Artificial axons as a biomimetic 3D myelination platform for the discovery and validation of promyelinating compounds.

Multiple sclerosis (MS), a chronic neurodegenerative disease driven by damage to the protective myelin sheath, is currently incurable. Today, all clinically available treatments modulate the immune-mediated symptoms of the disease but they fail to stop neurodegeneration in many patients. Remyelination, the regenerative process of myelin repair by oligodendrocytes, which is considered a necessary step to protect demyelinated axons and stop neuronal death, is impaired in MS patients. One of the major obstacles to finding effective remyelinating drugs is the lack of biomimetic drug screening platforms that enable quantification of compounds' potential to stimulate 3D myelination in the physiologically relevant axon-like environment. To address this need, we built a unique myelination drug discovery platform, by expanding our previously developed technology, artificial axons (AAs), which enables 3D-printing of synthetic axon mimics with the geometry and mechanical properties closely resembling those of biological axons. This platform allows for high-throughput phenotypic myelination assay based on quantification of 3D wrapping of myelin membrane around axons in response to compounds. Here, we demonstrate quantification of 3D myelin wrapping by rat oligodendrocytes around the axon mimics in response to a small library of known pro-myelinating compounds. This assay shows pro-myelinating activity for all tested compounds consistent with the published in vitro and in vivo data, demonstrating predictive power of AA platform. We find that stimulation of myelin wrapping by these compounds is dose-dependent, providing a facile means to quantify the compounds' potency and efficacy in promoting myelin wrapping. Further, the ranking of relative efficacy among these compounds differs in this 3D axon-like environment as compared to a traditional oligodendrocyte 2D differentiation assay quantifying area of deposited myelin membrane. Together, we demonstrate that the artificial axons platform and associated phenotypic myelin wrapping assay afford direct evaluation of myelin wrapping by oligodendrocytes in response to soluble compounds in an axon-like environment, providing a predictive tool for the discovery of remyelinating therapies.

Combined PARP and WEE1 inhibition triggers anti-tumor immune response in BRCA1/2 wildtype triple-negative breast cancer.

Novel therapeutic strategies that can effectively combine with immunotherapies are needed in the treatment of triple-negative breast cancer (TNBC). We demonstrate that combined PARP and WEE1 inhibition are synergistic in controlling tumour growth in BRCA1/2 wild-type TNBC preclinical models. The PARP inhibitor (PARPi) olaparib combined with the WEE1 inhibitor (WEE1i) adavosertib triggered increases in anti-tumour immune responses, including STING pathway activation. Combinations with a STING agonist resulted in further improved durable tumour regression and significant improvements in survival outcomes in murine tumour models of BRCA1/2 wild-type TNBC. In addition, we have identified baseline tumour-infiltrating lymphocyte (TIL) levels as a potential predictive biomarker of response to PARPi, WEE1i and immunotherapies in BRCA1/2 wild-type TNBC.

Tucatinib promotes immune activation and synergizes with programmed cell death-1 and programmed cell death-ligand 1 inhibition in HER2-positive breast cancer.

BACKGROUND: Programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors have poor efficacy in patients with trastuzumab-resistant advanced HER2-positive breast cancer. Tucatinib is a potent, selective anti-HER2 tyrosine kinase inhibitor with proven clinical benefit in the advanced setting in patients with trastuzumab resistance. We investigated if tucatinib can alter the tumor microenvironment and if this could be harnessed for therapeutic efficacy. METHODS: We investigated the antitumor efficacy and contribution of the immune response of tucatinib using 2 immunocompetent, HER2-positive murine breast cancer models (trastuzumab-sensitive H2N113; trastuzumab-resistant Fo5) and the efficacy of tucatinib with trastuzumab and PD-1 or PD-L1 checkpoint inhibitors. RESULTS: In both models, tucatinib statistically significantly inhibited tumor growth and demonstrated dose-dependent efficacy. Ex vivo analysis by flow cytometry of tumor-infiltrating lymphocytes in mice treated with tucatinib showed increased frequency, higher proliferation, and enhanced effector function of CD8+ effector memory T cells. Tucatinib treatment also increased frequency of CD8+PD-1+ and CD8+TIM3+ T cells, CD49+ natural killer cells, monocytes, and major histocompatibility complex II expression on dendritic cells and macrophages and a decrease in myeloid-derived suppressor cells. Gene expression analysis revealed statistically significant enrichment in pathways associated with immune activation, type I and II interferon response, adaptive immune response, and antigen receptor signaling. In vivo, tucatinib and α-PD-L1 or α-PD-1 demonstrated statistically significantly increased efficacy and improved survival of mice compared with tucatinib alone. CONCLUSION: Tucatinib modulates the immune microenvironment favorably, and combination treatment with α-PD-L1 or α-PD-1 demonstrated increased efficacy in preclinical HER2-positive tumor models. These findings provide a rationale for investigation of tucatinib and immune checkpoint inhibition in the clinic.

Intratumoral CD8+ T cells with a tissue-resident memory phenotype mediate local immunity and immune checkpoint responses in breast cancer.

CD8+ tumor-infiltrating lymphocytes with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, the relative contribution of CD8+ TRM cells to anti-tumor immunity and immune checkpoint blockade efficacy in breast cancer remains unknown. Here, we show that intratumoral CD8+ T cells in murine mammary tumors transcriptionally resemble those from TNBC patients. Phenotypic and transcriptional studies established two intratumoral sub-populations: one more enriched in markers of terminal exhaustion (TEX-like) and the other with a bona fide resident phenotype (TRM-like). Treatment with anti-PD-1 and anti-CTLA-4 therapy resulted in expansion of these intratumoral populations, with the TRM-like subset displaying significantly enhanced cytotoxic capacity. TRM-like CD8+ T cells could also provide local immune protection against tumor rechallenge and a TRM gene signature extracted from tumor-free tissue was significantly associated with improved clinical outcomes in TNBC patients treated with checkpoint inhibitors.

A Prospective Analysis of Opioid Use Following Outpatient Pediatric Urologic Surgery.

OBJECTIVE: To examine our outpatient urologic surgery cohort for trends in opioid consumption, given the lack of consensus on opioid prescription after outpatient urologic procedures. While opioids have a role in multimodal postoperative analgesia, there is emerging data that they may not be routinely required after pediatric surgery. METHODS: Data on opioid use was prospectively collected over 16 months via postoperative telephone calls to caregivers of patients undergoing outpatient urologic surgery. Patient characteristics, surgery type, analgesia, and opioid prescription and usage information were recorded. Patients were prescribed as needed oxycodone and scheduled acetaminophen and ibuprofen for 48 hours, then as needed. The relationships between the log mean of the number of opioid doses used and age, type of surgery, race, and opioid prescription were modelled using negative binomial regression with the robust standard errors. RESULTS: Two hundred sixty-five patients were included. They were predominantly male with median age 2.6 years. The mean number of opioid doses prescribed per patient was 5.8 (SD 2.8, range 3-20). Over half of patients used no opioids, and mean opioid use was one dose. Those prescribed >5 doses took on average 3.4 times more doses compared to those prescribed >5 (P = .0003), and this was the only factor significantly associated with the amount of opioid used. CONCLUSION: Our findings suggest that opioids are over-prescribed after outpatient pediatric urologic surgery, with 95% of patients having leftover medication and 54% not using any opioids at all. While opioid requirements were low across all sub-cohorts, patients who were prescribed more opioid doses used significantly more doses.

Internship commencement during COVID-19: the impact of an extended induction model.

BACKGROUND: The COVID-19 pandemic necessitated an enforced 8-week induction period (18 May to 12 July 2020) for all new interns in Ireland. These unprecedented circumstances presented a unique opportunity to assess this induction period. AIM: To assess the impact of a prolonged induction period on the technical abilities of interns embarking on their clinical careers. METHOD: We distributed a 12-item questionnaire to new interns at our institution during the COVID-19 pandemic. Section 1 of the questionnaire was designed to assess the rate of self-reported improvement in the successful and independent execution of practical 'intern' tasks. Section 2 of the questionnaire captured the subjective experience of interns during this time in relation to the effectiveness of an 8-week induction period with senior intern support available. Statistical analysis of categorical predictor and ordinal outcome variables was performed using the two-sample Wilcoxon rank-sum (Mann-Whitney) test. RESULTS: Our results demonstrated a statistically significant improvement in the proficiency at first attempt phlebotomy in week 8 compared with week 1 (p < 0.0001). There was a significant improvement in placing first-attempt peripheral IV lines in week 8 compared with week 1 (p < 0.001). Regarding the need for senior assistance, we demonstrated a statistically significant reduction in week 8 compared with week 1 (p = 0.046). There were 95.56% (n = 43) of interns that said they would recommend the induction period for future incoming interns. CONCLUSION: The COVID-19 pandemic has inadvertently identified a model of internship induction that benefits interns, their colleagues and their patients through the production of more technically capable interns.

Tissue-resident memory T cells in breast cancer control and immunotherapy responses.

The presence of tumour-infiltrating lymphocytes (TILs) is associated with favourable outcomes in patients with breast cancer as well as in those with other solid tumours. T cells make up a considerable proportion of TILs and current evidence suggests that CD8+ T cells are a crucial determinant of favourable clinical outcomes. Studies involving tumour material from numerous solid tumour types, including breast cancer, demonstrate that the CD8+ TILs include a subpopulation of tissue-resident memory T (TRM) cells. This subpopulation has features consistent with those of TRM cells, which have been described as having a role in peripheral immune surveillance and viral immunity in both humans and mice. Patients with early-stage triple-negative breast cancers harbouring greater numbers of TRM cells have a substantially improved prognosis and longer overall survival. Furthermore, patients with advanced-stage breast cancers with higher levels of TRM cells have increased response rates to anti-PD-1 antibodies. These findings have motivated efforts to explore whether CD8+ TRM cells include tumour-specific T cells, their functional responses to cognate antigens and their role in responses to immune checkpoint inhibition. In this Review, we focus on the clinical significance of CD8+ TRM cells and the potential ways that these cells can be targeted to improve the success of immunotherapeutic approaches in patients with breast cancer, as well as in those with other solid tumour types.

Scaphoid fractures: has magnetic resonance imaging become a cost-effective alternative to definitive diagnosis?

BACKGROUND: A clinical examination finding of anatomical snuffbox tenderness (AST) with plain film radiographs alone yields low sensitivity and specificity in diagnosing scaphoid fractures. International guidelines suggest immediate referral for magnetic resonance imaging (MRI) following one non-diagnostic radiograph in such patients. Perceived cost, high demand and limited capacity of MRI scanners have resulted in few suspected fractures following this pathway in our institution. AIMS: Our study aimed to audit cost-effectiveness of immediate MRI referral following one non-diagnostic radiograph in the patient with AST versus current local practice. METHODS: Retrospective analysis of all patients with suspected scaphoid fractures referred from the ED to the orthopaedic service over a six-month period was performed. Mean pricing per radiograph, casting, MRI and fracture clinic presentation was obtained from our hospital's Finance department. RESULTS: Ninety-seven patients were identified; 26 had scaphoid fractures (26.8%). Seventy-one patients with no fractures cost a mean €82,111.50 (IQR: €55,025, €98,335) having a mean of 3.1 clinic visits, 4.6 radiographs and 4.7 weeks casted, versus €40,115 for early MRI referral as per guidelines (p > 0.05). CONCLUSIONS: In conclusion, when compared with current local practice, immediate referral of the patient with AST for MRI following one non-diagnostic ED radiograph is potentially cost-effective in establishing efficient diagnosis of scaphoid fractures. We recommend the implementation of published international guidelines in the investigation of query scaphoid fractures as a pragmatic and cost-effective practice.

Valgus instability of the elbow: acute and chronic form.

There has been an increase in thrower-specific elbow injuries in recent years. High valgus stresses during the late cocking and acceleration phases of throwing need to be compensated by the flexor pronator muscles as these can exceed the tensile strength of the medial collateral ligament complex. Prevention of injuries is the priority, with a focus on strengthening, reducing throwing frequency, decreasing force, and promoting a technique. The spectrum of thrower injuries ranges from a simple sprain to complete failure of the valgus stabilizing factors. The medial collateral ligament can stretch, leading to posteromedial impingement and radiocapitellar compression forces. This in turn can result in arthrosis and the formation of osteophytes. Ligament failure may eventually occur, making it impossible for the athlete to continue their throwing activities. The outcome of conservative treatment with strengthening, improvement of technique, and relative rest is often disappointing. Direct repair may no longer be possible in these acute-on-chronic injuries and a reconstruction with a tendon graft may be necessary.

Publisher Correction: Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.

In the version of this article originally published, the institution in affiliation 10 was missing. Affiliation 10 was originally listed as Department of Surgery, Royal Melbourne Hospital and Royal Womens' Hospital, Melbourne, Victoria, Australia. It should have been Department of Surgery, Royal Melbourne Hospital and Royal Womens' Hospital, University of Melbourne, Melbourne, Victoria, Australia. The error has been corrected in the HTML and PDF versions of this article.

Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.

The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes1. Although T cells are the predominant TIL population2, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8+ T cells with features of tissue-resident memory T (TRM) cell differentiation and that these CD8+ TRM cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8+ TRM gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8+ TRM cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of TRM cells will be crucial for successful immunotherapeutic development in BC.

Corrective Osteotomy for Malunited Diaphyseal Forearm Fractures Using Preoperative 3-Dimensional Planning and Patient-Specific Surgical Guides and Implants.

PURPOSE: Three-dimensional planning based on computed tomography images of the malunited and the mirrored contralateral forearm allows preoperative simulations of corrective osteotomies, the fabrication of patient-specific osteotomy guides, and custom-made 3-dimensional printed titanium plates. This study aims to assess the precision and clinical outcome of this technique. METHODS: This was a prospective pilot study with 5 consecutive patients. The mean age at initial injury was 11 years (range, 4-16 years), and the mean interval from the time of injury to the time of corrective surgery was 32 months (range, 7-107 months). Patient-specific osteotomy guides and custom-made plates were used for multiplanar corrective osteotomies of both forearm bones at the distal level in 1 patient and at the middle-third level in 4 patients. Patients were assessed before and after surgery after a mean follow-up of 42 months (range, 29-51 months). RESULTS: The mean planned angular corrections of the ulna and radius before surgery were 9.9° and 10.0°, respectively. The mean postoperative corrections obtained were 10.1° and 10.8° with corresponding mean errors in correction of 1.8° (range, 0.3°-5.2°) for the ulna and 1.4° (range, 0.2°-3.3°) for the radius. Forearm supination improved significantly from 47° (range, 25°-75°) before surgery to 89° (range, 85°-90°) at final review. Forearm pronation improved from 68° (range, 45°-84°) to 87° (range, 82°-90°). In addition, there was a statistically significant improvement in pain and grip strength. CONCLUSIONS: This study demonstrates that 3-dimensional planned patient-specific guides and implants allow the surgeon to perform precise corrective osteotomies of complex multiplanar forearm deformities with satisfactory preliminary results. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

Surgeon delivered psoas compartment block in total hip arthroplasty.

Total hip arthroplasty is associated with significant postoperative pain. A psoas compartment block is superior to other regional techniques in analgesia post THA. However, traditional methods of delivery are associated with serious complications. We present a technique of a surgeon delivered lumbar plexus block through injection at the portion of the iliopsoas seen intraoperatively. We randomised fifty-three consecutive patients into two groups. The group that received the block had an increased period prior to requesting supplementary analgesia and lower overall pain scores. There were no adverse effects. We have demonstrated the analgesic efficacy of Psoas Compartment Block performed during surgical access for total hip arthroplasty. This technique should be considered in the analgesic regimen for total hip arthroplasty.

Paediatric talus fracture.

Paediatric talus fractures are rare injuries resulting from axial loading of the talus against the anterior tibia with the foot in dorsiflexion. Skeletally immature bone is less brittle, with higher elastic resistance than adult bone, thus the paediatric talus can sustain higher forces before fractures occur. However, displaced paediatric talus fractures and those associated with high-energy trauma have been associated with complications including avascular necrosis, arthrosis, delayed union, neurapraxia and the need for revision surgery. The authors present the rare case of a talar neck fracture in a skeletally immature young girl, initially missed on radiological review. However, clinical suspicion on the part of the emergency physician, repeat examination and further radiographic imaging revealed this rare paediatric injury.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence.

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

Saltwater nectotizing fasciitis following coral reef laceration possibly exacerbated by a long-haul flight: a case report.

INTRODUCTION: Necrotising fasciits is a rapidly progressive disease characterised by extensive necrosis of the fascia, skin, and subcutaneous tissue, with relative sparing of the underlying muscle. CASE PRESENTATION: We present the case of a 24-year old Irish male student who sustained a laceration to his right shin from contact with a coral reef while swimming in the Phuket region, off the west coast of Thailand. The following day, he returned to Ireland and presented with an aggressive and destructive variant of group A beta-hemolytic streptococcal necrotising fasciitis originating at the site of the coral reef injury, and exacerbated by the long-haul flight. He was successfully treated with aggressive surgical debridement, vacuum-assisted dressings, split skin grafting and broad spectrum antibiotics. CONCLUSION: Necrotising fasciitis can progress rapidly to systemic toxicity and even death without expedient diagnosis and aggressive treatment. Long-haul flights induce significant fluid accumulation in the lower extremity. These physiological fluid shifts may have contributed to the severity of our patient's necrotizing condition following his flight from Thailand.

Netrin-4 regulates angiogenic responses and tumor cell growth.

Netrin-4 is a 628 amino acid basement membrane component that promotes neurite elongation at low concentrations but inhibits neurite extension at high concentrations. There is a growing body of literature suggesting that several molecules, including netrins, are regulators of both neuronal and vascular growth. It is believed that molecules that guide neural growth and development are also involved in regulating morphogenesis of the vascular tree. Further, netrins have recently been implicated in controlling epithelial cell branching morphogenesis in the breast, lung and pancreas. Characterization of purified netrin-4 in in vitro angiogenesis assays demonstrated that netrin-4 markedly inhibits HMVEC migration and tube formation. Moreover, netrin-4 inhibits proliferation of a variety of human tumor cells in vitro. Netrin-4 has only modest effects on proliferation of endothelial and other non-transformed cells. Netrin-4 treatment results in phosphorylation changes of proteins that are known to control cell growth. Specifically, Phospho-Akt-1, Phospho-Jnk-2, and Phospho-c-Jun are reduced in tumor cells that have been treated with netrin-4. Together, these data suggest a potential role for netrin-4 in regulating tumor growth.

Protein tyrosine phosphatase PRL-3 in malignant cells and endothelial cells: expression and function.

Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor-stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy.

Infiltrating immune cells, but not tumour cells, express FasL in non-small cell lung cancer: No association with prognosis identified in 3-year follow-up.

Non-small cell lung cancer (NSCLC) remains a difficult disease to treat and independent prognostic markers other than tumour stage and histology have not emerged. The immune cell content of solid tumours has been associated with tumour regression and at times, tumour progression. The involvement of immune cells in prognosis of NSCLC is poorly described. Poor immune responses within solid tumours have been linked with tumour production of immunosuppressive cytokines. Tumour expression of FasL is thought to disarm responses through the transduction of a death signal in Fas-expressing T cells. The existence of the 'tumour counterattack' in vivo has been questioned. We undertook to measure T cell and macrophage infiltration of the tumour bed in NSCLC and report the association between immune cell content and prognosis in a limited, 3-year analysis of survival (n = 113). In addition we investigated FasL expression (n = 45). T cells and macrophages were found to frequently infiltrate lung tumours, albeit in small numbers. Generally there were more T cells infiltrating than macrophages. T cell and macrophage numbers were not associated with prognosis. Lung tumours were found not to express FasL, although occasional immune cells surrounding tumour cells were strongly positive. FasL expression was not associated with prognosis in this series. Thus, immune cells infiltrating NSCLC are not capable of suppressing tumour growth, nor are they associated with tumour progression. We report that lung tumours do not express the FasL, and that although some immune cells are FasL positive, this is not a reflection of general immune cell activation.