Metabolic reprogramming, through increased uptake of cholesterol in the form of low-density lipoproteins (LDL), is one way by which cancer cells, including high grade gliomas (HGG), maintain their rapid growth. In this study, we determined LDL receptor (LDLR) expression in HGGs using immunohistochemistry on tissue microarrays from intra- and inter tumour regions of 36 adult and 133 paediatric patients to confirm LDLR as a therapeutic target. Additionally, we analysed expression levels in three representative cell line models to confirm their future utility to test LDLR-targeted nanoparticle uptake, retention, and cytotoxicity. Our data show widespread LDLR expression in adult and paediatric cohorts, but with significant intra-tumour variation observed between the core and either rim or invasive regions of adult HGG. Expression was independent of paediatric tumour grade or identified clinicopathological factors. LDLR-expressing tumour cells localized preferentially within perivascular niches, also with significant adult intra-tumour variation. We demonstrated variable levels of LDLR expression in all cell lines, confirming their suitability as models to test LDLR-targeted nanotherapy delivery. Overall, our study reveals the LDLR pathway as a ubiquitous metabolic vulnerability in high grade gliomas across all ages, amenable to future consideration of LDL-mediated nanoparticle/drug delivery to potentially circumvent tumour heterogeneity. Gliomas are the most common primary brain tumours and originate from the glial cells of the brain. They are a heterogenous spectrum of slow-growing to highly aggressive infiltrating tumours and have been classified based on their histological features into four grades by the World Health Organization (WHO); grade 1 (pilocytic astrocytoma), grade 2 (diffuse astrocytoma), grade 3 (anaplastic astrocytoma), and grade 4  ,6,7.® wafer) placed at the affected tumour site during surgery [Since the mid-twentieth century, standard-of-care treatment has involved surgical resection of the tumour followed by external beam radiotherapy (except in children under 3 years) and more recently the use of adjuvant chemotherapeutic agents such as temozolomide, nitrosoureas and polymer-based carmustine  exists in the majority of cancers including malignant brain tumours, and it is in fact fundamental to our understanding of the variation in patient response to treatment and treatment failure . StudiesAbnormal cellular metabolism is one of the hallmarks of cancer, with most cancer cells including HGGs relying on glycolysis rather than oxidative phosphorylation for energy supply, a phenomenon known as the Warburg effect. Additionally, the increased requirement for cholesterol to maintain tumour proliferation and spread is an observed feature in many cancers ,16,17. COverexpression of FASN is associated with various pathological conditions such as cardiovascular diseases, diabetes and several different cancers including gliomas, breast, prostate, lung and colon cancer . SimilarHere, we test the hypothesis that invariably high levels of expression of LDLR protein across intra- and inter-tumour regions in adult and paediatric high-grade gliomas confirm LDLR as a therapeutic target. This was determined through immunohistochemistry (IHC) using an LDL-receptor targeting antibody carried out on patient-derived tissue microarrays (TMA), which incorporated varying intra-tumour regions in the adult GBM cohort and single region samples for the paediatric HGGs. Additionally, representative cell lines of adult (GIN28) and paediatric  glioblastoma were assessed for LDLR expression by immunofluorescence (IF) in vitro to confirm the future utility of these cell lines as models to test fluorescently labelled LDLR-targeted stealth nanoparticle uptake, retention, and cytotoxicity.Tissue microarrays (TMAs) were previously prepared in the Children’s Brain Tumour Research Centre (CBTRC) from adult and paediatric patients with confirmed diagnosis of high-grade glioma (HGG) from multi-centre trials, retrospective studies and cases registered with the Children’s Cancer and Leukaemia Group (CCLG). A total of 21 paediatric TMAs, each consisting of cores from different areas of a single biopsy specimen and 4 adult TMAs containing cores taken from three distinct intra-tumour regions were studied, with each TMA housing samples from 10–27 patients in triplicates. There was a total of 36 adult GBM and 133 paediatric high grade glioma cases, of which clinical diagnosis was available for 68 of paediatric cases. Of these, 13 were grade III and 55 grade IV.Primary glioblastoma multiforme (GBM) cell line (GIN28) was generated from invasive tumour margin of adult brain tumour patients treated at the Nottingham University Hospitals (NUH) while, paediatric GBM cell lines  were obtained from Professor Chris Jones of the Institute of Cancer Research (ICR), London, UK.This project was approved by the National Research Ethics Committee (NRES Committee East Midlands) and the granted Ethics Reference Number is 11/EM/0076.Low density lipoprotein receptor (LDLR) protein in human HGG samples was detected using indirect IHC for better signal amplification. Formalin-fixed paraffin-embedded (FFPE) slide sections were deparaffinized in xylene for 15 min and rehydrated in decreasing concentrations of alcohol and running water. Slides were placed in a pre-heated Sodium Citrate buffer solution (pH 6.0) for 40 min in a steamer for antigen retrieval, cooled for 10–20 min, and washed in phosphate-buffered saline (PBS). All incubations were carried out in a humidified box at room temperature and washes between stages in PBS. Sections were covered in 20% normal goat serum (NGS) in PBS for 20 min to minimize non-specific antibody binding and endogenous peroxidase activity was blocked by incubating slides in DAKO peroxidase blocking solution for 5 min to prevent background staining. The primary antibody used was Abcam  anti-LDLR rabbit monoclonal antibody (EP1553Y) ab52818 and was optimized on normal liver tissue (positive control) at dilutions ranging from 1:250 to 1:1400 incubated for 1 h at room temperature, subsequent experiments were performed at 1:1400 dilution. Slides were incubated in DAKO REAL Envision HRP Rabbit/Mouse secondary antibody for 30 min. Detection of antibody binding was enzyme-based following incubation in DAKO diaminobenzindine (DAB) chromogen substrate for 10–15 min. Slides were counterstained in Gills Haematoxylin, differentiated in lithium carbonate solution, washed in running water, and dehydrated in increasing alcohol concentrations prior to immersion in xylene for mounting. Slides were mounted with coverslips using DePeX mounting medium. Slides were scanned at 40× magnification using a Nanozoomer at the Histopathology Department, NUH and expression levels analysed using the NDPview2 software. Scoring was based on proportion  of cells stained. A double-blinded approach was implemented in scoring to produce fair and reproducible results.SF188 and KNS42 paediatric GBM cell lines were cultured in Dulbecco Modified Eagle’s Medium (DMEM)/F-12 nutrient mix (Gibco) supplemented with 10% Foetal Bovine Serum (FBS) and 2% L-glutamine. Adult GBM cell line, GIN28 was cultured in DMEM  containing 15% FBS, 1% L-glutamine and antibiotics (1% penicillin and streptomycin). HeLa cell line were used as positive control and grown in DMEM (Gibco) with 10% FBS and 2% L-glutamine. Cells were passaged at approximately 80% confluency by dissociating with trypsin-EDTA and seeded onto treated tissue culture plastic (polystyrene) and chamber slides. GBM cell lines were passaged twice a week to maintain exponential growth at varying seeding ratios of 1:3–1:5 and at cellular densities of 15,000–30,000 cells per well. HeLa cells were passaged daily due to their rapid doubling time and plated at 5000 cells per well. Optimal culture conditions of 37 °C, 21% Oxygen and 5% Carbon IV Oxide was maintained for all experiments.i fluorescent microscope and images were captured with Hamamatsu camera and Volocity 6.0 software at 40× magnification. ImageJ software was used to analyse images and correlate corrected total cell fluorescence (CTCF) with amount of LDLR protein expressed based on intensity of signal generated.SF188, KNS42, GIN28 and HeLa cells were plated on 8-well chamber slides at optimised densities and incubation periods. Cells were washed in PBS, fixed in 0.4% paraformaldehyde for 20 min, permeabilised with PBS-T  and blocked with 5% NGS in PBS-T for 1 h at room temperature. Primary anti-LDLR antibody was optimised on HeLa cells at dilutions ranging from 1:50–1:1000 and incubated overnight at 4 °C, subsequent experiments were performed at 1:250 dilution. Cells were incubated with goat anti-rabbit IgG secondary antibody labelled with Alexa Fluor 488  at 1:200 dilution for 1 h in the dark and washed in PBS-T. Slides were mounted with coverslips using DAPI-containing Vectastain mounting medium . Microscopy was carried out using a Nikon eclipse 90p < 0.05 was considered statistically significant.All statistical analysis was performed using SPSS 24.0 software. Friedman ANOVA and Wilcoxon signed rank tests were used to analyse the significance of adult intra-tumour results. Mann–Whitney U or Kruskal–Wallis tests were used to determine the relationship between LDLR expression and clinicopathological factors. The overall survival time was compared using Kaplan–Meier and log-rank methods. Localisation of positive tumour cells within peri-vascular niches and LDLR expression in GBM cell lines were assessed with the One-way ANOVA test and Tukey’s HSD post hoc test for multiple comparisons, and Patient diagnosis and clinico-pathological details of TMA samples are shown in p = 0.008) and further pairwise analysis revealed that the observed significant differences were between the core and either rim or invasive margins (p = 0.003 and 0.014 respectively). No significant difference was detected in LDLR expression between the rim and invasive margin .LDLR protein expression was assessed in three distinct GBM intra-tumour regions representing core, rim, and invasive margin, from 36 patients. A universal expression was observed across all the regions, where 92% of samples from the tumour core had a frequency of positive cells above 25%. Similar results were also observed in the rim and invasive margins where more than 70% of cases expressed a frequency of LDLR positive cells greater than 25% p = 0.285, Mann–Whitney U test). This suggests a ubiquitous role for LDLR in high grade glioma metabolism, which could be exploited in developing novel future therapeutic strategies for both adult and paediatric anaplastic astrocytoma and glioblastoma cases.The expression of LDLR protein in grade 3 (anaplastic astrocytoma) and grade 4 (GBM) paediatric samples  shows anp = 0.043, Positively stained tumour cells were repeatedly observed to localize preferentially in varying proportions near blood vessels compared to other areas of the tumour. We therefore examined the distribution of positive tumour cells in respect to vasculature in our adult and paediatric TMAs A–C. The p > 0.05, Mann–Whitney U and Kruskal–Wallis tests, with the exception of age in tumour core regions of in the adult cohort)  .p = 0.126, Log-rank test)  .p = 0.001 and p = 0.008 for SF188 vs. GIN28 and KNS42 vs. GIN28 respectively, One-way ANOVA test) and on pair-wise comparisons, no significant difference was observed between the paediatric cell lines . This was in harmony with previous studies showing variable LDLR expression between different cell lines [To ascertain that our cell lines expressed LDLR and were representative of results obtained from IHC of adult and paediatric TMAs, and therefore amenable to utilize as future in vitro models to test LDLR-targeted nanoparticle uptake/retention, expression levels were examined in three GBM cell lines. SF188, KNS42 (both paediatric) and GIN28 (adult). This revealed widespread staining confirming LDLR expression A. The coll lines .As metabolic reprogramming is a well-established hallmark of cancer , researcp-value of 0.126 (when comparing the <25% and >50% LDLR expression cohorts) certainly warrants re-examination in a larger cohort of high-grade glioma patients to refute a prognostic role more confidently for LDLR expression. Finally, we confirmed variable levels of detectable LDLR expression in adult (GIN28) and paediatric  GBM in vitro cell lines, thus rendering these lines as future tools to test LDLR-targeted nanotherapy delivery.We hypothesized that high-grade gliomas express invariably high levels of LDLR, despite the known genetic inter- and intra-tumour heterogeneity. Our results demonstrated widespread LDLR expression in the majority of the adult GBM cases with intra-tumour variation noted and a similar paediatric expression pattern which was independent of tumour grade, confirming LDLR as a valid therapeutic target across all ages for malignant glioma. Intriguingly, the localization of LDLR-expressing tumour cells within peri-vascular niches was observed in the vast majority of both adult and paediatric high-grade gliomas. Additionally, LDLR expression was discovered not to vary with clinicopathological factors, nor did it correlate with survival. However, a The intra-tumour variation observed in adult GBM cases, where the core had the highest LDLR expression and the invasive margin the least, is consistent with current literature on intra-tumour heterogeneity ,14. The p = 0.285) in our paediatric cohort points to a possible ubiquitous role of LDLR in paediatric high grade glioma metabolism. Based on this finding, we can predict that both anaplastic astrocytoma (grade 3) and glioblastoma (grade 4) may be amenable to anti-LDLR targeting to the same extent. However, re-examination in a larger paediatric cohort is required to validate a claim of LDLR as a broadly ubiquitous target. Considering that children are not able to tolerate the same levels of intense treatments as adults and that current standard-of-care treatment modalities, such as radiation therapy, are exempt in children under 3 years of age, paediatric patients might potentially benefit more from preferential LDLR targeting. Although our data did not show paediatric LDLR expression to be dependent on tumour grade, differences may be more subtle and require a larger sample size to interrogate.The non-significant difference in levels of LDLR expression between anaplastic astrocytoma and glioblastoma (p = 0.040) as confirmed by Tukey’s HSD test. This phenomenon can be explained by the higher cellular density and proliferation of the core compared to other regions which outstrips it of its blood supply, inducing a state of nutrient deficiency and hypoxia—the main driver of angiogenesis and neo-vascularization within the tumour. Similar comparison between paediatric anaplastic astrocytoma and glioblastoma was limited by disparity in sample sizes (13 and 55 respectively) which would have given a biased result. A larger sample size, particularly of grade 3 tumours, would have improved the power and validity of such analysis.LDLR expressing tumour cells were consistently observed to localize within perivascular areas, consistent with seminal studies by Calabrese et al., in 2007, across a host of primary brain tumours, notably glioblastoma, ependymoma, medulloblastoma and oligodendroglioma, where cancer stem cells (CSCs) were shown to associate in close proximity to tumour capillaries creating vascular niches. This tumour microenvironment is essential for the maintenance of tumour growth and proliferation and is one of the key rationales for several anti-angiogenic therapies . The oveThe association of positive tumour cells with the vasculature is of immense therapeutic significance. Hitherto, one of the greatest challenges of anti-cancer drug delivery to tumour sites is the difficulty in penetrating and accumulating to sufficient levels as to effect cell kill within the target organ, without incurring significant toxicity to surrounding normal tissues. In attempts to overcome this, novel drug delivery strategies such as macromolecular drug carriers have been developed ,37. A stTaken together, our results have clearly shown the LDLR pathway to represent a metabolic vulnerability in both paediatric and adult high-grade gliomas, which potentially circumvents intra- and inter-tumour heterogeneity and is therefore potentially amenable to future lipoprotein based anti-LDLR nanotherapy, ubiquitously in patient cohorts. To maximize the benefits from this novel therapeutic strategy, there may be a need for selection of cases likely to benefit the most based on demonstrated levels of LDLR expression, tumour vascularity and/or further lipidomic studies.


The unmet clinical need for effective treatments in ovarian cancer has yet to be addressed using monoclonal antibodies (mAbs), which have largely failed to overcome tumour-associated immunosuppression, restrict cancer growth, and significantly improve survival. In recent years, experimental mAb design has moved away from solely targeting ovarian tumours and instead sought to modulate the wider tumour microenvironment (TME). Tumour-associated macrophages (TAMs) may represent an attractive therapeutic target for mAbs in ovarian cancer due to their high abundance and close proximity to tumour cells and their active involvement in facilitating several pro-tumoural processes. Moreover, the expression of several antibody crystallisable fragment (Fc) receptors and broad phenotypic plasticity of TAMs provide opportunities to modulate TAM polarisation using mAbs to promote anti-tumoural phenotypes. In this review, we discuss the role of TAMs in ovarian cancer TME and the emerging strategies to target the contributions of these cells in tumour progression through the rationale design of mAbs. The unmet clinical need for effective treatments in ovarian cancer has yet to be addressed using monoclonal antibodies (mAbs). In this review we discuss the role of tumour-associated macrophages (TAMs) in ovarian cancer and consider how TAMs can be modulated by novel mAb therapies to provide unique opportunities for clinical efficacy.  Ovarian cancer has the highest mortality rate among gynaecological malignancies . This poOver the past 30 years, mAb therapies have become widely used in cancer treatment, offering significant advantages relative to conventional chemotherapy and radiotherapy, including high specificity and affinity for a single epitope target, which limits off-target effects . TherapeDespite significant successes in other tumour types, mAb therapies in ovarian cancer frequently report disappointing clinical trial results . In receMacrophages are highly abundant mononuclear phagocytic cells present in almost every human tissue .Macrophages are both important for inflammatory reactions and homeostatic functions. Monocyte-derived macrophages (MDMs) rapidly increase in number during inflammatory events such as infection, to aid the restoration of homeostasis through the promotion of pathogen clearance and subsequently tissue repair . MoreoveTAMs frequently constitute a highly abundant population within TMEs, typified in ovarian cancer, where they can account for over 50% of all cells in peritoneal tumours and ascites . InitialIn ovarian cancer, total TAM density exhibits no prognostic significance , 45. HowIn vivo, especially in TMEs, the binary M1/M2 model has proven oversimplified, with a spectrum model possibly offering a more accurate representation of macrophage polarisation . In. In77]. A key requirement for the development of both the primary and metastatic tumours is the establishment of access to the circulatory system via neo-angiogenesis. TAMs potently promote neo-angiogenesis, displaying enrichment at sites with high angiogenic requirements, including hypoxic tumour nests, nascent peritoneal tumours, and perivascular regions , 78, 79 The IgG1 mAb Bevacizumab blockades VEGF-A/VEGFR-mediated neo-angiogenesis through binding to VEGF-A  87]. Be. Be87]. TAMs may play a significant role in this resistance. In a murine model of Bevacizumab-resistant ovarian cancer, tumours exhibited restored response when treated with a TAM-depleting anti-CSF-1 mAb . SpecifiThe potential utility of combination therapy for targeting neo-angiogenesis has been indicated clinically. Firstly, in a Phase III trial, peptide-Fc fusion protein Trebananib, which targets Ang2, as well as additional Tie2 ligand, Ang1, was found to enhance PFS in ovarian cancer patients  23]. Se. Se23]. Due to the redundancy of neo-angiogenesis pathways and the centrality of TAMs in driving them, a more durable therapeutic strategy may be to specifically target pro-angiogenic TAM subsets via mAbs  and 3. TInstead of inhibiting the pro-tumoural activity of TAMs, specific mAbs have sought to promote the anti-tumour function of TAMs.One such approach is through an antibody’s simultaneous engagement of tumour-associated antigens (TAAs) on tumour cells and antibody Fc receptors on TAMs, to trigger tumouricidal TAM effector function . This caAlthough TAA-targeting IgG mAbs have displayed efficacy in solid tumours, clinical results in ovarian cancer have been disappointing . This diOne emerging strategy to enhance the efficacy of TAA-targeting IgG mAbs is through mAb-mediated blockade of ‘don’t eat me’ signals expressed on tumour cells, to enhance the contribution of TAM effector functions to their mechanism of action. For example, tumour cell CD47 and CD24 engage cognate receptors on TAMs, signal regulatory protein-α (SIRPα) and sialic acid-binding Ig-like lectin 10 (Siglec-10), respectively, to trigger signalling cascades which inhibit macrophage phagocytosis , 103 Fi. CD47 exin vitro, as well as inhibition of tumour growth in murine ovarian cancer models ..26].Moreover, in view of the enhanced pre-clinical efficacy of TAA-targeting mAbs when in combination with anti-CD47 mAbs, several trials are currently underway using this regimen in non-ovarian solid tumours. For example, a phase II study in colorectal cancer patients of anti-CD47 IgG4 mAb Hu5F9-G4 in combination with Cetuximab, reported stable disease in 45% of patients, as well as increased macrophage tumour infiltration . It remahighIL-12low secretome  upregulation and downregulation of M2-associated CSF1R. However, a Phase I/II trial of ABBV-428, a bispecific mAb targeting CD40 (as an agonist) and tumour cell marker mesothelin, was not able to replicate these promising anti-tumoural effects in ovarian cancer patients . De. De57]. Clinically, a Phase I trial using anti-CD40 IgG2 mAb CDX-1140 with anti-PD-1 IgG4 mAb Pembrolizumab (Keytruda) is currently recruiting ovarian cancer patients (NCT03329950). This will provide the first insight into whether anti-CD40 mediated TAM repolarisation can skew the ovarian TME immune landscape towards ICB efficacy .Ka for IgG of 108–109 M−1, the high-affinity IgE receptor, Fc ε receptor I (FcεRI), exhibits a substantially higher affinity for IgE (Ka = 1010–1011 M−1) [Currently, all licensed full-length mAbs are of the IgG isotype . IgE ant011 M−1) , 123. Th011 M−1) , 124. Mo011 M−1) . Further011 M−1) , 127. Ac011 M−1) . ConsequAmong several developed IgE mAbs targeting TAAs, MOv18 IgE, represents the first-in-class to enter clinical testing, in an ongoing phase I trial involving predominantly ovarian cancer patients (NCT02546921). MOv18 IgE is targeted against TAA, folate receptor α (FRα), which is overexpressed in 82% of serous epithelial ovarian cancers and inversely correlated with patient OS and disease-free interval (DFI) , 130.In pre-clinical models, MOv18 IgE exerted anti-tumoural effects via Fc engagement of monocytes and macrophages in a two-armed mechanism, comprising tumour killing and crucially, pro-inflammatory polarisation . In an oIn vitro interrogation of tumour cell killing determined that MOv18 IgE predominantly induces cancer cell ADCC by monocytes, as well as ADCP, when monocytes are IL-4-stimulated to induce expression of low-affinity IgE receptor, CD23 [or, CD23 . In compin vitro and in vivo studies have found that cross-linking of TAA-bound IgE initiates a TNF-α/CCL2-mediated monocyte and macrophage pro-inflammatory recruitment feedback loop, to potentiate tumour killing and drive pro-inflammatory polarisation [in vitro cross-linking of the TAA-specific IgE mAb SF-25 [Consistently, risation . Explorarisation . ConsistAb SF-25 .in vivo anti-tumour activity, may permit sustained efficacy, which has hitherto eluded TAA-targeting IgG mAbs in ovarian cancer. Recent interim results from the Phase I clinical trial of MOv18 IgE have demonstrated the therapy to be well tolerated and, although preliminary, anti-tumour activity was displayed by one patient [Collectively, TAA-targeting IgE mAbs may drive a unique monocyte and macrophage polarisation signature, which combined with an alternative tumour killing mechanism and superior  patient .High frequency, broad Fc receptor expression, extensive potential pro-tumoural activities and strong phenotypic plasticity, render TAMs and their associated functions as attractive mAb targets to meet the unmet clinical need in ovarian cancer . AntibodConsequently, instead of viewing TAMs as a deleterious population for removal, recent experimental mAb strategies have frequently sought to exploit their extensive plasticity and immunoregulatory function via phenotypic repolarisation, to mediate a TAM-driven shift in the TME towards anti-tumour immunostimulation  and 3. TOverall, mAb-mediated TAM repolarisation may facilitate a therapeutic window in which the ovarian TME is biased towards anti-tumour activity. Clinical exploitation of this opportunity therefore may best be achieved through combination with established cytotoxic therapies. For example, potential synergistic anti-tumour activity between TAM repolarisation and both tumour-targeting chemotherapy and adaptive immunity-targeting ICB have begun to emerge.Further investigation of these mAb candidates and possible combination regimens is warranted in larger clinical studies, to determine whether therapeutic targeting of TAMs may represent a viable pathway for efficacy, which has generally eluded mAbs in ovarian cancer.


Ewing’s sarcoma (ES) is a small round cell sarcoma arising in the bone or soft tissue. Ewing’s sarcoma/primitive neuroectodermal tumours (PNET) of the thoracopulmonary region is called Askin’s tumour. The common clinical presentations described for this extrapulmonary tumour are fever, chest wall mass with or without pain, dyspnea, and cough. Very few cases of Askin’s tumour have been reported with haemoptysis as the initial presentation, which is usually a presentation of intrapulmonary lesions. A 22-year-old male presented to the emergency department with complaints of haemoptysis, mild chest pain, and swelling on the right side of the chest wall. Radiological investigations showed a soft tissue mass measuring 13 cm × 11 cm × 10 cm in the right thoracic region, causing the destruction of the second rib. Histopathological examination showed the presence of a malignant small round cell tumour. Immunohistochemistry (IHC) analysis showed the tumour to be positive for CD99, NKX 2.2, and MIC2. The final diagnosis of Askin’s tumour of the thoracopulmonary region was given. The case is being reported in view of the rare type of clinical presentation. Ewing’s sarcoma (ES) is a malignant small round cell tumour with characteristic molecular findings and varying neuroectodermal differentiation. It is mostly skeletal but can be extraskeletal, with the incidence of the latter reported to be around 12-13% .,9.1,9].Askin’s tumour seldom presents with haemoptysis and, to the best of our knowledge, this is the second case to be reported with haemoptysis . In the Since the patient presented with haemoptysis, the initial suspicion was directed towards an intrapulmonary tumour. Studies reveal haemoptysis due to carcinoma of the lung to be still a common cause in patients aged less than 35 years . Hence, The CECT chest and PET CT scans helped in pointing out the origin of the tumour from chest wall. With subsequent correlation with histopathology and immunohistochemistry, helped the pathologist in arriving at the final diagnosis of Askin tumour, i.e., Ewing Sarcoma/PNET of the thoracopulmonary region.In a retrospective study of 114 patients with non-metastatic Ewing’s sarcoma of the chest wall who were recipients of various treatment modalities including polychemotherapy, local radiotherapy, surgery, or a combination of radiotherapy and surgery, the overall survival was found to be about 60%. The recipients of pre-operative irradiation, though small in number, had no evidence of any local or systemic relapse [Haemoptysis is a rare clinical presentation of Askin’s tumour which can misdirect the pulmonologist to think in terms of a lung tumour arising from an intrapulmonary location rather than the chest wall/thoracopulmonary region. A young patient presenting with haemoptysis with a mass in the chest wall and rib erosion should prompt the suspicion of Ewing sarcoma/PNET of the thoracopulmonary region. A combined diagnostic approach utilising clinicoradiological findings in the context of histomorphological and judicious immunohistochemical analysis can help in arriving at an accurate diagnosis.


Lumbar spinal stenosis, a narrowing of the spinal canal around the spinal neurovascular structures, is a common etiology for lower back and leg pain in older people. Sciatica, a frequent symptom of lumbar spinal stenosis, typically presents with sharp and/or aching pain that originates in the buttock, extends to the thigh, and radiates into the foot and toes; in addition, it can be accompanied by weakness of the associated lower extremity. In individuals with sciatica-related persistent symptoms or functional limitations or both, spinal decompression surgery may be necessary. A cerebrospinal fluid leak is a potential complication of lumbar spinal stenosis surgery; it is frequently--yet not always--accompanied by a postural headache. The cerebrospinal fluid leak can result from an intraoperative tear or postoperatively. Albeit a more common adverse event after body contouring surgery, seroma--a postoperative serous fluid collection that is usually detectable as a palpable or visible fluid wave on clinical examination--has also been observed as a complication following lumbar spinal stenosis surgery. A man who experienced an intra-operative accidental dural tear during lumbar spinal stenosis surgery is described. A large cerebrospinal fluid leak that involved both the laminectomy bed and the subcutaneous tissue of his back subsequently developed; the leak eventually presented as duro-cutaneous fistulas without headache. His doctors misinterpreted the cerebrospinal fluid leak as a seroma; this may have occurred since not only did the color of the persistent and continuously dripping fluid varied from being clear to slightly tinged pink, but also the patient never had a headache or any other symptoms associated with a cerebrospinal fluid leak. When his lower back was appropriately evaluated with magnetic resonance imaging, the diagnosis of a large cerebrospinal fluid leak was established. In conclusion, lumbar spinal stenosis back surgery can be associated with postoperative complications, including cerebrospinal fluid leak and--less frequently--seroma. However, following lumbar spinal stenosis surgery, the absence of a headache does not exclude the possibility of a cerebrospinal fluid leak. Also, the presence of fluid leaking from the surgical site after lumbar spinal stenosis back surgery should not only prompt the clinician to entertain the possibility of a surgery-associated cerebrospinal fluid leak but also to obtain additional diagnostic studies--such as magnetic resonance imaging--to establish the diagnosis. Lumbar spinal stenosis is a narrowing of the spinal canal around the spinal neurovascular structures. It has a prevalence of approximately 11 percent in United States adults and commonly presents with sciatica symptoms. Management ranges from conservative non-surgical modalities to operative intervention .A cerebrospinal fluid leak is a potential complication following back surgery; a dural tear during the procedure may place the patient at greater risk for this post-operative adverse event. Headache is a common symptom that typically accompanies a cerebrospinal fluid leak. However, not all patients with a cerebrospinal fluid leak have an associated headache -20.A man with bilateral and severe lumbar stenosis affecting multiple spinal disc levels had back surgery during which a dural tear experienced during the procedure was repaired; he never experienced any postoperative headache. However, his cerebrospinal fluid leak clinically presented on a postoperative day seven as continuous draining fluid onto the skin of his back within the area of his prior operative wound. Hence, the absence of a headache does not exclude the possibility of a cerebrospinal fluid leak--especially in the setting of an intraoperative tear of the dura and fluid draining onto the skin at the surgical site.The patient, a 58-year-old man, presented to the emergency center for an evaluation regarding the fluid that slowly yet continuously was seeping from the suture sites on his lower back. He was afebrile, and his vital signs  were normal. He had not experienced a headache during the prior 12 days after his back surgery and currently did not have a headache.Cutaneous examination of his lower back demonstrated two areas in which fluid was present. The fluid at the superior location had dried onto the skin surface. Clear fluid was sparsely present on the erythematous lower site Figure .Magnetic resonance imaging of the lumbar spine was performed--without and with ten milliliters of Gadavist intravenous contrast--on a 1.5 Tesla scanner, with not only sagittal short T1 inversion recovery, T1-weighted, and T2-weighted imaging Figure , but alsIn summary, the large cerebrospinal fluid collection located deep in the laminectomy bed communicated with another large collection of cerebrospinal fluid located more superficially in the subcutaneous tissue of the patient’s back; also, there was discontinuity of the dura. Correlation of the clinical history and magnetic resonance imaging findings established the diagnosis of a dural tear with a subsequent large cerebrospinal fluid leak. In addition, there was bilateral severe spinal canal stenosis at multiple disc levels with compression of the cauda equina nerve roots.The magnetic resonance imaging changes of his cerebrospinal fluid leak were readily apparent when compared to his initial magnetic resonance imaging, performed 18 months earlier when he presented with bilateral pain that originated in his thighs and extended distally towards his feet Figure ; the magA computed tomography-guided epidural corticosteroid injection localized to the site of the most severely affected nerves was performed. All symptoms resolved; the patient was able to continue running. However, nearly a year later, symptoms recurred, and right leg weakness had developed; the patient was no longer able to stand up on the toes of his right foot. Another computed tomography-guided epidural corticosteroid injection resulted in symptom relief; yet, within three months, left leg weakness--similar to that of his right leg--developed. He met with the surgeon and decided to have surgery.He had spinal surgery (consisting of decompression laminectomy without fusion) to treat the severe bilateral spinal stenosis that involved multiple vertebrae 12 days before his visit to the emergency center. During the operation, a surgical tear of the dura was repaired; he was restricted to the hospital bed and only allowed to lie on his back--with his feet slightly elevated--until the third post-operative day. He was able to urinate, defecate, and ambulate by the fifth postoperative day; he was discharged with a simple bandage covering the surgical site that was to remain in place for the next three days.The area covered by the dressing was moist with clear fluid when the visiting nurse removed the bandage. The nurse returned two days later--the ninth postoperative day--and expressed concern when she re-examined the surgical site and observed that it was still moist. That same day the patient returned to the surgery clinic to be assessed.During his evaluation in the clinic, fluid--now with a slight pink tinge--was noted at the surgical site on his back. Based on this finding--and an absence of any headache--the patient’s doctors diagnosed the fluid as a seroma. He was sent home.The fluid, predominantly clear in appearance, persisted dripping onto the skin of his back. Two days later, on postoperative day 11, the patient and his wife  independently contacted neurosurgeons whom they knew. The neurosurgeon contacted by the patient commented that although most patients with a cerebrospinal fluid leak presented with a headache, the absence of a headache did not exclude the possibility of a cerebrospinal fluid leak. The neurosurgeon contacted by the patient’s wife thought the diagnosis of a seroma was interesting and then adamantly stated that the patient had a cerebrospinal fluid leak. Both neurosurgeons insisted that the patient immediately return to the emergency center and not leave until the cerebrospinal fluid leak was diagnosed and treated.The surgeon who evaluated the patient in the emergency center recommended the placement of several percutaneous sutures placed deeply into the subcutaneous tissue to tamponade the skin leak of cerebrospinal fluid. Eight sutures were placed Figure . There wHowever, as documented by the magnetic resonance imaging findings Figures , 3, the Narrowing of the spinal canal results in lumbar spinal stenosis; this can result from degenerative changes in the facet spinal joints, the intervertebral spinal disks, and/or the ligamentum flavum. Indeed, the most common reason for spinal surgery in patients over age 65 years is lumbar spinal stenosis. In addition to decreased sensation and fatigue in the lower extremities, low back pain, and radiating pain in the legs (sciatica), additional symptoms of lumbar spinal stenosis may include impairment of urination and/or defecation and neurogenic--persistent or intermittent--claudication  or tumor. Sciatica is usually unilateral; however, central disk herniation, lumbar spinal stenosis, and spondylolisthesis can result in bilateral sciatica ,2.The diagnosis of lumbar spinal stenosis-related sciatica can be suspected based on symptoms and physical examination. For persistent or progressive sciatica, magnetic resonance imaging can be useful to define the etiology. Computed tomography and radiographs are usually not utilized. Although electrodiagnostic evaluation (such as needle electromyography and nerve conduction studies) can be used to determine whether sciatica is caused by radiculopathy instead of a musculoskeletal etiology, its role in the assessment of sciatica remains to be established ,2,8.Various conservative modalities have been used for the treatment of sciatica-associated lumbar spinal stenosis. These include exercise, medication, and physical therapy. In addition, there are several other non-operative treatments Table  3,4,8].,4,8.3,4,Management of lumbar spinal stenosis for patients with persistent symptoms, functional limitations, or both may require surgery. A recent meta-analysis showed that the optimal choice for interventional treatments for lumbar spinal stenosis included non-fusion methods (such as decompression and an interspinous process device). In addition, it noted that the use of an interspinous process device had both a low incidence of complications and a high rate of reoperation ,8.An epidural injection is a common non-surgical treatment for sciatica-associated lumbosacral spinal stenosis and radicular pain. In the early 1900s, the technique originated in Paris, and cocaine was injected into the sacral hiatus. Subsequently, in the 1950s, patients began to be treated with epidural corticosteroids .Nerve edema--associated with spinal stenosis--results from the chemical irritation by inflamed nerve roots and the structural stimulation of physically compressed nerve tissue. Indeed, congestion of venous blood around the nerve roots causes local ischemia that subsequently elicits not only nerve edema but also inflammation from phospholipase and/or leukotriene B release. The mechanism of action whereby epidural corticosteroid injection diminishes nerve edema, and its associated inflammation, is by inhibiting the production and release of cytokines, reducing the migration of leukocytes, and stabilizing the membranes of cells .An analysis was performed to assess whether epidural corticosteroid injections were effective for lumbosacral radicular pain. The study showed that, at short-term follow-up, the corticosteroid injection was slightly more effective than the placebo for leg pain; also, there were only minor adverse effects from the procedure. However, the long-term benefits of epidural corticosteroid injections have not been established. Therefore, epidural corticosteroid injection continues to be used for the management of patients whose daily life is interfered with by either acute radiating pain and/or neurological claudication ,7,8.The reported patient had an epidural corticosteroid injection on two occasions. After each of the injections, there was prompt relief of any sciatica-related pain. However, the lumbosacral stenosis-associated weakness did not resolve or improve following the injections; the lack of an efficacious response regarding the motor strength of his lower extremities prompted him to proceed with surgery.An intraoperative tear of the dura occurred during the patient’s lumbar spinal surgery. This is commonly referred to as either an accidental dural tear or an incidental durotomy. The incidence of a dural tear during spinal surgery has been observed to range from 0.4 percent to 15.8 percent; a summarized incidence is approximately 5.8 percent ,10,12.Advancing age , revision surgery, and lumbar stenosis have been recognized as significant risk factors for accidental dural tears. Diabetes mellitus was also weakly associated with incidental durotomy; however, neither the individual’s sex nor obesity were considered to be statistically significant . Hence, Similar to this patient, the dural tear may be noticed during the surgical procedure. Therefore, attempting to repair the tear, the iatrogenic defect in the dura was sutured during the operation. Subsequently, after the completion of surgery and for two additional days, he was placed in a slight Trendelenburg-like position with his feet higher than his head ,20. In other individuals, the cerebrospinal fluid leak from an intraoperative tear of the dura may not be recognized during the spinal surgery. However, the leak of cerebrospinal fluid into the surrounding space results in cranial hypovolemia and cranial hypotension with a corresponding reduction of intracranial pressure. Subsequently, a postural headache and/or other symptoms associated with the accidental dural tear with its related cerebrospinal fluid leak and spontaneous intracranial hypotension may occur Table  10-12].-12.10-12In addition to a postural headache and other symptoms associated with spontaneous intracranial hypotension, other clinical manifestations suggestive of a possible intraoperative dural tear may also occur following spinal surgery. These findings include clear fluid draining from the surgical wound, neurologic deficits, physical examination demonstrating a palpable fluid collection, symptoms experienced by the patient , and wound drainage systems with unexpectedly high output. The observation of any of these findings should prompt the clinician--optimally the surgeon who operated--to immediately assess the patient and manage the complication in a timely and effective manner by not only entertaining the possibility of a cerebrospinal fluid leak but also considering prompt imaging, such as magnetic resonance imaging, for evaluation . The reported patient only had persistent and continuous drainage from his surgical wound, which varied in presentation from being clear to slightly pink-tinged. When he returned to the surgery clinic for assessment, the physicians--based on the color of the dripping fluid--concluded that he had a seroma. Therefore, he was sent home and instructed to return to the clinic in a few weeks.A seroma is a postoperative serous fluid collection; it is usually detectable on clinical examination. Surgical procedures in which there is the creation of an anatomical dead space may be associated with seroma formation. Therefore, seroma is commonly observed as a complication of plastic surgery procedures such as body contouring surgery for massive weight loss; however, seromas have also been noted postoperatively in lumbar spine surgery patients -19. The diagnosis of seroma--in patients who had drains placed during surgery--may be suspected when a palpable or visible fluid wave is observed after the drains are removed. Radiologic examination--either utilizing ultrasonography or computed tomography--can also be used to establish the diagnosis; however, these modalities require ancillary equipment for evaluation and are expensive. Therefore, needle aspiration is not only a more cost-effective manner to consider for assessing the patient, but is also considered to be a conservative initial approach to the management of a seroma; however, aspiration of a subcutaneous collection of cerebrospinal fluid presenting as a draining duro-cutaneous fistula in the unsterile setting of a surgery clinic would not be recommended. If the seroma does not resolve after serial needle aspirations, treatment alternatives include inserting a seroma catheter for continuous drainage, injection of sclerosing agents, or surgical interventions such as marsupialization and packing the wound or returning to the operating room to excise the seroma cavity -19. Hence, the patient’s cerebrospinal fluid leak was initially misinterpreted as a seroma. This occurred since the fluid dripping from his surgical wound was slightly tinged pink--instead of being completely clear--and he had not experienced a headache or any other signs and symptoms typically associated with a postoperative cerebrospinal fluid leak. Fortunately for the patient, neither aspiration of the fluid nor injection of a sclerosant solution was performed during his surgery clinic visit. Indeed, the color of the fluid and the absence of any other symptoms resulted in a delay in the diagnosis of the patient’s cerebrospinal fluid leak his doctors; it was not until he decided to return to the emergency center and insisted on a neuroimaging evaluation of his lower back, that the correct diagnosis of a cerebrospinal fluid leak--from the intraoperative dural tear--was established.Serious complications can be associated with an accidental dural tear. Several of these potential consequences are summarized in Table The patient’s cerebrospinal fluid leak was easily diagnosed after viewing the magnetic resonance imaging of his lower back. Indeed, several imaging techniques can be used to diagnose a cerebrospinal fluid leak following an accidental dural tear. These include magnetic resonance imaging (brain or spine), cisternography (radioisotope), and myelography  ,11.Other methods for diagnosing a cerebrospinal fluid leak include analysis of the fluid. The biochemical tests evaluate the fluid for the presence of either glucose oxidase, beta-2 transferrin, or beta-trace protein. The glucose oxidase test is inexpensive, rapid, and easy to perform; however, both the sensitivity and specificity of the test are low secondary to false positives  and false negatives (in diabetic patients). Although the beta-trace protein test is highly sensitive, rapid (taking only 20 minutes to perform on a nephelometer), and inexpensive; yet, the diagnostic usefulness is limited not only by associated problems with assessing accurate cut-off values but also by increased serum and decreased cerebrospinal fluid beta-trace protein values are observed in a patient with renal insufficiency and bacterial meningitis ,15.Detecting beta-2 transferrin is the gold standard for confirming a cerebrospinal fluid leak; however, the gel electrophoresis technique is difficult and labor-intensive, expensive, and requires three to six hours to perform. Yet, a new platform for the detection of beta-2 transferrin has been developed that enables the rapid identification of cerebrospinal fluid leakage. None of these tests were needed to be performed to confirm the cerebrospinal fluid leak in the reported patient after the evaluation of his magnetic resonance imaging ,16.Direct suture repair is a common treatment approach for a cerebrospinal fluid leak after spine surgery; however, there is nearly a ten percent failure rate. Therefore, some surgeons will use adjuvant dural closure material such as polyethylene glycol as a hydrogel sealant. If the dural defect cannot be repaired directly, an augmented closure with either fat, muscle tissue, or fascial graft may be performed ,13,20.Management based on fluid flow mechanics can also be incorporated into the approach to treating a dural tear with accompanying cerebrospinal fluid leak. This involves decreasing the difference between the pressure of the subarachnoid fluid and the epidural fluid. Hence, the cerebrospinal fluid leak can be slowed by either reducing the subarachnoid fluid pressure or by increasing the epidural fluid pressure, or both ,13,20.Adjusting the position of the patient, using a subarachnoid catheter to shunt the cerebrospinal fluid, or inhibiting cerebrospinal fluid formation (by using acetazolamide) can reduce subarachnoid fluid pressure. For example, patients with a lumbar or thoracic cerebrospinal fluid leak should be kept in either Trendelenburg or prone position. The reported patient was placed on his back in the Trendelenburg position for at least two-and-a-half days after his surgery was completed ,13,20.An increase in epidural fluid pressure--accompanied by slowing of cerebrospinal fluid flow and facilitation of dural flap adherence--can result from a tight fascial closure. In addition, elimination of the dead space can result by incorporating a subfascial drain and discharging the excess cerebrospinal fluid. Another alternative to seal the cerebrospinal fluid leak and concurrently increase the epidural fluid pressure is an epidural blood patch ,13,20. The patient refused to return to the operating room for a surgical evacuation of the cerebrospinal fluid in an attempt at an operative repair of the torn dura. Therefore, the patient’s duro-cutaneous fistulas were successfully treated with tamponade sutures that compressed the affected areas. Subsequently, computed tomography-guided partial removal of the extra-dural collections of cerebrospinal fluid provided relief of the recurrent sciatica symptoms from the compression of his nerve roots from the fluid. Follow-up magnetic resonance imaging shortly after the procedure confirmed that some of the cerebrospinal fluid had been removed without the accumulation of new fluid; another magnetic resonance imaging, three months later, demonstrated less cerebrospinal fluid, indicating some spontaneous absorption of the residual fluid.Four and a half years after surgery, the patient remains free of sciatica nerve symptoms. However, paresthesia affecting both his left distal foot and right middle three toes appeared on the evening of his surgery and continues to persist with altered proprioception of the affected areas. In addition, he did not regain the motor strength of his legs; it takes him at least 15 minutes to ambulate a mile.Before his back surgery, the patient had completed 20 marathons in Houston, Texas Figure . He realLumbar spinal stenosis is a significant problem for ageing individuals. Non-operative treatments  may be effective for the associated lower back and leg symptoms. Epidural corticosteroid injections are also commonly used; although they often provide short-term pain relief, the long-term benefit has not been demonstrated. Spinal decompression surgery may be necessary if symptoms persist and/or functional limitations develop despite non-operative interventions. A potential complication of lumbar spinal stenosis surgery is a cerebrospinal fluid leak. A man (who is the first author of this paper) is described who experienced an intra-operative accidental dural tear during lumbar spinal stenosis surgery and subsequently developed a large cerebrospinal fluid leak that involved both the laminectomy bed and the subcutaneous tissue of his back that presented as duro-cutaneous fistulas without the headache which his doctors misinterpreted as a seroma. In summary, the absence of a headache does not exclude the possibility of a cerebrospinal fluid leak following lumbar spinal stenosis surgery. And although a seroma may occur following lumbar spinal stenosis back surgery, the presence of fluid leaking from the surgical site should not only prompt consideration of a surgery-associated cerebrospinal fluid leak but also additional evaluation--such as imaging studies--to confirm the diagnosis.


Nature, Chou and colleagues define a new evolutionarily conserved class of tumour-elicited immune response mediated by a distinct population of T cell receptor (TCR)-positive FCER1G-expressing innate-like T cells with high cytotoxic potential (αβILTCKs).1In a recent study published in 2 These ICBs target inhibitory receptors such as PD-1 on tumour infiltrating CD8+ cytotoxic T lymphocytes (CTLs) that can recognise mutated cancer cell antigens and thereby enable tumour cell killing. Yet, a significant cohort of cancer patients are non-responsive to ICB treatment and therefore, there is a strong need to discover additional anti-cancer immunomechanisms. Recent work in Nature by Chou and colleagues identifies a population of αβILTCKs that exhibit reactivity to unmutated tumour antigens.1Targeted immunotherapies and most prominently immune checkpoint blockade (ICB) therapies, brought clinical benefits to tumour patients that were inconceivable 15 years ago.+TCRβ+CD8α+ cells directly isolated from the breast cancer tissues of MMTV-PyMT (PyMT) mice.1 Bioinformatic clustering identified five distinct T cell populations including naïve/recently activated, exhausted and proliferative cells, as well as αβILTCKs characterised by high NK1.1 expression.3 These cells are characterised by a unique transcriptome that is distinct from that of conventional tumour-infiltrating CD8+ T cells. They belong to the unconventional type-1-like innate lymphoid cells and previous work by Dadi and colleagues had shown that αβILTCKs can in principle, exhibit innate cytotoxicity towards tumour cells.3 Chou and colleagues also detected the αβILTCKs in murine prostate cancer and human colorectal cancer tissues, indicating that they might represent evolutionarily conserved regulators of tumour-elicited immunosurveillance . From these analyses they could unequivocally conclude that these two tumour infiltrating T cell subsets do not originate from a shared progenitor cell, but represent two mutually exclusive cell fate choices.Because the reactivity, ontogeny and cancer cell sensing mechanisms of αβILTCKs remained unknown,+ T cells. These conventional CTLs typically recognise tumours with a high tumour mutational burden due to the ability of their TCRs to detected mutated neoantigens as “non-self”.4 In contrast, elegant TCR reporter assays demonstrated that the majority of the TCRs from NK1.1+CD8α+ αβILTCKs showed reactivity against unmuted antigens from heterologous cancer cell populations.1 In this setting, the αβILTCK derived TCRs recognise the cancer-specific peptide in the context of major histocompatibility complex class I (MHC-I) complexes.As indicated above, immunotherapies that target the PD-1 axis require the activity of conventional PD-1Fgd5-creER-Rosa26LSL-tdTomato PyMT mice, in which a pulse of tamoxifen injection stably labels Lin−c-KIT+SCA1+ haematopoietic stem cells to allow fate-mapping of their progenitors. The intratumoural αβILTCKs were fate-mapped which demonstrated that this population is replenished by thymic progenitors and in situ proliferation as their primary means of population.To define the developmental origin and dynamics of the tumour infiltrating αβILTCKs, the authors utilised tumour bearing +TCRβ+CD8α+ cells from breast tumour tissues of PyMT mice. Interestingly, expression of Fcer1g which encodes for the Fc Epsilon Receptor Ig protein (FCER1G), was differentially expressed within the αβILTCK cluster compared to other tumour infiltrating CD8+ T cells, including PD-1+ T cells, regardless of their activation status. In primary human colon cancer tissue, FCER1G expression was also detected in TCRβ+ cells with a co-receptor expression profile similar to the mouse αβILTCK counterparts. Moreover, these FCER1G+ T cells were enriched in cancerous compared to adjacent normal tissue,1 indicating that FCER1G is a lineage-defining marker that characterises tumour infiltrating T cells committed to the αβILTCK lineage. Together, these data suggest that the αβILTCK programme in the tumour tissue appears to represent an evolutionarily conserved immune response in both mouse and human.To gain insight into the specifications of the αβILTCK lineage, Chou and colleagues next compared the gene expression profiles of the tumour infiltrating αβILTCKs to that of all other CD453 However, the origin of IL-15 that drives the expansion and activation of intratumoural αβILTCKs was previously unknown. Therefore, Chou and colleagues next generated a conditional transgenic breast cancer mouse model in which IL-15 production was specifically ablated within the transformed tumour cells, but not in healthy tissue. Intriguingly, in these S100a8-cre-Il15fl/flPyMT mice, the loss of cancer-cell derived IL-15 resulted in reduced αβILTCK recruitment into the tumour tissue and impaired cancer immunosurveillance, with accelerated tumour growth in comparison to wild-type controls. Next, the authors constitutively activated IL-15 receptor (IL-15R) signalling by genetically expressing a gain of function STAT5B variant (STAT5B-CA) selectively in αβILTCK progenitors. Adoptive transfer of these engineered αβILTCKs into lymphocyte-deficient tumour-bearing PyMT mice, strongly suppressed tumour growth compared to control αβILTCKs. Even upon transfer into lymphocyte-replete PyMT hosts, these STAT5B-CA expressing αβILTCK progenitors colonised tumour tissue, expanded, differentiated into αβILTCK effector cells, and diminished tumour growth.The immunosurveillance programme of αβILTCKs is critically dependent on the pro-inflammatory cytokine IL-15.+CD8+ CTLs. Therefore, utilising these αβILTCKs could be particularly useful against tumours that are refractory to current checkpoint inhibitor therapies. Nevertheless, since the majority of these experiments were completed in murine models, future investigations will need to validate the applicability of αβILTCK-based adoptive cellular therapy in a human model. Moreover, as αβILTCKs recognise still undefined non-mutated antigens which might also be expressed on normal tissue cells, additional studies are required to define potential autoimmune side effects and related toxicities of engineered or IL-15 triggered overactivated αβILTCKs.In summary, Chou and colleagues identified FCER1G expressing αβILTCKs as a unique and evolutionarily conserved tumour infiltrating cytotoxic T cell population that mediates tumour immune surveillance. Importantly, these cells can be engineered ex vivo and manipulated in vivo to enhance their activity via engagement of the IL-15 signalling pathway. As such, they represent interesting targets for novel tumour immunotherapies. These αβILTCKs can recognise tumour cells with a low mutational burden and their mode of action is distinct from that of conventional PD-1


Passive leg raise (PLR) can be used as a reversible preload challenge to stratify patients according to preload response. We aim to evaluate the accuracy of PLR, monitored by a non-invasive cardiac output monitor in predicting to response to fluid resuscitation in emergency department (ED).We recruited adult patients planned to receive a resuscitation fluid bolus. Patients were monitored using a thoracic electrical bioimpedance (TEB) cardiac output monitor . A 3-min PLR was carried out before and after fluid infusion. Stroke volume changes (ΔSV) were calculated and a positive response was defined as ≥ 15% increase.We recruited 39 patients, of which 37 were included into the analysis. The median age was 63 (50–77) years and 19 patients were females. 17 patients (46%) were fluid responders compared to 11 (30%) with positive response to PLR1. ΔSV with PLR1 and fluid bolus showed moderate correlation  and 62% concordance rate. For the prediction of the response to a fluid bolus the PLR test had a sensitivity of 41% (95% CI 22–64) and specificity of 80% (95% CI 58–92) with an area under the curve of 0.59 (95% CI 0.41–0.78). None of the standard parameters showed a better predictive ability compared to PLR.Using TEB, ΔSV with PLR showed a moderate correlation with fluid bolus, with a limited accuracy to predict fluid responsiveness. The PLR test was a better predictor of fluid responsiveness than the parameters commonly used in emergency care (such as heart rate and blood pressure). These data suggest the potential for a clinical trial in sepsis comparing TEB monitored, PLR directed fluid management with standard care. What is already known on this subject?Both over- and under-resuscitation are associated with harm.Passive leg raise showed high accuracy in predicting fluid response in ICU and peri-operative settings.There is little evidence on the accuracy of passive leg raise in Emergency Department.What this study adds?Stroke volume changes with passive leg raise are moderately correlated with fluid bolus with limited accuracy.Patients who had a >15% reduction in stroke volume with fluid bolus were accurately predicted by a negative passive leg raise.Passive leg raise showed better accuracy than commonly used standard parameters (e.g. heart rate and blood pressure).Fluid resuscitation is often the first line of treatment for circulatory failure in emergency care, often guided by resuscitation protocols to ensure timely delivery. However, one-size-fits-all protocols have not consistently demonstrated benefit over standard care . The varall patients, however ‘suspected sepsis’ covers such a wide range of patients that this ‘one size fits all’ approach is unlikely to be correct. In Emergency Department (ED), monitoring the response to treatment relies on standard parameters (e.g. blood pressure and heart rate). These are often insensitive to changes in circulatory volume, cardiac preload or the cardiac output response to fluid resuscitation—and potentially misleading [This challenge is most evident in sepsis resuscitation. Protocols commonly recommend a fixed volume of resuscitation fluids followed by escalation of treatment to vasopressors or inotropes . This prsleading .Standard monitoring parameters are used as proxies for cardiac output (CO) to determine whether patients are responsive following a preload challenge (preload/fluid responsiveness). A positive response means that patients operate on the steep part of Frank-Sterling curve and could tolerate  fluid resuscitation . A negatWith no risk of overzealous infusion, Passive leg raise (PLR), a reversible self-fluid challenge, can be used to stratify patients into responders and non-responders . StandarIn this study, we investigated the accuracy of stroke volume response to PLR, monitored by a non-invasive bioimpedance cardiac output monitor, in predicting the stroke volume response to a subsequent fluid bolus in ED.This was a prospective observational study of a convenience sample of adult ED patients (≥ 18 years old) planned to receive at least one resuscitation bolus of IV fluids by the treating team. The study was carried out at Leicester Royal Infirmary, an inner city acute hospital at Leicester, UK. The study was sponsored by the University of Leicester, UK and ethical approvals were obtained from Essex Research Ethics Committee (16/EE/0145).Exclusion criteria were: clinical condition preventing the performance of a PLR (e.g. trauma), mental health presentations, alcohol intoxication, patients deemed unable to consent due to a pre-existing medical problem (e.g. dementia) and prisoners.Patients were screened for eligibility by the clinical team or a member of the research team. Recruitment was carried out following a 2-stage consent process. Study procedure was initiated following initial verbal ascent  following a brief explanation. This was followed by a full informed consent.On recruitment, we recorded demographic data, presenting complaint, past medical history, and pre-inclusion fluid administered . Patients were followed up to record ED diagnosis, final discharge diagnosis, length of stay and mortality data. All biochemical results were recorded.• Heart rate (HR)• Respiratory rate• Temperature• Systolic blood pressure (SBP)• Diastolic blood pressure (DBP)• Oxygen saturation (SpO2)After the initial consent process standard monitoring was used to record standard physiologic parameters throughout the patients’ stay. From the standard usual care monitoring system the following parameters were recorded at baseline, and at the end of the study:• Patients were monitored for at least 3 min in a 45° head-up position (baseline recording)• A 3-min PLR test was carried out by trolley manipulation for up to 45° (PLR1)• Return to baseline position for at least 3 min (baseline 2)• Fluid bolus administration • PLR was repeated following at least 10 min from fluid bolus end (PLR2)The TEB monitoring electrodes were applied following manufacturers recommendations—two to each side of the neck and two to each side of the patient’s lower thorax. The study procedure was carried out as follows:• Baseline1: the minute immediately before leg raise• PLR1: the middle 1-min of PLR1• Baseline2: the minute immediately before fluid bolus• Fluid bolus (FB): immediately following the end of fluid bolus• Baseline 3: 10 min following the end of fluid bolus• PLR2: the middle 1-min of the second PLR2`• Baseline4: at least 2 min following PLR2TEB data were transferred to Microsoft Excel  and data on CO, SV and HR were extracted. A minute by minute average was calculated for each parameter across the whole time series. To evaluate the overall trend, each parameter was averaged for each minute across the study cohort. To estimate preload responsiveness we considered the haemodynamic variables at seven time periods:Previous studies used a timeframe for assessing the response to PLR ranging 1–10 min  A 3-min Preload responsiveness was defined as ≥ 15% increase in SV. Accordingly, patients were classified into PLR + /PLR- (responders and non-responders to PLR), and R/NR (responders and non-responders to fluid bolus).Descriptive data were presented as means with 95% confidence interval (CI), medians with interquartile ranges (IQR) and proportions as appropriate. Data analysis was performed using Graphpad Prism 7 . Correlation between the stroke volume changes (∆SV) with PLR1 and FB was calculated using Pearson’s r. Categorical agreement between PLR1 and FB was evaluated using Cohen Kappa. Parametric and non-parametric tests were used as appropriate to evaluate statistical significance of baseline variables between responders and non-responders.Patients and public were involved in the design stage through a meeting with the Leicester Cardiovascular Research Review Group. The research protocol was presented to the group and the burden of the intervention was discussed. Feedback from the group was considered during the design and conduct of the study.Thirty-nine patients met the inclusion criteria. One patient did not tolerate PLR and another had missing data due to technical failure of data transfer, so 37 patients were included in the analysis. The median age was 63 (IQR 50–77) years and 19 patients (51%) were females. At baseline, the median early warning score (NEWS) was 5 (IQR 3–6) and the majority of patients had a normal blood pressure and a tachycardia. 22 patients (60%) had an infection-related/sepsis as their ED diagnosis . Overall, patient characteristics by response to a fluid bolus are shown in Table All patients received a crystalloid fluid bolus with the majority of patients receiving 500 mL with a median of 7.9 mL/kg , IQR 6.1–11. The median time of fluid bolus infusion was 12 min (IQR 9–15.5), with a median fluid infusion rate of 53 mL/min (IQR 33–73).p = 0.252). One patient died in hospital.Thirty-five patients were admitted to a hospital ward (level 0), one patient to intensive care unit (level 3) and another patient to enhanced acute ward (level 1). Patients had a median hospital stay of 5.5 days (IQR 2.25–8), with no significant difference between those who responded to the initial fluid bolus and those who did not  immediately following fluid infusion and then fell back to a median of 67 mL at 10 min and was 63 mL (near baseline) by 20 min. The baseline PLR1 Test gave a median increase in SV of 10%, with the PLR2 test (after fluid infusion) giving the same result  showed a positive SV response (≥ 15%) to the baseline PLR test. with a median SV increase of 30% following the PLR manoeuvre, which then rapidly fell back to baseline. In these patients, the median SV increased following fluid bolus by 22% with partial resolution at 10 min after infusion. When the PLR test was repeated after fluid infusion in this patient group there was a median increase of SV by 18% again rapidly reversed after the manoeuvre.In the patients with a negative baseline PLR test there was no change in median SV with the fluid bolus and again no change in SV with the PLR test after fluid infusion Fig. .Fig. 2StSeventeen patients (46%) responded to the fluid bolus (≥ 15% SV change). In these ‘In the patients who did not have a change in SV  increase in SV (15%)in response to the PLR test performed after the fluid bolus Fig. .Change in SV (ΔSV) during the baseline PLR1 test showed a moderate correlation with ΔSV after fluid bolus—Pearson r of 0.47 (95% CI 0.17–0.69) and 62% concordance 23 patients), Fig.  patientsTo predict fluid responsiveness (ΔSV ≥ 15% with fluid bolus), the Baseline PLR test had a sensitivity of 41% (95% CI 22–64) and specificity of 80% (95% CI 58–92). This gave a positive predictive value of 64% (95% CI 35–85) and a negative predictive value of 62% (95% CI 43–78). The positive likelihood ratio was 2.06 (95% CI 0.72–5.85), while the negative likelihood ratio was 0.74 (95% CI 0.47–1.16). PLR1 had an area under the curve of 0.59 (95% CI 0.41–0.78) – P value 0.33.We analysed the degree of concordance between PLR1 and FB using different thresholds (0–15%) to define ‘positive response’ to both the baseline PLR test and the fluid bolus. The highest concordance 62%) was achieved using the 15% threshold for both PLR1 and FB . Fluid challenge is the gold standard for assessing preload responsiveness, however variation in the challenge technique (volume and rate) may affect the outcome. A meta-analysis by Toscani et al. showed heterogenicity in technique and that an infusion time > 30 min was associated with a lower proportion of responsiveness . A systeStroke volume change (ΔSV) in both PLR tests (before and after fluid bolus) was positive in 30% of patients, which is within the range reported in previous literature . HoweverStroke volume changes (ΔSV) with baseline PLR showed a moderate correlation with ΔSV following fluid bolus with 62% positive concordance Fig. . Past reWhile PLR had limited diagnostic performance in predicting FR, none of the standard monitoring parameters showed better performance. Notably, Baseline SV and CO showed a higher predictive ability than any of the currently used parameters (such as pulse rate and blood pressure), which is consistent with our volunteer results . This geIn our experiment, PLR was able to stratify patients into two different groups according to fluid responsiveness. However, it seemed to be testing a cardiovascular response that was related to, but different from the response to a fluid bolus. Using a diagnostic threshold of an increase in SV > 15% failed to show adequate accuracy for PLR alone as a definitive diagnostic test to predict fluid responsiveness, however as it performed better than any of the currently used parameters.Our study included a convenience sample of ED patients and may be liable for selection bias. A single operator (ME) carried out the procedure so we cannot present any data on inter-rater reliability. Operator was not blinded to SV changes during PLR and fluid bolus. However, this is less likely to have introduced bias as the analysis was carried out using a standardised code blinded to individual SV changes.In our study, data analysis did not happen in real-time and was based on summary numbers over time windows. This is not strictly comparable to the clinical situation where the clinician at the bedside is able to look at the continuous SV trace. This gives additional information about the reliability of the data, such as the stability of signal and the degree of artefacts observed (similar to the interpretation of pulse oximetry at the bedside). This additional information (which could not be used in this study without introducing observer bias) might change the utility of SV monitoring in real life.A potential limitation of the study is that, as expected in older people, five patients had atrial fibrillation. The TEB monitoring technique is known to be less accurate in these patients, however they were included in the study to ensure that the results were generalisable to a normal ED sepsis population. A further limitation is that compared with previous ICU studies the ED patients had relatively little fluid before the PLR test – this may have reduced the fluid shift caused by the PLR, and given a false negative result. However, most ED patients have little fluid resuscitation on presentation, so the study population is representative of normal practice.Another caveat is the specific time windows for evaluating a dynamic process . While this method has some evidence base, there is a disadvantage in artificially imposing time windows . In realThe PLR test showed a moderate correlation with SV changes following a subsequent fluid bolus, with a limited accuracy to predict fluid responsiveness. Based on our data, the low test sensitivity, does not allow for a safe prediction of over-resuscitation. However, from our observations, a group of patients who have an adverse response to the conventional sepsis management protocol might be predicted by a negative SV change on baseline PLR test. The PLR test was a better predictor of fluid responsiveness than the parameters commonly used in emergency care (such as heart rate and blood pressure) which had poor accuracy. In the context of what is already known about cardiac monitoring and fluid challenges, these data suggest the potential for a clinical trial in sepsis comparing TEB monitored, PLR directed fluid management with standard care.


Chlamydia is an obligate intracellular bacterium where most species are pathogenic and infectious, causing various infectious diseases and complications in humans and animals. Antibiotics are often recommended for the clinical treatment of chlamydial infections. However, extensive research has shown that antibiotics may not be sufficient to eliminate or inhibit infection entirely and have some potential risks, including antibiotic resistance. The impact of chlamydial infection and antibiotic misuse should not be underestimated in public health. This study explores the possibility of new therapeutic techniques, including a review of recent studies on preventing and suppressing chlamydial infection by non-antibiotic compounds. Chlamydia is an obligate intracellular, multi-host, and gram-negative pathogen with a unique developmental cycle   reported that the number of C. trachomatis infections had reached 1,579,885 cases in the United States  and increase this misuse  and IFN-γ during replication, it may enter a particular state called “chlamydial persistence” , which have a high degree of sensitivity and specificity , inhibits the development of Moreover, gonorrhea and the HIV infection rate appeared to be higher in women using the N-9 gel  and polyanetholsulfonic acid sodium salt (SPS), can suppress the formation of chlamydial inclusion in a concentration-dependent manner  and nuclear factor-κB (NF-κB) signaling pathways in genital tract cervical tissue infected with C. trachomatis in mice . In fact, experiments have shown that parts of lipids have anti-chlamydia activity and can destroy the membrane, which may be the primary mechanism . After exposing EBs to 50 mM 1-O-hexyl-sn-glycerol for 90 min, EBs appeared to have a hollow shell, ruptured cell membranes, and cytoplasmic contents leaking from the cell , and PG-1 have also been shown to reduce C. trachomatis and C. pneumoniae inclusion at a concentration of 10 μg/mL , bovine lactoferrin (bLF), and ovoTF, can stop C. psittaci from attaching to and entering the cell  can inhibit intravaginal C. trachomatis infection and reduce the number of inclusions and the overall replication of C. suis in McCoy cells with C. suis-spiked semen samples  are pathogenic intracellular pathogens that cause a Th1-type immune response, and IFN-γ plays a significant role in resistance to Chlamydia and Mtb infections  family is an effective group of cytokines that helps promote or inhibit chlamydial infection. Studies have shown that macrophages, Jurkat cells, and THP-1 cells infected with Cytokines play a critical role in the fight against tumor cells and pathogens. However, many barriers, such as toxicity-related inherent characteristics, including short half-lives in circulation, inherent pleiotropic functions, and off-target effects, have previously blocked the development of cytokines as immunotherapy drugs  modeling and simulation is an innovative technique that links PK profiles with the corresponding PD to improve drug development. Collecting and analyzing PK/PD information for designing optimal dosing strategies, evaluating animal models, and planning clinical studies is crucial. Moreover, some studies suggest that In conclusion, antibacterial agents must provide good PK/PD support data. Other critical issues of drug development, such as the mechanism of action, specificity, and toxic side effects, should be addressed. Finally, we will only comprehend the control and effectiveness of drugs with ongoing clinical trials.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the Natural Science Foundation of Hunan Province (Grant No. 2021JJ40455), the Projects of the Hunan Health Committee (Grant No. 202112071537), the National Innovation and Entrepreneurship Training program for college students (Grant No. 202110555102), the Innovation and Entrepreneurship Training program for college students of the University of South China (Grant Nos. X202010555371 and X202010555372), and Graduate Student Research Innovation Project of Hunan Province (Grant No. CX20221007).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.