Single cell spatial analysis reveals inflammatory foci of immature neutrophil and CD8 T cells in COVID-19 lungs.

Single cell spatial interrogation of the immune-structural interactions in COVID -19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we develop a suite of mathematical tools to search for statistically significant co-locations amongst immune and structural cells identified using 37-plex imaging mass cytometry. This unbiased method reveals a cellular map interleaved with an inflammatory network of immature neutrophils, cytotoxic CD8 T cells, megakaryocytes and monocytes co-located with regenerating alveolar progenitors and endothelium. Of note, a highly active cluster of immature neutrophils and CD8 T cells, is found spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings offer further insights into how immune cells interact in the lungs of severe COVID-19 disease. We provide our pipeline [Spatial Omics Oxford Pipeline (SpOOx)] and visual-analytical tool, Multi-Dimensional Viewer (MDV) software, as a resource for spatial analysis.

A rare case of Ovarian Juvenile Granulosa Cell Tumor in an Infant with Isosexual Pseudo Puberty and a Revision of Literature.

Juvenile ovarian granulosa cell tumors (JGCTs) are described infrequently in pediatrics, and their finding in infants is exceptional. We highlight the presenting symptoms, radiologic images, operative management, and histopathologic findings of a 9-month-old female with isosexual pseudopuberty. An updated revision of literature in infants below the age of 12 months is also reported.

Multi-Modal Characterization of Monocytes in Idiopathic Pulmonary Fibrosis Reveals a Primed Type I Interferon Immune Phenotype.

Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. Circulating monocytes have been implicated in immune pathology in IPF but their phenotype is unknown. In this work, we determined the immune phenotype of monocytes in IPF using multi-colour flow cytometry, RNA sequencing and corresponding serum factors, and mapped the main findings to amount of lung fibrosis and single cell transcriptomic landscape of myeloid cells in IPF lungs. We show that monocytes from IPF patients displayed increased expression of CD64 (FcγR1) which correlated with amount of lung fibrosis, and an amplified type I IFN response ex vivo. These were accompanied by markedly raised CSF-1 levels, IL-6, and CCL-2 in serum of IPF patients. Interrogation of single cell transcriptomic data from human IPF lungs revealed increased proportion of CD64hi monocytes and "transitional macrophages" with higher expression of CCL-2 and type I IFN genes. Our study shows that monocytes in IPF patients are phenotypically distinct from age-matched controls, with a primed type I IFN pathway that may contribute to driving chronic inflammation and fibrosis. These findings strengthen the potential role of monocytes in the pathogenesis of IPF.

A Pilot Feasibility Study in Establishing the Role of Ultrasound-Guided Pleural Biopsies in Pleural Infection (The AUDIO Study).

BACKGROUND: Pleural infection is a common complication of pneumonia associated with high mortality and poor clinical outcome. Treatment of pleural infection relies on the use of broad-spectrum antibiotics because reliable pathogen identification occurs infrequently. We performed a feasibility interventional clinical study assessing the safety and significance of ultrasound (US)-guided pleural biopsy culture to increase microbiological yield. In an exploratory investigation, the 16S ribosomal RNA technique was applied to assess its utility on increasing speed and accuracy vs standard microbiological diagnosis. METHODS: Twenty patients with clinically established pleural infection were recruited. Participants underwent a detailed US scan and US-guided pleural biopsies before chest drain insertion, alongside standard clinical management. Pleural biopsies and routine clinical samples (pleural fluid and blood) were submitted for microbiological analysis. RESULTS: US-guided pleural biopsies were safe with no adverse events. US-guided pleural biopsies increased microbiological yield by 25% in addition to pleural fluid and blood samples. The technique provided a substantially higher microbiological yield compared with pleural fluid and blood culture samples (45% compared with 20% and 10%, respectively). The 16S ribosomal RNA technique was successfully applied to pleural biopsy samples, demonstrating high sensitivity (93%) and specificity (89.5%). CONCLUSIONS: Our findings demonstrate the safety of US-guided pleural biopsies in patients with pleural infection and a substantial increase in microbiological diagnosis, suggesting potential niche of infection in this disease. Quantitative polymerase chain reaction primer assessment of pleural fluid and biopsy appears to have excellent sensitivity and specificity.

Myocardial steatosis and left ventricular contractile dysfunction in patients with severe aortic stenosis.

BACKGROUND: Aortic stenosis (AS) leads to left ventricular (LV) hypertrophy and dysfunction. We hypothesized that cardiac steatosis is involved in the pathophysiology and also assessed whether it is reversible after aortic valve replacement. METHODS AND RESULTS: Thirty-nine patients with severe AS (symptomatic=25, asymptomatic=14) with normal LV ejection fraction and no significant coronary artery disease and 20 age- and sex-matched healthy controls underwent cardiac 1H-magnetic resonance spectroscopy and imaging for the determination of steatosis (myocardial triglyceride content) and cardiac function, including circumferential strain (measured by magnetic resonance tagging). Strain was lower in both symptomatic and asymptomatic AS (-16.4 ± 2.5% and -18.1 ± 2.9%, respectively, versus controls -20.7 ± 2.0%, both P<0.05). Myocardial steatosis was found in both symptomatic and asymptomatic patients with AS (0.89 ± 0.42% in symptomatic AS; 0.75 ± 0.36% in asymptomatic AS versus controls 0.45 ± 0.17, both P<0.05). Importantly, multivariable analysis indicated that steatosis was an independent correlate of impaired LV strain. Spectroscopic measurements of myocardial triglyceride content correlated significantly with histological analysis of biopsies obtained during aortic valve replacement. At 8.0 ± 2.1 months after aortic valve replacement, steatosis and strain had recovered toward normal. CONCLUSIONS: Pronounced myocardial steatosis is present in severe AS, regardless of symptoms, and is independently associated with the degree of LV strain impairment. Myocardial triglyceride content measured by magnetic resonance spectroscopy correlates with histological quantification. Steatosis and strain impairment are reversible after aortic valve replacement. Our findings suggest a novel pathophysiological mechanism in AS, myocardial steatosis, which may be amenable to treatment, thus potentially delaying onset of LV dysfunction.

Pivotal Advance: Invariant NKT cells reduce accumulation of inflammatory monocytes in the lungs and decrease immune-pathology during severe influenza A virus infection.

Little is known of how a strong immune response in the lungs is regulated to minimize tissue injury during severe influenza A virus (IAV) infection. Here, using a model of lethal, high-pathogenicity IAV infection, we first show that Ly6C(hi)Ly6G(-) inflammatory monocytes, and not neutrophils, are the main infiltrate in lungs of WT mice. Mice devoid of iNKT cells (Jα18(-/-) mice) have increased levels of inflammatory monocytes, which correlated with increased lung injury and mortality (but not viral load). Activation of iNKT cells correlated with reduction of MCP-1 levels and improved outcome. iNKT cells were able to selectively lyse infected, MCP-1-producing monocytes in vitro, in a CD1d-dependent process. Our study provides a detailed profile and kinetics of innate immune cells in the lungs during severe IAV infection, highlighting inflammatory monocytes as the major infiltrate and identifying a role for iNKT cells in control of these cells and lung immune-pathology.

Reduction of natural killer but not effector CD8 T lymphocytes in three consecutive cases of severe/lethal H1N1/09 influenza A virus infection.

BACKGROUND: The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We present the cellular immunology profile in the blood, and detailed clinical (and post-mortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. CONCLUSION/SIGNIFICANCE: Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype.

Analysis of outcome in women with borderline glandular change on cervical cytology.

OBJECTIVE: To review the outcome of women presenting with borderline glandular smear on cervical cytology and to investigate correlation between cytology, colposcopy and histology and subsequent smear history. STUDY DESIGN: A 5 year retrospective analysis of outcome in all women reported with borderline glandular changes on cytology between 2001 and 2005 at the John Radcliffe Hospital, Oxford, and Cheltenham General Hospital, Gloucestershire, was performed. Cytology, colposcopy, histology and follow-up cytology data at 6, 12 and 24 months after index smear were collated. RESULTS: Of 92 women with borderline glandular smears, 56 were referred for colposcopy. This accounts for 0.9% of the total referral (6293) to the colposcopy units. A significant rate of abnormal histology was noted, with CIN 1, 2, 3, CGIN or worse in 20 women (36%) and benign pathology in 18 women (32%). Colposcopy had a sensitivity of 82%, specificity of 39%, negative predictive value of 80% and positive predictive value of 49% in predicting abnormal (premalignant/malignant) histology. Only one woman with normal histology at presentation had subsequent abnormal cytology (1/31), whereas abnormal histology at presentation was strongly associated with subsequent abnormality on follow-up cytology over 24 months (8/21) with a p value=0.0058. CONCLUSION: Colposcopic examination and biopsy of colposcopically identified abnormalities is reliable, with negative colposcopy having a high negative predictive value. We also recommend thorough colposcopic examination before any invasive investigation as over treatment may impact on the future reproductive outcome [1].

Use of lipoteichoic acid-T for pleurodesis in malignant pleural effusion: a phase I toxicity and dose-escalation study.

BACKGROUND: Bacterial infection of the pleural space often causes adherence of the pleural membranes by fibrous tissue, probably mediated by inflammation initiated by bacterial cell-wall motifs, including lipoteichoic acid-T (LTA-T). We postulated that therapeutically administered LTA-T might produce a similar effect, achieving control of malignant pleural effusion (pleurodesis). METHODS: Patients with histocytologically proven symptomatic malignant pleural effusions were included in this phase I toxicity and dose-escalation study, An indwelling pleural catheter was placed in the pleural effusion to drain the fluid fully. A control dose of intrapleural saline was administered after complete drainage (day 1) and pleural-fluid production was recorded for 7 days. On day 7 a single dose of intrapleural LTA-T (increasing in each patient) was administered and pleural-fluid production was monitored for a further 7 days. Long-term fluid control was recorded. This study is registered as an International Standard Randomised Controlled Trial, ISRCTN44367564. FINDINGS: Between November, 2004, and November, 2005, 14 patients were enrolled on the trial at the Oxford Centre for Respiratory Medicine (Oxford, UK). 13 of 14 patients received escalated doses of LTA-T. A dose-limiting toxic effect (ie, systemic inflammation) occurred at 3000 microg, and a therapeutic dose of 750-1500 microg was established. Toxic effects were mild and had no consistent pattern at the therapeutic dose. Pleural-fluid production decreased significantly after a dose of at least 750 microg LTA-T, compared with saline control (mean fluid production after saline control 1244 mL [SD 933], mean fluid production after LTA-T 394 mL [SD 375], mean difference -850 mL [SD 699], p=0.028), and six of seven (86%) patients achieved pleural-fluid control at 1 month with no further intervention. INTERPRETATION: The toxic effects of intrapleural LTA-T seem to be mild and favourable when compared with the toxicity profiles of standard pleurodesis agents. There is early evidence of LTA-T-induced pleurodesis efficacy, suggesting that this might be a viable therapeutic strategy for the control of malignant pleural effusion.

Activation of invariant NKT cells enhances the innate immune response and improves the disease course in influenza A virus infection.

Invariant NKT (iNKT) cells have an indubitable role in antiviral immunity, although the mechanisms by which these cells exert their functions are not fully elucidated. With the emerging importance of high-pathogenicity influenza A virus infections in humans, we questioned whether iNKT cells contribute to immune defence against influenza A virus and whether activation of these cells influences outcome. We show that activation of iNKT cells with alpha-galactosylceramide (alpha-GC) during influenza virus infection transiently enhanced early innate immune response without affecting T cell immunity, and reduced early viral titres in lungs of C57BL/6 mice. This is accompanied by a better disease course with improved weight loss profile. Temporal changes in iNKT cells in the liver, blood and lungs suggest activation and migration of iNKT cells from the liver to the lungs in mice that were administered alpha-GC. Improvement in viral titres appears dependent on activation of iNKT cells via the intraperitoneal route since intranasal administration of alpha-GC did not have the same effect. We conclude that activation of iNKT cells enhances early innate immune response in the lungs and contribute to antiviral immunity and improved disease course in influenza A virus infection.

Recurrent localized fibrous tumor of the pleura.

Localized fibrous tumors of the pleura are rare. They are often asymptomatic and may have symptoms based on size, bronchial invasion, or hormone production, or a combination of these. Complete resection offers the best chance of cure. However, recurrence is reported in a significant number of patients and can often be treated by repeated resection, albeit with increasing difficulty. We present a case in which delayed recurrence occurred after excision of such a tumor. This required a chest-wall resection and reconstruction after which a second recurrence occurred. Further thoracotomy including a latissimus dorsi free flap procedure was needed for a third-time recurrence.

Seeding of stage I thymoma into the chest wall 12 years after needle biopsy.

It is known that benign encapsulated thymoma can pursue an aggressive clinical course on rare occasions. It may recur locally, it may be invasive, or it may metastasize. We present a case of local seeding into the chest wall, presenting 12 years after core needle biopsy and complete excision of the mediastinal tumor. We draw attention to the malignant clinical behavior of some benign stage I thymomas.

End-organ function during chronic nonpulsatile circulation.

BACKGROUND: Evolving blood pump technology has produced user-friendly continuous flow left ventricular assist devices, but uncertainty exists about the safety of chronic nonpulsatile circulation. We established consistently nonpulsatile blood flow in a sheep model using the Terumo magnetically suspended centrifugal pump. We then compared end-organ function between pulseless and control animals. METHODS: Fifteen healthy sheep (65 to 85 kg) were allocated to either left ventricular assist device (n = 9) or control (n = 6) groups. We implanted the device through a left thoracotomy and determined the flow rate at which pulse pressure was absent. The flow rate was then adjusted to exceed that rate (4.2 +/- 1.5 L/min), and all variables of pump function were continuously monitored by computer. Blood tests were taken serially for hepatic and renal function and plasma renin levels. The sheep were sacrificed electively at 30 (n = 3), 90 (n = 4), 180 (n = 1), and 340 (n = 1) days. Detailed histologic examination was made of the brain, liver, kidney, myocardium, and major arteries. RESULTS: All animals remained in good condition until sacrifice. All measures of end-organ function remained within normal limits for both groups. There were no histologic differences between the organs of pulsatile and nonpulsatile animals. Although there was no significant difference in mean blood pressure, plasma renin levels were substantially elevated in pulseless animals (1.4 +/- 0.3 pg/mL versus 2.9 +/- 0.3 pg/mL; p < 0.05). We also identified thinning of the medial layer of the ascending aorta in nonpulsatile sheep (1.8 +/- 0.4 mm in left ventricular assist device animals versus 2.6 +/- 0.6 mm in control sheep; p < 0.05). CONCLUSIONS: Chronic nonpulsatile circulation was well tolerated, and we found neither functional nor histologic changes in major end organs. The renin-angiotensin system was upregulated, but this did not provide a significant rise in blood pressure. The changes in the aortic wall merit further investigation. As a result of these findings, we consider that nonpulsatile devices can be used safely for long-term circulatory support.