemical properties. These results suggest that despite the extreme conditions, diverse microbial communities exist in these putative refugia that have presumably remained undisturbed at least through the LGM. We suggest that similarities in microbial communities can be interpreted as evidence for historical climate legacies on an ecosystem-wide scale.

Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses.

Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.

Structural mapping of antibody landscapes to human betacoronavirus spike proteins.

Preexisting immunity against seasonal coronaviruses (CoVs) represents an important variable in predicting antibody responses and disease severity to severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infections. We used electron microscopy-based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against ß-CoV spike proteins in prepandemic (PP) sera or SARS-CoV-2 convalescent (SC) sera. We observed that most PP sera had antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the SC sera showed reactivity across all human ß-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by preexisting antibodies and SC serum antibodies. Our studies provide an antigenic landscape to ß-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-CoV-2-infected individuals.

Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit.

Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs.

One dose of COVID-19 nanoparticle vaccine REVC-128 protects against SARS-CoV-2 challenge at two weeks post-immunization.

ABSTRACTA COVID-19 vaccine that can give early protection is needed to eliminate the viral spread efficiently. Here, we demonstrate the development of a nanoparticle vaccine candidate, REVC-128, in which multiple trimeric spike ectodomains with glycine (G) at position 614 were multimerized onto a nanoparticle. In-vitro characterization of this vaccine confirms its structural and antigenic integrity. In-vivo immunogenicity evaluation in mice indicates that a single dose of this vaccine induces potent serum neutralizing antibody titre at two weeks post-immunization. This is significantly higher than titre caused by trimeric spike protein without nanoparticle presentation. The comparison of serum binding to spike subunits between animals immunized by a spike with and without nanoparticle presentation indicates that nanoparticle prefers the display of spike RBD (Receptor-Binding Domain) over S2 subunit, likely resulting in a more neutralizing but less cross-reactive antibody response. Moreover, a Syrian golden hamster in-vivo model for the SARS-CoV-2 virus challenge was implemented two weeks post a single dose of REVC-128 immunization. The results showed that vaccination protects hamsters against the SARS-CoV-2 virus challenge with evidence of steady body weight, suppressed viral loads and alleviation of tissue damage for protected animals, compared with ∼10% weight loss, high viral loads and tissue damage in unprotected animals. Furthermore, the data showed that vaccine REVC-128 is thermostable at up to 37°C for at least 4 weeks. These findings, along with a history of safety for protein vaccines, suggest that the REVC-128 is a safe, stable and efficacious single-shot vaccine to give the earliest protection against SARS-CoV-2 infection.

Wood ash and water: Cause of superficial alkaline burns in a toddler.

Though seemingly innocuous, the combination of wood ash and water can result in an alkaline solution with a pH up to 12, making it a burn risk. A 2-year-old boy developed a significant irritant reaction to his arms and legs while playing outdoors in wood ashes with a water gun. This case is novel because an unintentional, trivial exposure resulted in ulcerative irritant dermatitis. It is necessary to raise awareness of the corrosive risk from wood ash and water in the medical community, especially in primary and emergency care settings, to improve recognition of ash-water burns and irritant contact dermatitis and to ensure appropriate treatment.