The immune checkpoint TIGIT is upregulated on T cells during bacterial infection and is a potential target for immunotherapy.

Antibiotic resistance is a major public health threat, and alternatives to antibiotic therapy are urgently needed. Immunotherapy, particularly the blockade of inhibitory immune checkpoints, is a leading treatment option in cancer and autoimmunity. In this study, we used a murine model of Salmonella Typhimurium infection to investigate whether immune checkpoint blockade could be applied to bacterial infection. We found that the immune checkpoint T-cell immunoglobulin and ITIM domain (TIGIT) was significantly upregulated on lymphocytes during infection, particularly on CD4+ T cells, drastically limiting their proinflammatory function. Blockade of TIGIT in vivo using monoclonal antibodies was able to enhance immunity and improve bacterial clearance. The efficacy of anti-TIGIT was dependent on the capacity of the antibody to bind to Fc (fragment crystallizable) receptors, giving important insights into the mechanism of anti-TIGIT therapy. This research suggests that targeting immune checkpoints, such as TIGIT, has the potential to enhance immune responses toward bacteria and restore antibacterial treatment options in the face of antibiotic resistance.

TGF-ß signalling limits effector function capacity of NK cell anti-tumour immunity in human bladder cancer.

BACKGROUND: Natural killer (NK) cells are important innate immunity players and have unique abilities to recognize and eliminate cancer cells, particularly in settings of antibody-opsonization and antibody-dependant cellular cytotoxicity (ADCC). However, NK cell-based responses in bladder cancers to therapeutic antibodies are typically immunosuppressed, and these immunosuppressive mechanisms are largely unknown. METHODS: Single cell RNA sequencing (scRNA-seq) and high-dimensional flow cytometry were used to investigate the phenotype of tumour-infiltrating NK cells in patients with bladder cancer. Further, in vitro, and in vivo models of this disease were used to validate these findings. FINDINGS: NK cells within bladder tumours displayed reduced expression of FcγRIIIa/CD16, the critical Fc receptor involved in ADCC-mediated cytotoxicity, on both transcriptional and protein levels. Transcriptional signatures of transforming growth factor (TGF)-ß-signalling, a pleiotropic cytokine known for its immunosuppressive and tissue residency-inducing effects, were upregulated in tumour-infiltrating NK cells. TGF-ß mediated CD16 downregulation on NK cells, was further validated in vitro, which was accompanied by a transition into a tissue residency phenotype. This CD16 downregulation was also abrogated by TGF-ßR signalling inhibition, which could also restore the ADCC ability of NK cells subject to TGF-ß effects. In a humanized mouse model of bladder cancer, mice treated with a TGF-ß inhibitor exhibited increased ADCC activity compared to mice treated only with antibodies. INTERPRETATION: This study highlights how TGF-ß-rich bladder cancers inhibit NK cell-mediated ADCC by downregulating CD16. TGF-ß inhibition represents new avenues to reverse immunosuppression and enhance the tumoricidal capacity of NK cells in bladder cancer. FUNDING: The Guimaraes Laboratory is funded by a US Department of Defense-Breast Cancer Research Program-Breakthrough Award Level 1 (#BC200025), a grant (#2019485) awarded through the Medical Research Future Fund (MRFF, with the support of the Queensland Children's Hospital Foundation, Microba Life Sciences, Richie's Rainbow Foundation, Translational Research Institute (TRI) and UQ), and a grant (#RSS_2023_085) funded by a Metro South Health Research Support Scheme. J.K.M.W. is funded by a UQ Research Training Program PhD Scholarship and N.O. is funded by a NHMRC Postgraduate Scholarship (#2021932).

Proposal for standardized ultrasound analysis of the salivary glands: Part 1 submandibular gland.

Objectives: The Salivary Gland Committee of the American Academy of Otolaryngology-Head and Neck Surgery seeks to standardize terminology and technique for ultrasonograpy used in the evaluation and treatment of salivary gland disorders. Methods: Development of expert opinion obtained through interaction with international practitioners representing multiple specialties. This committee work includes a comprehensive literature review with presentation of case examples to propose a standardized protocol for the language used in ultrasound salivary gland assessment. Results: A multiple segment proposal is initiated with this focus on the submandibular gland. We provide a concise rationale for recommended descriptive language highlighted by a more extensive supplement that includes an extensive literature review with additional case examples. Conclusion: Recommendations are provided to improve consistency both in performing and reporting submandibular gland ultrasound.

Normative High-Resolution Pharyngeal Manometry: Impact of Age, Size of System, and Sex on Primary Metrics and Pressure Stability.

There have been many reports of normative pharyngeal swallowing pressures using high-resolution pharyngeal manometry, but there is a fair amount of between-subject variance in reported pressure parameters. The purpose of this study was to put forward normative pharyngeal high-resolution manometry measures across the lifespan and investigate the effects of age, size of system, and sex. High-resolution pharyngeal manometry was performed on 98 healthy adults (43 males) between the ages 21 and 89. Pressure duration, maxima, integral, and within-individual variability metrics were averaged over 10 swallows of 10-ml thin liquid. Multiple linear and logistic regressions with model fitting were used to examine how pharyngeal pressures relate to age, pharyngeal size, and sex. Age was associated with tongue base maximum pressure, tongue base maximum variability, and upper esophageal sphincter-integrated relaxation pressure (F3,92 = 6.69; p < 0.001; adjusted R2 = 0.15). Pharyngeal area during bolus hold was associated with velopharynx integral (F1,89 = 5.362; p = 0.02; adjusted R2 = 0.05), and there was no significant model relating pharyngeal pressures to C2-C4 length (p < 0.05). Sex differences were best described by tongue base integral and hypopharynx maximum variability (χ2 = 10.27; p = 0.006; pseudo R2 = 0.14). Normative data reveal the distribution of swallow pressure metrics which need to be accounted for when addressing dysphagia patients, the importance of pressure interactions in normal swallow, and address the relative stability of swallow metrics with normal aging.

The Manometric Representation of the Upper Esophageal Sphincter During the Resting State: A Descriptive Study.

The upper esophageal sphincter (UES) is the high-pressure zone marking the transition between the hypopharynx and esophagus. There is limited research surrounding the resting UES using pharyngeal high-resolution manometry (HRM) and existing normative data varies widely. This study describes the manometric representation of the resting UES using a clinically accessible method of measurement. Data were obtained from 87 subjects in a normative database of pharyngeal HRM with simultaneous videofluoroscopy. The resting UES manometric region was identified and ten measurement segments of this region were taken throughout the duration of the study using the Smart Mouse function within the manometry software. Intraclass correlation coefficients (ICC) were used to analyze within-subject reliability across measurements. Linear mixed-effects regression models were used to analyze how subject characteristics and manometric conditions influence resting UES pressure. There was excellent within-subject reliability between resting UES mean pressures (ICC = 0.96). In bivariate analysis, there were significant effects of age, number of sensors contained within the resting UES, and preceding swallow volume on mean resting UES pressure. For every 1 unit increase in age, there was a 0.19 unit decrease in resting UES pressure (p = 0.008). For every 1 unit increase in number of sensors contained within the resting UES, there was a 3.71 unit increase in resting UES pressure (p < 0.001). This study presents normative data for the resting UES, using a comprehensive and clinically accessible protocol that can provide standard comparison for the study of populations with swallowing disorders, particularly UES dysfunction, and provides support for UES-directed interventions.

Depth of anaesthesia monitoring: time to reject the index?

Depth of anaesthesia monitors can fail to detect consciousness under anaesthesia, primarily because they rely on the frontal EEG, which does not arise from a neural correlate of consciousness. A study published in a previous issue of the British Journal of Anaesthesia showed that indices produced by the different commercial monitors can give highly discordant results when analysing changes in the frontal EEG. Anaesthetists could benefit from routinely assessing the raw EEG and its spectrogram, rather than relying solely on an index produced by a depth of anaesthesia monitor.

Interstitial Brachytherapy for Lip Cancer: Technical Aspects to Individualize Treatment Approach and Optimize Outcomes.

Primary radiation therapy using interstitial brachytherapy (IBT) provides excellent local tumor control for early-stage squamous cell carcinoma of the lip. Technical aspects of treatment are important to optimize outcomes. In this report, we discuss patient selection criteria, procedural details, and dosimetric considerations for performing IBT for cancers of the lip. Catheters are inserted across the length of tumor entering and exiting approximately 5 mm beyond the palpable tumor extent. A custom mouthpiece is fabricated to facilitate normal tissue sparing. Patients undergo computed tomography imaging, the gross tumor volume is contoured based on physical examination and computed tomography findings, and an individualized brachytherapy plan is generated with the goals of achieving gross tumor volume D90% ≥ 90% and minimizing V150%. Ten patients with primary (n = 8) or recurrent (n = 2) cancers of the lip who received high-dose-rate lip IBT using 2.0- to 2.5-week treatment regimens are described (median prescription: 47.6 Gy in 14 fractions of 3.4 Gy). Local tumor control was 100%. There were no cases of acute grade ≥4 or late grade ≥2 toxicity, and cosmesis scores were graded as good to excellent in all patients. IBT represents an excellent treatment option for patients with lip squamous cell carcinoma. With careful attention to technical considerations furthered described in the present report, high rates of tumor control, low rates of toxicity, and favorable esthetic and functional outcomes can be achieved with IBT for lip cancer.

Stress Effects on Pharyngeal Air Pressure and Upper Esophageal Sphincter Pressure During Phonation: The Influence of Vocal Tract Semi-Occlusion.

OBJECTIVES/HYPOTHESIS: The objective of this study was to determine whether vocal tract semi-occlusion (SOVT) influenced stress effects on pharyngeal air pressure and upper esophageal sphincter (UES) pressure during phonation. Relationships between dysphonia and stress are well recognized but poorly understood. Stress effects act globally on the body, and may be observed beyond intrinsic laryngeal muscles to include pharyngeal muscles and the UES, which contribute to voice modulation. Phonation with SOVT may provide resistance to stress effects on the vocal tract. We hypothesized that stress effects on pharyngeal air pressure and UES pressure would be measurable with a high-resolution, 360° pressure catheter, and that stress effects would be impacted differently by occlusal and non-occlusal phonatory tasks. METHODS: Ten healthy adults performed sustained vowel tasks (comfortable /a/, and loud /a/), and SOVT tasks (bilabial fricative and straw phonation). Each task was performed during a baseline condition, and during stress induced through a cold pressor task. Pharyngeal air pressure and UES pressure were measured via high-resolution manometry. Changes in pressure between baseline and stress were compared among phonatory tasks. RESULTS: Stress-induced changes to UES pressure differed by phonatory task (P < 0.01). Stress increased UES pressures during vowels, but had no effect during bilabial fricative, and decreased UES pressures during straw phonation. Change in UES pressure with stress was greater for comfortable /a/ and loud /a/ than straw phonation (P = 0.048 and P = 0.019, respectively), and was not significantly different between comfortable /a/ or loud /a/ and bilabial fricative. Stress-induced changes in pharyngeal air pressure were not significantly different among tasks. CONCLUSIONS: These findings help identify possible mechanisms underlying the relationship between stress and voice, and point to the utility of SOVT tasks for training vocal tract resistance to stress. This methodology provides a foundation for measuring changes to extra-laryngeal components of the vocal tract during phonation.

TGFß and CIS Inhibition Overcomes NK-cell Suppression to Restore Antitumor Immunity.

Antibodies targeting "immune checkpoints" have revolutionized cancer therapy by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8+ T cells. Interest in targeting analogous pathways in other cytotoxic lymphocytes is growing. Natural killer (NK) cells are key to cancer immunosurveillance by eradicating metastases and driving solid tumor inflammation. NK-cell antitumor function is dependent on the cytokine IL15. Ablation of the IL15 signaling inhibitor CIS (Cish) enhances NK-cell antitumor immunity by increasing NK-cell metabolism and persistence within the tumor microenvironment (TME). The TME has also been shown to impair NK-cell fitness via the production of immunosuppressive transforming growth factor ß (TGFß), a suppression which occurs even in the presence of high IL15 signaling. Here, we identified an unexpected interaction between CIS and the TGFß signaling pathway in NK cells. Independently, Cish- and Tgfbr2-deficient NK cells are both hyperresponsive to IL15 and hyporesponsive to TGFß, with dramatically enhanced antitumor immunity. Remarkably, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development, suggesting that combining suppression of these two pathways might represent a novel therapeutic strategy to enhance innate anticancer immunity.

CIS and TGF-ß regulatory pathways influence immunity to bacterial infection.

Immunotherapy has revolutionized cancer therapy by reactivating tumour-resident cytotoxic lymphocytes. More recently, immunotherapy has emerged to restore immunity against infectious agents, including bacterial infections. Immunotherapy primarily targets inhibitory pathways in T cells, however interest in other effector populations, such as natural killer (NK) cells, is growing. We have previously discovered that NK cell metabolism, proliferation and activation can be neutralized through the immunosuppressive transforming growth factor (TGF)-ß pathway by inducing plasticity of NK cells and differentiation into innate lymphoid cell (ILC)1-like subsets. NK cells are also regulated through cytokine-inducible SH2-containing protein (CIS), which is induced by interleukin (IL)-15 and is a potent intracellular checkpoint suppressing NK cell survival and function. Targeting these two distinct pathways to restore NK cell function has shown promise in cancer models, but their application in bacterial infection remains unknown. Here, we investigate whether enhancement of NK cell function can improve anti-bacterial immunity, using Salmonella Typhimurium as a model. We identified conversion of NK cells to ILC1-like for the first time in the context of bacterial infection, where TGF-ß signalling contributed to this plasticity. Future study should focus on identifying further drivers of ILC1 plasticity and its functional implication in bacterial infection model. We further describe that CIS-deficient mice displayed enhanced pro-inflammatory function and dramatically enhanced anti-bacterial immunity. Inhibition of CIS may present as a viable therapeutic option to enhance immunity towards bacterial infection.

Effects of sevoflurane versus propofol on cerebral autoregulation during anaesthesia for robot-assisted laparoscopic prostatectomy.

Robot-assisted laparoscopic prostatectomy requires a pneumoperitoneum combined with steep Trendelenburg positioning, and these conditions can be associated with impairment of cerebral autoregulation. The objective of this study was to determine if choice of anaesthetic agent affects the preservation of cerebral autoregulation during robot-assisted laparoscopic prostatectomy. We randomly assigned 30 patients to maintenance of general anaesthesia with either propofol or sevoflurane. Cerebral autoregulation was tested by administration of intravenous phenylephrine to increase mean arterial pressure from approximately 80 mmHg to 100 mmHg while assessing cerebral blood flow using transcranial Doppler ultrasonography. Autoregulation was first tested in the supine position and then approximately once every hour after Trendelenburg positioning. The main outcome measure was the result of the final autoregulation test prior to completion of surgery. At that time, we found cerebral autoregulation to be significantly impaired in six of the 15 patients receiving sevoflurane and none of the 15 patients receiving propofol (P = 0.02). However, it should be noted that some patients in the propofol group had impaired autoregulation on earlier tests. In conclusion, we found that autoregulation during robot-assisted laparoscopic prostatectomy is less likely to be impaired with propofol compared to sevoflurane anaesthesia, particularly towards the end of the surgery.

The radial forearm snake flap: An underutilized technique for fasciocutaneous and osteocutaneous forearm flaps with primary closure.

BACKGROUND: The radial forearm free flap (RFFF) is associated with troublesome donor site morbidity related to split thickness skin grafting (STSG). The radial forearm snake flap with primary closure of the donor site may reduce donor site complications. METHODS: Single institution, retrospective cohort study comparing rates of delayed donor site wound healing and tendon exposure in 52 patients undergoing radial forearm snake flap and 95 patients undergoing conventional RFFF with STSG closure of the donor site. RESULTS: Tendon exposure occurred in zero (0%) patients undergoing snake flap and four (4.2%) patients undergoing conventional RFFF (0/52 vs. 4/95; p = 0.297). Delayed wound healing occurred in zero (0%) patients undergoing snake flap and 19 (20.0%) patients undergoing conventional RFFF (0/52 vs. 19/95; p < 0.001). CONCLUSIONS: The radial forearm snake flap provides an alternative to conventional RFFF harvest, which enables primary donor site closure with reduced rates of delayed donor site healing.

The Natural Swallow: Factors Affecting Subject Choice of Bolus Volume and Pharyngeal Swallow Parameters in a Self-selected Swallow.

Predetermined volumes are used extensively throughout clinical assessment of swallowing physiology, but bolus volumes selected by an individual in their natural swallow can vary greatly from those used in structured assessment. This study aims to identify factors influencing self-selected volume and how the mechanics of self-selected volume swallows differ from predetermined volume swallows. We used pharyngeal high-resolution manometry (HRM) with simultaneous videofluoroscopy to measure swallowing pressures in the velopharynx, hypopharynx, and upper esophageal sphincter (UES). Data were collected from 95 healthy adults during thin liquid swallows of 10 mL and a self-selected comfortable volume. An intraclass correlation coefficient (ICC) was calculated to analyze within-subject self-selected volume reliability. Linear mixed effects regression models were used to examine the association of subject characteristics with self-selected swallow volume and of self-selected volumes on pharyngeal swallowing pressures and timing events. Mean self-selected volume was 16.66 ± 7.70 mL. Increased age (p = 0.002), male sex (p = 0.021), and increased pharyngeal hold area (p = 0.007) were significantly associated with increase in self-selected bolus volume. There was good reliability between subjects' individual swallow volumes (ICC = 0.80). Velopharyngeal maximum pressure and pressure integral, tongue base duration and maximum pressure, UES pre- and post-swallow maximum pressure, and overall pharyngeal contractile integral decreased significantly with self-selected boluses. Understanding a patient's natural swallow volume, and how their natural swallow functions, will be important for designing clinical evaluations that place stress on the patient's natural swallowing mechanics in order to assess for areas of dysfunction.

Towards efficient immunotherapy for bacterial infection.

The emergence of multiantibiotic-resistant bacteria, often referred to as superbugs, is leading to infections that are increasingly difficult to treat. Further, bacteria have evolved mechanisms by which they subvert the immune response, meaning that even antibiotic-sensitive bacteria can persist through antibiotic therapy. For these reasons, a broad range of viable therapeutic alternatives or conjunctions to traditional antimicrobial therapy are urgently required to reduce the burden of disease threatened by antibiotic resistance. Immunotherapy has emerged as a leading treatment option in cancer, and researchers are now attempting to apply this to infectious disease. This review summarizes and discusses the recent advances in the field and highlights current and future perspectives of using immunotherapies to treat bacterial infections.

Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host responses and gene signatures.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in late 2019 has spread globally, causing a pandemic of respiratory illness designated coronavirus disease 2019 (COVID-19). A better definition of the pulmonary host response to SARS-CoV-2 infection is required to understand viral pathogenesis and to validate putative COVID-19 biomarkers that have been proposed in clinical studies. METHODS: Here, we use targeted transcriptomics of formalin-fixed paraffin-embedded tissue using the NanoString GeoMX platform to generate an in-depth picture of the pulmonary transcriptional landscape of COVID-19, pandemic H1N1 influenza and uninfected control patients. RESULTS: Host transcriptomics showed a significant upregulation of genes associated with inflammation, type I interferon production, coagulation and angiogenesis in the lungs of COVID-19 patients compared to non-infected controls. SARS-CoV-2 was non-uniformly distributed in lungs (emphasising the advantages of spatial transcriptomics) with the areas of high viral load associated with an increased type I interferon response. Once the dominant cell type present in the sample, within patient correlations and patient-patient variation, had been controlled for, only a very limited number of genes were differentially expressed between the lungs of fatal influenza and COVID-19 patients. Strikingly, the interferon-associated gene IFI27, previously identified as a useful blood biomarker to differentiate bacterial and viral lung infections, was significantly upregulated in the lungs of COVID-19 patients compared to patients with influenza. CONCLUSION: Collectively, these data demonstrate that spatial transcriptomics is a powerful tool to identify novel gene signatures within tissues, offering new insights into the pathogenesis of SARS-COV-2 to aid in patient triage and treatment.

IFI27 transcription is an early predictor for COVID-19 outcomes, a multi-cohort observational study.

Purpose: Robust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness. Methods: We conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients. Results: We show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients. Conclusion: These data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus.

A Rare Complication of Fine-Needle Aspiration of Neck Structures.

Fine-needle aspiration (FNA) is a generally accepted tool for safe diagnostic evaluation in the workup of lesions and masses. Aside from the commonly discussed risks of infection and minor bleeding related to skin puncture, other more serious complications have been reported sparingly. We present two cases of pneumothorax from FNA of neck structures, which have been theorized but not previously reported to our knowledge. Discussion of cases of this complication rather than solely a theoretical understanding of it will aid in diagnosis and management of this complication.

MAIT cells regulate NK cell-mediated tumor immunity.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

Measurement of Pharyngeal Air Pressure During Phonation Using High-Resolution Manometry.

Purpose The study of air pressure in the vocal tract is essential to understanding vocal function. Changes in vocal tract shape during different phonatory gestures are hypothesized to produce nonuniform air pressure across lower vocal tract locations. Current methods of air pressure measurement, however, are limited to a single location in the anterior oral cavity. The purposes of this study were (a) to assess the feasibility of a novel method of simultaneously measuring phonatory air pressure at multiple locations across the lower vocal tract using high-resolution pharyngeal manometry (HRM) and (b) to compare pressure across locations and among phonatory tasks. Method Two subjects underwent HRM while performing phonatory tasks. A catheter was passed transnasally and air pressure was measured simultaneously at five locations between the velopharyngeal port and the upper esophageal sphincter. Descriptive statistics were calculated for each location by task, and for each task averaged across locations. Results HRM was well tolerated, and air pressures from multiple locations in the lower vocal tract were able to be obtained simultaneously. During vocal tract semi-occlusion tasks, air pressures differed by location. Pressures averaged across locations demonstrated a pattern of increasing pressure with increasing semi-occlusion. Conclusions HRM is feasible for measuring air pressure simultaneously at multiple locations in the lower vocal tract during phonation with high spatial and temporal resolution, providing rich data to augment understanding of vocal function. The high spatial and temporal resolution yielded by this new method, paired with preliminary evidence that pressures change by location as a function of phonatory task, may be useful in future assays exploring differences in lower vocal tract air pressures between normal and disordered populations.