Implementing L-DNA analogs as mirrors of PCR reactant hybridization state: theoretical and practical guidelines for PCR cycle control.

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm (N = 6) or preset cycling conditions (N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.

Acceptability of magnetic resonance imaging for prostate cancer diagnosis with patients and GPs: a qualitative interview study.

BACKGROUND: Magnetic resonance imaging (MRI) of the prostate is a new, more accurate, non-invasive test for prostate cancer diagnosis. AIM: To understand the acceptability of MRI for patients and GPs for prostate cancer diagnosis. DESIGN AND SETTING: Qualitative study of men who had undergone a prostate MRI for possible prostate cancer, and GPs who had referred at least one man for possible prostate cancer in the previous 12 months in West London and Devon. METHOD: Semi-structured interviews, conducted in person or via telephone, were audio-recorded and transcribed verbatim. Deductive thematic analysis was undertaken using Sekhon's Theoretical Framework of Acceptability, retrospectively for patients and prospectively for GPs. RESULTS: Twenty-two men (12 from Devon, age range 47-80 years), two patients' partners, and 10 GPs (6 female, age range 36-55 years) were interviewed. Prostate MRI was broadly acceptable for most patient participants, and they reported that it was not a significant undertaking to complete the scan. GPs were more varied in their views on prostate MRI, with a broad spectrum of knowledge and understanding of prostate MRI. Some GPs expressed concerns about additional clinical responsibility and local availability of MRI if direct access to prostate MRI in primary care were to be introduced. CONCLUSION: Prostate MRI appears to be acceptable to patients. Some differences were found between patients in London and Devon, mainly around burden of testing and opportunity costs. Further exploration of GPs' knowledge and understanding of prostate MRI could inform future initiatives to widen access to diagnostic testing in primary care.

A genome-wide association study provides insights into the genetic etiology of 57 essential and non-essential trace elements in humans.

Trace elements are important for human health but may exert toxic or adverse effects. Mechanisms of uptake, distribution, metabolism, and excretion are partly under genetic control but have not yet been extensively mapped. Here we report a comprehensive multi-element genome-wide association study of 57 essential and non-essential trace elements. We perform genome-wide association meta-analyses of 14 trace elements in up to 6564 Scandinavian whole blood samples, and genome-wide association studies of 43 trace elements in up to 2819 samples measured only in the Trøndelag Health Study (HUNT). We identify 11 novel genetic loci associated with blood concentrations of arsenic, cadmium, manganese, selenium, and zinc in genome-wide association meta-analyses. In HUNT, several genome-wide significant loci are also indicated for other trace elements. Using two-sample Mendelian randomization, we find several indications of weak to moderate effects on health outcomes, the most precise being a weak harmful effect of increased zinc on prostate cancer. However, independent validation is needed. Our current understanding of trace element-associated genetic variants may help establish consequences of trace elements on human health.

Persistence of Contact Lens-Induced Corneal Parainflammation Following Lens Removal.

Purpose: Contact lens wear induces corneal parainflammation involving increased immune cell numbers after 24 hours' (CD11c+, Lyz2+, γδ-T cells) and six days' (Ly6G+ cells) wear. We investigated the time course of onset and resolution of these responses. Methods: LysMcre or C57BL/6J mice were fitted with a contact lens (four to 48 hours). Contralateral eyes did not wear lenses. After lens removal, Lyz2+, MHC-II+ or Ly6G+ cells were examined by quantitative imaging. RT-qPCR determined cytokine gene expression. Results: Lens wear for 24 hours increased corneal Lyz2+ cells versus contralateral eyes approximately two-fold. Corneas remained free of visible pathology. The Lyz2+ response was not observed after four or 12 hours' wear, nor after 12 hours' wear plus 12 hours' no wear. Lens removal after 24 hours' wear further increased Lyz2+ cells (∼48% after one day), which persisted for four days, returning to baseline by seven days. Lyz2+ cells in contralateral eyes remained at baseline. MHC-II+ cells showed a similar response but without increasing after lens removal. Lens wear for 48 hours showed reduced Lyz2+ cells versus 24 hours' wear with one day discontinuation, correlating with reduced IL-1ß and IL-18 gene expression. Lens wear for 24 hours did not induce Ly6G+ responses six days after removal. Conclusions: Lens-induced corneal parainflammation involving Lyz2+ cells requires 24 hours' wear but persists after lens discontinuation, requiring seven days for reversal. Lens wear for 48 hours may suppress initial Lyz2+ cell and cytokine responses. The significance of parainflammation during and after lens wear remains to be determined.

The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.

Community Pharmacy Needle Exchange Programme: What Can Analysis of the Data Tell Us about the Changing Drug Market in Ireland?

Community Pharmacy Needle Exchanges are a harm reduction measure that have been established in a number of countries to provide access to sterile injecting equipment for people who inject drugs (PWID). To ensure that they are meeting needs, it is important to monitor the use of the services. This study aimed to determine patterns of needle distribution and return in community pharmacies in Ireland over time. The number of pharmacies, needle packs, clean needles and returned packs was obtained from the Health Service Executive (HSE) Planning and Business Information Unit (PBI). Yearly totals were calculated to show patterns from 2015 to 2022. There has been an 18% decline in the number of pharmacies providing the service since 2015, with a 19% decline in the number of packs provided and a 21% decline in the number of packs returned. The proportion of packs returned was 23% in 2015 and 18% in 2022. There has been a 16% decline in the number of sterile needles provided and a 6% reduction in the average number of needles per individual since 2017. Declining needle use and low rates of used needle return (against a backdrop of large numbers of PWID that have not significantly reduced over time) suggest that there is a need to investigate if community pharmacies in Ireland have the scope to improve their harm reduction impact. This raises questions in terms of the need to both improve and adapt the service against a backdrop of changing drug markets. Key recommendations include the need to review the harm reduction services employed by participating pharmacies when providing new equipment and organising the return of used equipment.

Commercial Tobacco Endgame Goals: Early experiences from six countries.

INTRODUCTION: Tobacco use is a major threat to health globally. A number of countries have adopted 'endgame goals' to minimise smoking prevalence. The INSPIRED project aims to describe and compare the experiences of the first six countries to adopt an endgame goal. METHODS: Data were collected on the initial experiences of endgame goals in Canada, Finland, Ireland, New Zealand (Aotearoa), Scotland, and Sweden up to 2018. Information was collated on the nature of the endgame goals, associated interventions and strategies, potential enablers and barriers, and perceived advantages and disadvantages. RESULTS: The INSPIRED countries had relatively low smoking prevalences and moderate to strong smokefree policies. Their endgame goals aimed for smoking prevalences of 5% or less. Target dates ranged from 2025 to 2035. Except for New Zealand (Aotearoa), all countries had an action plan to support their goal by 2018. However, none of the plans incorporated specific endgame measures. Lack of progress in reducing inequities was a key concern, despite the consideration of equity in all of the country's goals and/or action plans. Experience with endgame goals was generally positive, however participants thought additional interventions would be required to equitably meet their endgame goal. CONCLUSIONS: There was variation in the nature and approach to endgame goals. This suggests that countries should consider adopting endgame goals and strategies to suit their social, cultural, and political contexts. The experiences of the INSPIRED countries suggest that further and more significant interventions will be required for the timely and equitable achievement of endgame goals. IMPLICATIONS: By 2018, six countries (Canada, Finland, Ireland, New Zealand (Aotearoa), Scotland, and Sweden) had introduced government-endorsed 'endgame goals', to rapidly reduce smoking prevalence to very low levels by a specified date. The nature and implementation of endgame goals was variable. Early experiences with the goals were generally positive, but progress in reducing smoking prevalence was insufficient, particularly for priority groups. This finding suggests more significant interventions ('endgame interventions') and measures to reduce inequities need to be implemented to achieve endgame goals. Variation in the nature and experience of endgame goals demonstrates the importance of designing endgame strategies that suit distinct social, cultural, and political contexts.

Oncolytic vaccinia virus immunotherapy antagonizes image-guided radiotherapy in mouse mammary tumor models.

Ionizing radiation (IR) and oncolytic viruses are both used to treat cancer, and the effectiveness of both agents depends upon stimulating an immune response against the tumor. In this study we tested whether combining image guided ionizing radiation (IG-IR) with an oncolytic vaccinia virus (VACV) could yield a better therapeutic response than either treatment alone. ΔF4LΔJ2R VACV grew well on irradiated human and mouse breast cancer cells, and the virus can be combined with 4 or 8 Gy of IR to kill cells in an additive or weakly synergistic manner. To test efficacy in vivo we used immune competent mice bearing orthotopic TUBO mammary tumors. IG-IR worked well with 10 Gy producing 80% complete responses, but this was halved when the tumors were treated with VACV starting 2 days after IG-IR. VACV monotherapy was ineffective in this model. The antagonism was time dependent as waiting for 21 days after IG-IR eliminated the inhibitory effect but without yielding any further benefits over IR alone. In irradiated tumors, VACV replication was also lower, suggesting that irradiation created an environment that did not support infection as well in vivo as in vitro. A study of how four different treatment regimens affected the immune composition of the tumor microenvironment showed that treating irradiated tumors with VACV altered the immunological profiles in tumors exposed to IR or VACV alone. We detected more PD-1 and PD-L1 expression in tumors exposed to IR+VACV but adding an αPD-1 antibody to the protocol did not change the way VACV interferes with IG-IR therapy. VACV encodes many immunosuppressive gene products that may interfere with the ability of radiotherapy to induce an effective anti-tumor immune response through the release of danger-associated molecular patterns. These data suggest that infecting irradiated tumors with VACV, too soon after exposure, may interfere in the innate and linked adaptive immune responses that are triggered by radiotherapy to achieve a beneficial impact.

Estimating disease heritability from complex pedigrees allowing for ascertainment and covariates.

We propose TetraHer, a method for estimating the liability heritability of binary phenotypes. TetraHer has five key features. First, it can be applied to data from complex pedigrees that contain multiple types of relationships. Second, it can correct for ascertainment of cases or controls. Third, it can accommodate covariates. Fourth, it can model the contribution of common environment. Fifth, it produces a likelihood that can be used for significance testing. We first demonstrate the validity of TetraHer on simulated data. We then use TetraHer to estimate liability heritability for 229 codes from the tenth International Classification of Diseases (ICD-10). We identify 107 codes with significant heritability (p < 0.05/229), which can be used in future analyses for investigating the genetic architecture of human diseases.

The impact of COVID-19 on medication reviews in English primary care. An OpenSAFELY-TPP analysis of 20 million adult electronic health records.

AIMS: The COVID-19 pandemic caused significant disruption to routine activity in primary care. Medication reviews are an important primary care activity ensuring safety and appropriateness of prescribing. A disruption could have significant negative implications for patient care. Using routinely collected data, our aim was first to describe codes used to record medication review activity and then to report the impact of COVID-19 on the rates of medication reviews. METHODS: With the approval of NHS England, we conducted a cohort study of 20 million adult patient records in general practice, in-situ using the OpenSAFELY platform. For each month, between April 2019 and March 2022, we report the percentage of patients with a medication review coded monthly and in the previous 12 months with breakdowns by regional, clinical and demographic subgroups and those prescribed high-risk medications. RESULTS: In April 2019, 32.3% of patients had a medication review coded in the previous 12 months. During the first COVID-19 lockdown, monthly activity decreased (-21.1% April 2020), but the 12-month rate was not substantially impacted (-10.5% March 2021). The rate of structured medication review in the last 12 months reached 2.9% by March 2022, with higher percentages in high-risk groups (care home residents 34.1%, age 90+ years 13.1%, high-risk medications 10.2%). The most used medication review code was Medication review done 314530002 (59.5%). CONCLUSIONS: There was a substantial reduction in the monthly rate of medication reviews during the pandemic but rates recovered by the end of the study period. Structured medication reviews were prioritized for high-risk patients.

Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis.

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Complication reporting in trauma: An environmental scan and comparison of nationwide trauma registry data.

BACKGROUND: To explore variability in quality measurement, this study aimed to compare abstraction and definitions of complications reported across trauma registries in Canada. METHODS: A literature search was performed to identify active trauma registries used in Canadian hospitals. Registry characteristics, data abstraction, and reported complications and definitions based on registry data dictionaries were compared. RESULTS: Nine registries were included, most of which were provincial-level registries (67 â€‹%). A total of 53 individual complications were identified. Twenty-one (40 â€‹%) were recorded by only one registry each whereas 5 (9 â€‹%) were collected by all. Of the 32 complications collected by â€‹> â€‹1 registry, 18 (56 â€‹%) had different definitions. Of the 18 with different definitions, 12 (67 â€‹%), 5 (28 â€‹%), and 1 (6 â€‹%) had 2, 3, and 4 different definitions across registries, respectively. CONCLUSIONS: Complications reported by trauma registries are variable. Reliable benchmarking is likely challenging, and efforts to standardize complication reporting may be a valuable undertaking.

Accurate measurement of K/Ca in low-[K] carbonate samples using laser-ablation sector-field inductively coupled plasma mass spectrometry.

RATIONALE: Potassium (K) is a major component of several silicate minerals and seawater, and, therefore, constraining past changes in the potassium cycle is a promising way of tracing large-scale geological processes on Earth. However, [K] measurement using inductively coupled plasma mass spectrometry (ICP-MS) is challenging due to an ArH+ interference, which may be of a similar magnitude to the K+ ion beam in samples with <0.1% m/m [K]. METHODS: In this work, we investigated the effect of the ArH+ interference on K/Ca data quality by comparing results from laser-ablation (LA)-ICP-MS measured in medium and high mass resolution modes and validating our LA results via solution ICP-optical emission spectroscopy (OES) and solution ICP-MS measurements. To do so, we used a wide range of geological reference materials, with a particular focus on marine carbonates, which are potential archives of past changes in the K cycle but are typically characterised by [K] < 200 µg/g. In addition, we examine the degree to which trace-element data quality is driven by downhole fractionation during LA-ICP-MS measurements. RESULTS: Our results show that medium mass resolution (MR) mode is sufficiently capable of minimising the effect of the ArH+ interference on K+ . However, the rate of downhole fractionation for Na and K varies between different samples as a result of their differing bulk composition, resulting in matrix-specific inaccuracy. We show how this can be accounted for via downhole fractionation corrections, resulting in an accuracy of better than 1% and a long-term reproducibility (intermediate precision) of <6% (relative standard deviation) in JCp-1NP using LA-ICP-MS in MR mode. CONCLUSION: Our [K] measurement protocol is demonstrably precise and accurate and applicable to a wide range of materials. The measurement of K/Ca in relatively low-[K] marine carbonates is presented here as a key example of a new application opened up by these advances.

Prescription Drug Prices: An AAN Position Statement.

This position statement serves to establish the AAN's stance on the methods to address the cost of prescription drugs being considered by state and federal policymakers so that the AAN can continue to advocate effectively for its members. Neurologists seek to provide high-value care for patients with neurologic diseases at the lowest cost possible. However, many therapies for neurologic diseases are among the most expensive in the United States. The 3 major cost challenges include (1) unjustified increases in the pricing for drugs used to treat neurologic disorders, (2) the high cost of medications used to treat rare diseases where there are limited or no therapeutic options available, and (3) the high cost of noninnovative (already FDA-approved) therapies that used accelerated FDA approval pathways or Orphan Drug Act designated to expedite approvals in neurologic disorders. In each of these cases, AAN is concerned that the high cost does not deliver sufficient value to patients or society. The AAN's position is that action must be taken to ensure that effective prescription medications are accessible for patients with complex, chronic neurologic conditions. Potential solutions should be affordable, simple, and transparent. Cost-containment efforts must also address the burden on the entire healthcare system because high prescription drug prices may be shifted and absorbed in ways that negatively affect patient and prescriber access to important medications. AAN supports price negotiations, the cost saving potential of generics and biosimilars, development of novel therapeutics, price transparency, and importation.

Induction of durable remission by dual immunotherapy in SHIV-infected ART-suppressed macaques.

The eradication of the viral reservoir represents the major obstacle to the development of a clinical cure for established HIV-1 infection. Here, we demonstrate that the administration of N-803 (brand name Anktiva) and broadly neutralizing antibodies (bNAbs) results in sustained viral control after discontinuation of antiretroviral therapy (ART) in simian-human AD8 (SHIV-AD8)-infected, ART-suppressed rhesus macaques. N-803+bNAbs treatment induced immune activation and transient viremia but only limited reductions in the SHIV reservoir. Upon ART discontinuation, viral rebound occurred in all animals, which was followed by durable control in approximately 70% of all N-803+bNAb-treated macaques. Viral control was correlated with the reprogramming of CD8+ T cells by N-803+bNAb synergy. Thus, complete eradication of the replication-competent viral reservoir is likely not a prerequisite for the induction of sustained remission after discontinuation of ART.

Omidenepag Isopropyl Versus Timolol in Patients With Glaucoma or Ocular Hypertension: Two Randomized Phase 3 Trials (SPECTRUM 4 and 3).

PURPOSE: The SPECTRUM 4 and 3 studies assessed the intraocular pressure (IOP)-lowering efficacy and safety of omidenepag isopropyl (OMDI) 0.002% vs timolol 0.5% in patients with glaucoma or ocular hypertension (OHT). DESIGN: Phase 3, randomized, controlled, double-masked, noninferiority studies. METHODS: Multicenter studies in the US. Inclusion criteria for adults ≥ 18 years (SPECTRUM 4 [N = 409] and 3 [N = 413]) were open-angle glaucoma or OHT, and IOP ≥ 22 mm Hg and ≤ 34 mm Hg; and for pediatric patients < 18 years (N = 13, SPECTRUM 3) were pediatric glaucoma or OHT. The primary objective in both studies was OMDI noninferiority to timolol in reducing IOP (3 months). SPECTRUM 3 included an additional 9 months of OMDI treatment. Safety evaluations were of ocular/non-ocular adverse events (AEs). RESULTS: The IOP-lowering range of OMDI remained consistent in SPECTRUM 4 and 3 (-5.6 to -5.9 vs -5.3 to -5.7 mm Hg, respectively); however, timolol efficacy varied (-5.4 to -6.1 vs -6.4 to -7.0 mm Hg, respectively). OMDI noninferiority was achieved in SPECTRUM 4. Efficacy was maintained with 12-month treatment in SPECTRUM 3. Both studies reported more ocular AEs with OMDI, but lower rates of appearance-altering AEs vs timolol. No new safety concerns were identified. Rates of macular edema in pseudophakic patients increased with prolonged OMDI exposure. CONCLUSIONS: SPECTRUM 4 and 3 demonstrated consistent 3-month IOP-lowering efficacy and safety of OMDI vs timolol in patients with glaucoma or OHT. The 12-month data from SPECTRUM 3 suggest OMDI may have long-term benefits in patients with glaucoma or OHT.

Nutritional Support for the Trauma and Emergency General Surgery Patient: What You Need to Know.

LEVEL OF EVIDENCE: Level V, literature synthesis and expert opinion.

Codelivery of TGFß and Cox2 siRNA inhibits HCC by promoting T-cell penetration into the tumor and improves response to Immune Checkpoint Inhibitors.

Upregulation of TGFß and Cox2 in the tumor microenvironment results in blockade of T-cell penetration into the tumor. Without access to tumor antigens, the T-cell response will not benefit from administration of the immune checkpoint antibodies. We created an intravenous polypeptide nanoparticle that can deliver two siRNAs (silencing TGFß and Cox2). Systemic administration in mice, bearing a syngeneic orthotopic hepatocellular carcinoma (HCC), delivers the siRNAs to various cells in the liver, and significantly reduces the tumor. At 2 mg/kg (BIW) the nanoparticle demonstrated a single agent action and induced tumor growth inhibition to undetectable levels after five doses. Reducing the siRNAs to 1mg/kg BIW demonstrated greater inhibition in the presence of PD-L1 mAbs. After only three doses BIW, we could still recover a smaller tumor and, in tumor sections, showed an increase in penetration of CD4+ and CD8+ T-cells deeper into the remaining tumor that was not evident in animals treated with non-silencing siRNA. The combination of TGFß and Cox2 siRNA co-administered in a polypeptide nanoparticle can act as a novel therapeutic alone against HCC and may augment the activity of the immune checkpoint antibodies. Silencing TGFß and Cox2 converts an immune excluded (cold) tumor into a T-cell inflamed (hot) tumor.

Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization.