Nivolumab treatment beyond RECIST-defined progression in recurrent or metastatic squamous cell carcinoma of the head and neck in CheckMate 141: A subgroup analysis of a randomized phase 3 clinical trial.

BACKGROUND: Response patterns with immune checkpoint inhibitors may be different from those with chemotherapy. Therefore, assessment of response to immunotherapy with the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, could result in premature treatment termination. The randomized, open-label, phase 3 CheckMate 141 trial (NCT02105636), which evaluated nivolumab in recurrent/metastatic squamous cell carcinoma of the head and neck after platinum therapy, allowed treatment beyond first RECIST-defined progression (TBP) according to protocol-specified criteria. METHODS: In CheckMate 141, patients with RECIST-defined progression who had a stable performance status and demonstrated clinical benefit without rapid disease progression were permitted to receive TBP with nivolumab at 3 mg/kg every 2 weeks until further progression, which was defined as an additional ≥10% increase in tumor volume. This post hoc analysis evaluated outcomes for patients who received TBP with nivolumab. RESULTS: Of 240 patients randomized to nivolumab, 146 experienced RECIST-defined progression. Sixty-two of these patients received TBP, and 84 discontinued treatment (no TBP). Among the 60 TBP patients evaluable for response, 15 (25%) had no change in their tumor burden, and 15 (25%) had reductions in target lesion size; 3 patients (5%) had reductions >30%. The median overall survival among TBP patients was 12.7 months (95% confidence interval, 9.7-14.6 months). No new safety signals were observed with TBP. Exploratory analyses of immune cell biomarkers suggested a potential relationship with initial and TBP responses. CONCLUSIONS: Tumor burden reduction was noted in a proportion of patients who received TBP with nivolumab in CheckMate 141. Additional research is warranted to identify factors predictive of a TBP benefit in this population.

Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck.

BACKGROUND: Patients with recurrent or metastatic squamous-cell carcinoma of the head and neck after platinum chemotherapy have a very poor prognosis and limited therapeutic options. Nivolumab, an anti-programmed death 1 (PD-1) monoclonal antibody, was assessed as treatment for this condition. METHODS: In this randomized, open-label, phase 3 trial, we assigned, in a 2:1 ratio, 361 patients with recurrent squamous-cell carcinoma of the head and neck whose disease had progressed within 6 months after platinum-based chemotherapy to receive nivolumab (at a dose of 3 mg per kilogram of body weight) every 2 weeks or standard, single-agent systemic therapy (methotrexate, docetaxel, or cetuximab). The primary end point was overall survival. Additional end points included progression-free survival, rate of objective response, safety, and patient-reported quality of life. RESULTS: The median overall survival was 7.5 months (95% confidence interval [CI], 5.5 to 9.1) in the nivolumab group versus 5.1 months (95% CI, 4.0 to 6.0) in the group that received standard therapy. Overall survival was significantly longer with nivolumab than with standard therapy (hazard ratio for death, 0.70; 97.73% CI, 0.51 to 0.96; P=0.01), and the estimates of the 1-year survival rate were approximately 19 percentage points higher with nivolumab than with standard therapy (36.0% vs. 16.6%). The median progression-free survival was 2.0 months (95% CI, 1.9 to 2.1) with nivolumab versus 2.3 months (95% CI, 1.9 to 3.1) with standard therapy (hazard ratio for disease progression or death, 0.89; 95% CI, 0.70 to 1.13; P=0.32). The rate of progression-free survival at 6 months was 19.7% with nivolumab versus 9.9% with standard therapy. The response rate was 13.3% in the nivolumab group versus 5.8% in the standard-therapy group. Treatment-related adverse events of grade 3 or 4 occurred in 13.1% of the patients in the nivolumab group versus 35.1% of those in the standard-therapy group. Physical, role, and social functioning was stable in the nivolumab group, whereas it was meaningfully worse in the standard-therapy group. CONCLUSIONS: Among patients with platinum-refractory, recurrent squamous-cell carcinoma of the head and neck, treatment with nivolumab resulted in longer overall survival than treatment with standard, single-agent therapy. (Funded by Bristol-Myers Squibb; CheckMate 141 ClinicalTrials.gov number, NCT02105636 .).

Integrated Fluidic Circuits for Single-Cell Omics and Multi-omics Applications.

Single-cell genomics plays a crucial role in several aspects of biology, from developmental biology to mapping every cell in the human body through the Cell Atlas initiative. To meet these various applications, single-cell methods are rapidly evolving to increase throughput; improve sensitivity, quantification accuracy, and usability; and reduce nucleic-acid amplification bias and cost. In addition to improvement in single-cell methods, there is a huge interest in analyzing multiple analytes such as genome, epigenome, transcriptome, and protein from the same single cell. This approach is generalized as single-cell multi-omics. Automation of multi-step single-cell methods is highly desired to achieve a reproducible workflow; reduce human error and avoid contamination; and introduce technical variability to an existing stochastic process. Typically single-cell reactions start with a low level of nucleic acid, in the range of picograms. Miniaturization in microfluidic devices leads to a gain in reaction efficiency in Nanoliter or picoliter reaction volumes and active mixing help ensure that solid-state microfluidic devices provide the broadest flexibility and best sensitivity in single-cell reactions, compared to other methods. In this chapter, we will present integrated fluidic circuit (IFC) microfluidics for various single-cell multi-omics applications, and show how this technology fits into the current single-cell technology portfolio available from various vendors. We will then discuss possible uses for IFCs in multi-omics applications that are on the horizon.

Elotuzumab monotherapy in patients with smouldering multiple myeloma: a phase 2 study.

Smouldering multiple myeloma (SMM) is associated with increased risk of progression to multiple myeloma within 2 years, with no approved treatments. Elotuzumab has been shown to promote natural killer (NK) cell stimulation and antibody-dependent cellular cytotoxicity (ADCC) in vitro. CD56dim (CD56dim /CD16+ /CD3- /CD45+ ) NK cells represent the primary subset responsible for elotuzumab-induced ADCC. In this phase II, non-randomized study (NCT01441973), patients with SMM received elotuzumab 20 mg/kg intravenously (cycle 1: days 1, 8; monthly thereafter) or 10 mg/kg (cycles 1, 2: weekly; every 2 weeks thereafter). The primary endpoint was the relationship between baseline proportion of bone marrow-derived CD56dim NK cells and maximal M protein reduction; secondary endpoints included overall response rate (ORR) and progression-free survival (PFS). Fifteen patients received 20 mg/kg and 16 received 10 mg/kg; combined data arepresented. At database lock (DBL, September 2014), no association was found between baseline CD56dim NK cell proportion and maximal M protein reduction. With minimum 28 months' follow-up (DBL: January 2016), ORR (90% CI) was 10% (2·7-23·2) and 2-year PFS rate was 69% (52-81%). Upper respiratory tract infections occurred in 18/31 (58%) patients. Four (13%) patients experienced infusion reactions, all grade 1-2. Elotuzumab plus lenalidomide/dexamethasone is under investigation for SMM.

CheckMate 141: 1-Year Update and Subgroup Analysis of Nivolumab as First-Line Therapy in Patients with Recurrent/Metastatic Head and Neck Cancer.

Nivolumab significantly improved overall survival (OS) vs investigator's choice (IC) of chemotherapy at the primary analysis of randomized, open-label, phase 3 CheckMate 141 in patients with recurrent or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN). Here, we report that OS benefit with nivolumab was maintained at a minimum follow-up of 11.4 months. Further, OS benefit with nivolumab vs IC was also noted among patients who received first-line treatment for R/M SCCHN after progressing on platinum therapy for locally advanced disease in the adjuvant or primary (i.e., with radiation) setting.

Randomized phase 2 study: elotuzumab plus bortezomib/dexamethasone vs bortezomib/dexamethasone for relapsed/refractory MM.

In this proof-of-concept, open-label, phase 2 study, patients with relapsed/refractory multiple myeloma (RRMM) received elotuzumab with bortezomib and dexamethasone (EBd) or bortezomib and dexamethasone (Bd) until disease progression/unacceptable toxicity. Primary endpoint was progression-free survival (PFS); secondary/exploratory endpoints included overall response rate (ORR) and overall survival (OS). Two-sided 0.30 significance level was specified (80% power, 103 events) to detect hazard ratio (HR) of 0.69. Efficacy and safety analyses were performed on all randomized patients and all treated patients, respectively. Of 152 randomized patients (77 EBd, 75 Bd), 150 were treated (75 EBd, 75 Bd). PFS was greater with EBd vs Bd (HR, 0.72; 70% confidence interval [CI], 0.59-0.88; stratified log-rank P = .09); median PFS was longer with EBd (9.7 months) vs Bd (6.9 months). In an updated analysis, EBd-treated patients homozygous for the high-affinity FcγRIIIa allele had median PFS of 22.3 months vs 9.8 months in EBd-treated patients homozygous for the low-affinity allele. ORR was 66% (EBd) vs 63% (Bd). Very good partial response or better occurred in 36% of patients (EBd) vs 27% (Bd). Early OS results, based on 40 deaths, revealed an HR of 0.61 (70% CI, 0.43-0.85). To date, 60 deaths have occurred (28 EBd, 32 Bd). No additional clinically significant adverse events occurred with EBd vs Bd. Grade 1/2 infusion reaction rate was low (5% EBd) and mitigated with premedication. In patients with RRMM, elotuzumab, an immunostimulatory antibody, appears to provide clinical benefit without added clinically significant toxicity when combined with Bd vs Bd alone. Registered to ClinicalTrials.gov as NCT01478048.

Predicting Nucleation of Isonicotinamide from the Solvent-Solute Interactions of Isonicotinamide in Common Organic Solvents.

The interactions of isonicotinamide (INA) with seven common solvents (acetic acid, acetonitrile, acetone, chloroform, ethyl acetate, and methanol) have been studied to examine solute-solvent effects on the nucleation of INA from these solvents. In a simple model of 1:1 solute-solvent interactions, the strongest INA-solvent interaction is with acetic acid (binding energy, Δ Ebind = -64.05 kJ mol-1) and the weakest is with chloroform (Δ Ebind = -24.85 kJ mol-1). This arises since acetic acid and INA form a hydrogen-bonding motif containing two moderate strength N-H···O hydrogen bonds, while chloroform and INA have a single weak C-H···O hydrogen bond. Taking acetic acid, chloroform, and methanol, the solvents with the strongest, the weakest, and an intermediate strength INA-solvent binding energy, the solvation of INA was studied to compare it with the 1:1 model. Acetic acid has the strongest binding energy (-872.24 kJ mol-1) and solvation energy (-341.20 kJ mol-1) with chloroform binding energy (-517.72 kJ mol-1) and solvation energy (-199.05 kJ mol-1). Methanol has intermediate binding energy (-814.19 kJ mol-1) and solvation energies (-320.81 kJ mol-1). These results further confirm the recent the findings which indicate that the key trends in solvent-solute interactions can be determined from a simple and efficient 1:1 dimer model and can be used to predict ease of nucleation with stronger binding energies correlating to slower, more difficult nucleation. A limit of this model is revealed by considering alcohol and acid solvents with longer alkyl chains.

Nivolumab versus standard, single-agent therapy of investigator's choice in recurrent or metastatic squamous cell carcinoma of the head and neck (CheckMate 141): health-related quality-of-life results from a randomised, phase 3 trial.

BACKGROUND: Patients with platinum-refractory recurrent or metastatic squamous cell carcinoma of the head and neck have few treatment options and poor prognosis. Nivolumab significantly improved survival of this patient population when compared with standard single-agent therapy of investigator's choice in Checkmate 141; here we report the effect of nivolumab on patient-reported outcomes (PROs). METHODS: CheckMate 141 was a randomised, open-label, phase 3 trial in patients with recurrent or metastatic squamous cell carcinoma of the head and neck who progressed within 6 months after platinum-based chemotherapy. Patients were randomly assigned (2:1) to nivolumab 3 mg/kg every 2 weeks (n=240) or investigator's choice (n=121) of methotrexate (40-60 mg/m2 of body surface area), docetaxel (30-40 mg/m2), or cetuximab (250 mg/m2 after a loading dose of 400 mg/m2) until disease progression, intolerable toxicity, or withdrawal of consent. On Jan 26, 2016, the independent data monitoring committee reviewed the data at the planned interim analysis and declared overall survival superiority for nivolumab over investigator's choice therapy (primary endpoint; described previously). The protocol was amended to allow patients in the investigator's choice group to cross over to nivolumab. All patients not on active therapy are being followed for survival. As an exploratory endpoint, PROs were assessed at baseline, week 9, and every 6 weeks thereafter using the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire-Core 30 (QLQ-C30), the EORTC head and neck cancer-specific module (EORTC QLQ-H&N35), and the three-level European Quality of Life-5 Dimensions (EQ-5D) questionnaire. Differences within and between treatment groups in PROs were analysed by ANCOVA among patients with baseline and at least one other assessment. All randomised patients were included in the time to clinically meaningful deterioration analyses. Median time to clinically meaningful deterioration was analysed by Kaplan-Meier methods. CheckMate 141 was registered with ClinicalTrials.org, number NCT02105636. FINDINGS: Patients were enrolled between May 29, 2014, and July 31, 2015, and subsequently 361 patients were randomly assigned to receive nivolumab (n=240) or investigator's choice (n=121). Among them, 129 patients (93 in the nivolumab group and 36 in the investigator's choice group) completed any of the PRO questionnaires at baseline and at least one other assessment. Treatment with nivolumab resulted in adjusted mean changes from baseline to week 15 ranging from -2·1 to 5·4 across functional and symptom domains measured by the EORTC QLQ-C30, with no domains indicating clinically meaningful deterioration. By contrast, eight (53%) of the 15 domains in the investigator's choice group showed clinically meaningful deterioration (10 points or more) at week 15 (change from baseline range, -24·5 to 2·4). Similarly, on the EORTC QLQ-H&N35, clinically meaningful worsening at week 15 was seen in no domains in the nivolumab group and eight (44%) of 18 domains in the investigator's choice group. Patients in the nivolumab group had a clinically meaningful improvement (according to a difference of 7 points or greater) in adjusted mean change from baseline to week 15 on the EQ-5D visual analogue scale, in contrast to a clinically meaningful deterioration in the investigator's choice group (7·3 vs -7·8). Differences between groups were significant and clinically meaningful at weeks 9 and 15 in favour of nivolumab for role functioning, social functioning, fatigue, dyspnoea, and appetite loss on the EORTC QLQ-C30 and pain and sensory problems on the EORTC QLQ-H&N35. Median time to deterioration was significantly longer with nivolumab versus investigator's choice for 13 (37%) of 35 domains assessed across the three questionnaires. INTERPRETATION: In this exploratory analysis of CheckMate 141, nivolumab stabilised symptoms and functioning from baseline to weeks 9 and 15, whereas investigator's choice led to clinically meaningful deterioration. Nivolumab delayed time to deterioration of patient-reported quality-of-life outcomes compared with single-agent therapy of investigator's choice in patients with platinum-refractory recurrent or metastatic squamous cell carcinoma of the head and neck. In view of the major unmet need in this population and the importance of maintaining or improving quality of life for patients with recurrent or metastatic squamous cell carcinoma of the head and neck, these data support nivolumab as a new standard-of-care option in this setting. FUNDING: Bristol-Myers Squibb.

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response.

BACKGROUND: Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear. RESULTS: We used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS. CONCLUSIONS: These results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Elotuzumab in combination with thalidomide and low-dose dexamethasone: a phase 2 single-arm safety study in patients with relapsed/refractory multiple myeloma.

Elotuzumab is an immunostimulatory, humanized immunoglobulin G1 monoclonal antibody that selectively targets and kills signalling lymphocytic activation molecule family member 7-expressing myeloma cells. We evaluated the safety and tolerability of elotuzumab 10 mg/kg combined with thalidomide 50-200 mg and dexamethasone 40 mg (with/without cyclophosphamide 50 mg) in patients with relapsed/refractory multiple myeloma (RRMM). The primary endpoint was the proportion of grade ≥3 non-haematological adverse events (AEs); other endpoints included the number of dose reductions/discontinuations and efficacy. Forty patients were treated, who had a median of three previous therapies, including bortezomib (98%) and lenalidomide (73%). Grade ≥3 non-haematological AEs were reported in 63% of patients, most commonly asthenia (35%) and peripheral oedema (25%). Six (15%) patients had an infusion reaction. Twenty-six (65%) patients had ≥1 dose reduction/discontinuation due to an AE, none related to elotuzumab. Overall response rate was 38%; median progression-free survival was 3·9 months. Median overall survival was 16·3 months and the 1-year survival rate was 63%. Minimal incremental toxicity was observed with addition of elotuzumab to thalidomide/dexamethasone with or without cyclophosphamide, and efficacy data suggest clinical benefit in a highly pre-treated population. Elotuzumab combined with thalidomide may provide an additional treatment option for patients with RRMM.

Modified transurethral resection of the prostate (TURP) for men with moderate lower urinary tract symptoms (LUTS) before brachytherapy is safe and feasible.

OBJECTIVE: To report the urinary toxicity outcomes for patients at greater risk of voiding symptoms and retention who received a modified limited transurethral resection of the prostate (TURP) before low-dose rate (LDR) brachytherapy. PATIENTS AND METHOD: Data were analysed from patients receiving the above procedures between 2006 to present, taken from the prospective brachytherapy database of 2000 patients at the St. Luke's Cancer Centre. The limited TURP (TURP(BXT) ) was performed at a median (range) of 64 (25-205) days before seed implantation with a median resection weight of 1.15 g. Selection criteria were based on patients with moderate lower urinary tract symptoms, poor flow or post-void residual urine volume (PVR), or a prominent middle lobe or high bladder neck on transrectal ultrasonography. Baseline prostate cancer characteristics, uroflowmetry, International Prostate Symptom Score (IPSS) and quality-of-life QoL scores were collected and compared with follow-up IPSS and QoL scores. RESULTS: Data for 112 patients was gathered from the database. The TURP(BXT) resulted in statistically significant improvements before LDR brachytherapy in maximum urinary flow rate (Qmax ) and PVR, IPSS and QoL scores (the mean Qmax before vs after the TURP(BXT) was 11.3 vs 16.7 mL/s). The IPSS and QoL scores at 6 months after seed implantation were increased compared with baseline values before the TURP(BXT) (mean IPSS at 6 months 11.7 vs 9.2 before TURP(BXT) ), but no difference at 1 year (mean IPSS 9), and improved scores at 2, 3, 4 and 5 years follow-up (mean IPSS of 7.9, 5.6, 5.3 and 7.4, respectively). CONCLUSION: The present study suggests patients at increased risk of deteriorating voiding symptoms, including urinary retention, are no longer contraindicated against LDR brachytherapy if they receive a modified TURP before seed implantation. This procedure does not appear to carry the risk of urinary incontinence thought to be associated with a conventional TURP before LDR brachytherapy.

Clinical and diagnostic findings in patients with elevated cerebrospinal bilirubin.

INTRODUCTION: Cerebrospinal fluid (CSF) spectroscopy can identify subarachnoid haemorrhage (SAH) when CT is negative in patients presenting with acute severe headache. The primary objective of this study was to evaluate the clinical use and usefulness of CSF spectrophotometry. Secondary objectives were to identify other causes of elevated CSF bilirubin, to analyse headache descriptions and to compare clinical features in patients with an elevated CSF bilirubin among those with and without an intracranial vascular cause of SAH (avSAH). METHODS: Consecutive patients admitted to two hospitals in Enniskillen and Londonderry between 1 January 2004 and 30 September 2014 with CSF spectroscopy bilirubin results were identified from a clinical chemistry laboratory dataset. Patients with elevated CSF bilirubin were studied. Clinical demographics, delays to investigation and final diagnoses were recorded. Patients with avSAH were compared with patients without avSAH. RESULTS: Among 1813 patients with CSF spectrophotometry results, requests increased more than threefold during the study (p<0.001). Fifty-six patients had elevated CSF bilirubin. Ten (17.9%) had avSAH, of which 8 (14.3%) had aneurysmal SAH. Non-vascular causes of elevated CSF bilirubin included meningitis, spontaneous intracranial hypotension and carcinomatous meningitis. Headache descriptions varied. Time from headache onset to admission, CT scan and lumbar puncture did not differ significantly for patients with avSAH and non-avSAH. CSF red cell counts were higher among patients with avSAH than patients with non-avSAH (p=0.005). CONCLUSIONS: CSF bilirubin measurement has an important role in identifying avSAH in CT-negative patients presenting with a thunderclap headache. Better clinical selection of patients is required as CSF spectrophotometry, although sensitive, is not specific for SAH.

Analysis of SARS-CoV-2 antibody seroprevalence in Northern Ireland during 2020-2021.

Background: With the spread of SARS-CoV-2 impacting upon public health directly and socioeconomically, further information was required to inform policy decisions designed to limit virus spread during the pandemic. This study sought to contribute to serosurveillance work within Northern Ireland to track SARS-CoV-2 progression and guide health strategy. Methods: Sera/plasma samples from clinical biochemistry laboratories were analysed for anti-SARS-CoV-2 antibodies. Samples were assessed using an Elecsys anti-SARS-CoV-2 or anti-SARS-CoV-2 S ECLIA (Roche) on an automated cobas e 801 analyser. Samples were also assessed via an anti-SARS-CoV-2 ELISA (Euroimmun). A subset of samples assessed via the Elecsys anti-SARS-CoV-2 ECLIA were subsequently analysed in an ACE2 pseudoneutralisation assay using a V-PLEX SARS-CoV-2 Panel 7 for IgG and ACE2 (Meso Scale Diagnostics). Results: Across three testing rounds (June-July 2020, November-December 2020 and June-July 2021 (rounds 1-3 respectively)), 4844 residual sera/plasma specimens were assayed for anti-SARS-CoV-2 antibodies. Seropositivity rates increased across the study, peaking at 11.6 % (95 % CI 10.4 %-13.0 %) during round 3. Varying trends in SARS-CoV-2 seropositivity were noted based on demographic factors. For instance, highest rates of seropositivity shifted from older to younger demographics across the study period. In round 3, Alpha (B.1.1.7) variant neutralising antibodies were most frequently detected across age groups, with median concentration of anti-spike protein antibodies elevated in 50-69 year olds and anti-S1 RBD antibodies elevated in 70+ year olds, relative to other age groups. Conclusions: With seropositivity rates of <15 % across the assessment period, it can be concluded that the significant proportion of the Northern Ireland population had not yet naturally contracted the virus by mid-2021.

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

Synergy Assessment of Four Antimicrobial Bioactive Compounds for the Combinational Treatment of Bacterial Pathogens.

Antimicrobial resistance (AMR) has become a topic of great concern in recent years, with much effort being committed to developing alternative treatments for resistant bacterial pathogens. Drug combinational therapies have been a major area of research for several years, with modern iterations using combining well-established antibiotics and other antimicrobials with the aim of discovering complementary mechanisms. Previously, we characterised four GRAS antimicrobials that can withstand thermal polymer extrusion processes for novel medical device-based and therapeutic applications. In the present study, four antimicrobial bioactive-silver nitrate, nisin, chitosan and zinc oxide-were assessed for their potential combined use as an alternative synergistic treatment for AMR bacteria via a broth microdilution assay based on a checkerboard format. The bioactives were tested in arrangements of two-, three- and four-drug combinations, and their interactions were determined and expressed in terms of a synergy score. Results have revealed interesting interactions based on treatments against recognised test bacterial strains that cause human and animal infections, namely E. coli, S. aureus and S. epidermidis. Silver nitrate was seen to greatly enhance the efficacy of its paired treatment. Combinations with nisin, which is a lantibiotic, exhibited the most interesting results, as nisin has no effect against Gram-negative bacteria when used alone; however, it demonstrated antimicrobial effects when combined with silver nitrate or chitosan. This study constitutes the first study to both report on practical three- and four-drug combinational assays and utilise these methods for the assessment of established and emerging antimicrobials. The novel methods and results presented in this study show the potential to explore previously unknown drug combination compatibility measures in an ease-of-use- and high-throughput-based format, which can greatly help future research that aims to identify appropriate alternative treatments for AMR, including the screening of potential new bioactives biorefined from various sources.

Colon cancer associated genes exhibit signatures of positive selection at functionally significant positions.

BACKGROUND: Cancer, much like most human disease, is routinely studied by utilizing model organisms. Of these model organisms, mice are often dominant. However, our assumptions of functional equivalence fail to consider the opportunity for divergence conferred by ~180 Million Years (MY) of independent evolution between these species. For a given set of human disease related genes, it is therefore important to determine if functional equivalency has been retained between species. In this study we test the hypothesis that cancer associated genes have different patterns of substitution akin to adaptive evolution in different mammal lineages. RESULTS: Our analysis of the current literature and colon cancer databases identified 22 genes exhibiting colon cancer associated germline mutations. We identified orthologs for these 22 genes across a set of high coverage (>6X) vertebrate genomes. Analysis of these orthologous datasets revealed significant levels of positive selection. Evidence of lineage-specific positive selection was identified in 14 genes in both ancestral and extant lineages. Lineage-specific positive selection was detected in the ancestral Euarchontoglires and Hominidae lineages for STK11, in the ancestral primate lineage for CDH1, in the ancestral Murinae lineage for both SDHC and MSH6 genes and the ancestral Muridae lineage for TSC1. CONCLUSION: Identifying positive selection in the Primate, Hominidae, Muridae and Murinae lineages suggests an ancestral functional shift in these genes between the rodent and primate lineages. Analyses such as this, combining evolutionary theory and predictions - along with medically relevant data, can thus provide us with important clues for modeling human diseases.

Microfluidic live tracking and transcriptomics of cancer-immune cell doublets link intercellular proximity and gene regulation.

Cell-cell communication and physical interactions play a vital role in cancer initiation, homeostasis, progression, and immune response. Here, we report a system that combines live capture of different cell types, co-incubation, time-lapse imaging, and gene expression profiling of doublets using a microfluidic integrated fluidic circuit that enables measurement of physical distances between cells and the associated transcriptional profiles due to cell-cell interactions. We track the temporal variations in natural killer-triple-negative breast cancer cell distances and compare them with terminal cellular transcriptome profiles. The results show the time-bound activities of regulatory modules and allude to the existence of transcriptional memory. Our experimental and bioinformatic approaches serve as a proof of concept for interrogating live-cell interactions at doublet resolution. Together, our findings highlight the use of our approach across different cancers and cell types.

Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity.

Angiogenesis, a critical driver of tumor development, is controlled by interconnected signaling pathways. Vascular endothelial growth factor receptor (VEGFR) 2 and tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 play crucial roles in the biology of normal and tumor vasculature. Regorafenib (BAY 73-4506), a novel oral multikinase inhibitor, potently inhibits these endothelial cell kinases in biochemical and cellular kinase phosphorylation assays. Furthermore, regorafenib inhibits additional angiogenic kinases (VEGFR1/3, platelet-derived growth factor receptor-ß and fibroblast growth factor receptor 1) and the mutant oncogenic kinases KIT, RET and B-RAF. The antiangiogenic effect of regorafenib was demonstrated in vivo by dynamic contrast-enhanced magnetic resonance imaging. Regorafenib administered once orally at 10 mg/kg significantly decreased the extravasation of Gadomer in the vasculature of rat GS9L glioblastoma tumor xenografts. In a daily (qd)×4 dosing study, the pharmacodynamic effects persisted for 48 hr after the last dosing and correlated with tumor growth inhibition (TGI). A significant reduction in tumor microvessel area was observed in a human colorectal xenograft after qd×5 dosing at 10 and 30 mg/kg. Regorafenib exhibited potent dose-dependent TGI in various preclinical human xenograft models in mice, with tumor shrinkages observed in breast MDA-MB-231 and renal 786-O carcinoma models. Pharmacodynamic analyses of the breast model revealed strong reduction in staining of proliferation marker Ki-67 and phosphorylated extracellular regulated kinases 1/2. These data demonstrate that regorafenib is a well-tolerated, orally active multikinase inhibitor with a distinct target profile that may have therapeutic benefit in human malignancies.