Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL.

After treatment with chimeric antigen receptor (CAR) T cells, interleukin-15 (IL-15) elevation and CAR T-cell expansion are associated with non-Hodgkin lymphoma (NHL) outcomes. However, the association of preinfusion CAR product T-cell functionality with clinical outcomes has not been reported. A single-cell analysis of the preinfusion CD19 CAR product from patients with NHL demonstrated that CAR products contain polyfunctional T-cell subsets capable of deploying multiple immune programs represented by cytokines and chemokines, including interferon-γ, IL-17A, IL-8, and macrophage inflammatory protein 1α. A prespecified T-cell polyfunctionality strength index (PSI) applied to preinfusion CAR product was significantly associated with clinical response, and PSI combined with CAR T-cell expansion or pretreatment serum IL-15 levels conferred additional significance. Within the total product cell population, associations with clinical outcomes were greater with polyfunctional CD4+ T cells compared with CD8+ cells. Grade ≥3 cytokine release syndrome was associated with polyfunctional T cells, and both grade ≥3 neurologic toxicity and antitumor efficacy were associated with polyfunctional IL-17A-producing T cells. The findings in this exploratory study show that a preinfusion CAR product T-cell subset with a definable polyfunctional profile has a major association with clinical outcomes of CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT00924326.

Return to Sport, Re-injury and Performance After the Anterior Cruciate Ligament Reconstruction: Evaluating the Role of International Knee Documentation Committee (IKDC) and Knee Injury and Osteoarthritis Outcome Score (KOOS) Scoring Systems.

Introduction Anterior cruciate ligament (ACL) tears are common injuries that can considerably impact an individual's quality of life and athletic performance. In these cases, surgical reconstruction of the ligament can be considered to restore stability to the knee. This study aims to investigate the time taken for individuals to return to sport post-ACL reconstruction, assess the rate of re-injury and evaluate the reliability of the International Knee Documentation Committee (IKDC) and Knee Injury and Osteoarthritis Outcome Score (KOOS) scoring systems in predicting a return to sport at the pre-injury level. Methods In this retrospective study, a total of 104 patients who underwent ACL reconstruction between January 2016 and December 2022 by one surgical team at Mater Dei Hospital, Malta were evaluated using a self-administered questionnaire. The questionnaire was based on different components including the sport practised at the time of injury, sport engagement classification, return to sport, the ability to return to pre-injury levels of performance and re-injury. The participants then had to fill in IKDC and KOOS evaluation forms. Results In this study, 73% (n=76) of individuals successfully returned to sport after ACL reconstruction, with no significant difference being found between professional and recreational athletes (Chi-squared=0.00455, p=0.95). After reconstruction, 31.7% (n=33) of participants experienced an ipsilateral or contralateral ACL tear, with those returning to sport within six months showing a fivefold increase in re-injury risk compared to individuals who returned at eight or 12 months, suggesting a significant association between return duration and re-injury. The relationship between scoring systems and return to sport at the pre-injury level of performance was analysed using binary logistic regression, revealing that achieving scores of 85.6 or higher in IKDC or 89 or higher in KOOS meant having a 95% probability of returning to sport at the pre-injury level. Conclusions By considering these scoring systems with other post-operative criteria, clinicians can offer a more customised rehabilitation plan tailored to each patient who undergoes ACL reconstruction.

Subcutaneous Versus Transvenous Implantable Defibrillator Therapy: A Systematic Review and Meta-Analysis of Randomized Trials and Propensity Score-Matched Studies.

Background Subcutaneous implantable cardioverter-defibrillators (S-ICDs) have been of great interest as an alternative to transvenous implantable cardioverter-defibrillators (TV-ICDs). No meta-analyses synthesizing data from high-quality studies have yet been published. Methods and Results An electronic literature search was conducted to retrieve randomized controlled trials or propensity score-matched studies comparing S-ICD against TV-ICD in patients with an implantable cardioverter-defibrillator indication. The primary outcomes were device-related complications and lead-related complications. Secondary outcomes were inappropriate shocks, appropriate shock, all-cause mortality, and infection. All outcomes were pooled under random-effects meta-analyses and reported as risk ratios (RRs) and 95% CIs. Kaplan-Meier curves of device-related complications were digitized to retrieve individual patient data and pooled under a 1-stage meta-analysis using Cox models to determine hazard ratios (HRs) of patients undergoing S-ICD versus TV-ICD. A total of 5 studies (2387 patients) were retrieved. S-ICD had a similar rate of device-related complications compared with TV-ICD (RR, 0.59 [95% CI, 0.33-1.04]; P=0.070), but a significantly lower lead-related complication rate (RR, 0.14 [95% CI, 0.07-0.29]; P<0.0001). The individual patient data-based 1-stage stratified Cox model for device-related complications across 4 studies yielded no significant difference (shared-frailty HR, 0.82 [95% CI, 0.61-1.09]; P=0.167), but visual inspection of pooled Kaplan-Meier curves suggested a divergence favoring S-ICD. Secondary outcomes did not differ significantly between both modalities. Conclusions S-ICD is clinically superior to TV-ICD in terms of lead-related complications while demonstrating comparable efficacy and safety. For device-related complications, S-ICD may be beneficial over TV-ICD in the long term. These indicate that S-ICD is likely a suitable substitute for TV-ICD in patients requiring implantable cardioverter-defibrillator implantation without a pacing indication.

Surgical challenges, novel techniques, and systemic treatment of giant cell tumour of bone of the distal radius : clinical outcomes and systematic review of the literature.

AIMS: Giant cell tumour of bone (GCTB) treatment changed since the introduction of denosumab from purely surgical towards a multidisciplinary approach, with recent concerns of higher recurrence rates after denosumab. We evaluated oncological, surgical, and functional outcomes for distal radius GCTB, with a critically appraised systematic literature review. METHODS: We included 76 patients with distal radius GCTB in three sarcoma centres (1990 to 2019). Median follow-up was 8.8 years (2 to 23). Seven patients underwent curettage, 38 curettage with adjuvants, and 31 resection; 20 had denosumab. RESULTS: Recurrence rate was 71% (5/7) after curettage, 32% (12/38) after curettage with adjuvants, and 6% (2/31) after resection. Median time to recurrence was 17 months (4 to 77). Recurrences were treated with curettage with adjuvants (11), resection (six), or curettage (two). Overall, 84% (38/45) was cured after one to thee intralesional procedures. Seven patients had 12 months neoadjuvant denosumab (5 to 15) and sixmonths adjuvant denosumab; two recurred (29%). Twelve patients had six months neoadjuvant denosumab (4 to 10); five recurred (42%). Two had pulmonary metastases (2.6%), both stable after denosumab. Complication rate was 18% (14/76, with 11 requiring surgery). At follow-up, median MusculoSkeletal Tumour Society score was 28 (18 to 30), median Short Form-36 Health Survey was 86 (41 to 95), and median Disability of Arm, Shoulder, and Hand was 7.8 (0 to 58). CONCLUSION: Distal radius GCTB treatment might deviate from general GCTB treatment because of complexity of wrist anatomy and function. Novel insights on surgical treatment are presented in this multicentre study and systematic review. Intralesional surgery resulted in high recurrence-rate for distal radius GCTB, also with additional denosumab. The large majority of patients however, were cured after repeated curettage. Cite this article: Bone Jt Open 2022;3(7):515-528.

Single-Cell Multiplexed Proteomics on the IsoLight Resolves Cellular Functional Heterogeneity to Reveal Clinical Responses of Cancer Patients to Immunotherapies.

Cancer immunotherapies, in particular adoptive T cell therapy and immune-checkpoint blockade therapy have demonstrated a remarkable success in the treatment of cancer. However, due to heterogeneous functionality and complex immune response of immune cells, it remains challenging to identify predictive biomarkers which have the potential to correlate with efficacy and adverse effects of immunotherapies and help selecting patients who might benefit from the therapy, developing more personalized therapeutics as well as reducing clinical trial cost. The single-cell IsoCode chip in conjunction with fluorescent ELISA-based assay enables a simultaneous detection up to 40+ proteins secreted from live single immune cells, providing a large portion of the assayable functions for each immune cell type, and thus precise assessment of multifunctional, or polyfunctional, heterogeneity of each immune cell type.This unique functional detection capability provides a powerful solution to unmet needs in immunotherapy patient profiling today. Recently, the single-cell metric termed polyfunctional strength index (PSI™) by IsoCode chip computed from preinfusion anti-CD19 chimeric antigen receptor (CAR)-T cell products has demonstrated a significant association with clinical response and cytokine release syndrome (CRS) of cancer patient to the therapy after cell product infusion. This chapter elucidates IsoPlexis single-cell highly multiplexed proteomic platform and provides technical details for characterizing cell products and various cell subsets from peripheral blood, bone marrow, or tumor tissues using this assay.

Ultrafast Three-Dimensional Printing of Optically Smooth Microlens Arrays by Oscillation-Assisted Digital Light Processing.

A microlens array has become an important micro-optics device in various applications. Compared with traditional manufacturing approaches, digital light processing (DLP)-based printing enables fabrication of complex three-dimensional (3D) geometries and is a possible manufacturing approach for microlens arrays. However, the nature of 3D printing objects by stacking successive 2D patterns formed by discrete pixels leads to coarse surface roughness and makes DLP-based printing unsuccessful in fabricating optical components. Here, we report an oscillation-assisted DLP-based printing approach for fabrication of microlens arrays. An optically smooth surface (about 1 nm surface roughness) is achieved by mechanical oscillation that eliminates the jagged surface formed by discrete pixels, and a 1-3 s single grayscale ultraviolet (UV) exposure that removes the staircase effect. Moreover, computationally designed grayscale UV patterns allow us to fabricate microlenses with various profiles. The proposed approach paves a way to 3D print optical components with high quality, fast speed, and vast flexibility.

Bilateral Total Hip and Unilateral Knee Arthroplasties in a Young Adult with Arthropathy-Associated Intestinal Epithelial Dysplasia (Tufting Enteropathy).

Intestinal epithelial dysplasia (tufting enteropathy) is an uncommon congenital disorder. Furthermore, its association with chronic inflammatory arthropathy is rarely documented in the literature. Low prevalence rates of 1 in 100,000 live births in Western Europe exist, with higher rates in North Africa and Middle Eastern countries. Malta, being a small Mediterranean island at the cusp between Europe and North Africa, has an anecdotal sevenfold prevalence rate. This is the first documented case report of a patient with both intestinal epithelial dysplasia and severe bilateral hip and knee arthropathy that required simultaneous bilateral hip followed by, after a short interval, unilateral knee arthroplasties. Our aim is to highlight the rapid progression of associated arthropathy as well as the successful treatment with joint arthroplasties in such extreme cases. Surgical treatment may be a necessity despite best medical efforts to halt the disease.

Aligned fibers direct collective cell migration to engineer closing and nonclosing wound gaps.

Cell emergence onto damaged or organized fibrous extracellular matrix (ECM) is a crucial precursor to collective cell migration in wound closure and cancer metastasis, respectively. However, there is a fundamental gap in our quantitative understanding of the role of local ECM size and arrangement in cell emergence-based migration and local gap closure. Here, using ECM-mimicking nanofibers bridging cell monolayers, we describe a method to recapitulate and quantitatively describe these in vivo behaviors over multispatial (single cell to cell sheets) and temporal (minutes to weeks) scales. On fiber arrays with large interfiber spacing, cells emerge (invade) either singularly by breaking cell-cell junctions analogous to release of a stretched rubber band (recoil), or in groups of few cells (chains), whereas on closely spaced fibers, multiple chains emerge collectively. Advancing cells on fibers form cell streams, which support suspended cell sheets (SCS) of various sizes and curvatures. SCS converge to form local gaps that close based on both the gap size and shape. We document that cell stream spacing of 375 µm and larger hinders SCS advancement, thus providing abilities to engineer closing and nonclosing gaps. Altogether we highlight the importance of studying cell-fiber interactions and matrix structural remodeling in fundamental and translational cell biology.

CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay.

Malaria is a severe infectious disease with relatively high mortality, thus having been a scourge of humanity. There are a few candidate malaria vaccines that have shown a protective efficacy in humans against malaria. One of the candidate human malaria vaccines, which is based on human malaria sporozoites and called PfSPZ Vaccine, has been shown to protect a significant proportion of vaccine recipients from getting malaria. PfSPZ Vaccine elicits a potent response of hepatic CD8+ T cells that are specific for malaria antigens in non-human primates. To further characterize hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model, we have used a cutting-edge Single-cell Barcode (SCBC) assay, a recently emerged approach/method for investigating the nature of T-cells responses during infection or cancer. Using the SCBC technology, we have identified a population of hepatic CD8+ T cells that are polyfunctional at a single cell level only in a group of vaccinated mice upon malaria challenge. The cytokines/chemokines secreted by these polyfunctional CD8+ T-cell subsets include MIP-1α, RANTES, IFN-γ, and/or IL-17A, which have shown to be associated with protective T-cell responses against certain pathogens. Therefore, a successful induction of such polyfunctional hepatic CD8+ T cells may be a key to the development of effective human malaria vaccine. In addition, the SCBC technology could provide a new level of diagnostic that will allow for a more accurate determination of vaccine efficacy.

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response.

BACKGROUND: It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. METHODS: We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. RESULTS: We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. CONCLUSIONS: Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.

Coronary surgery is superior to drug eluting stents in multivessel disease. Systematic review and meta-analysis of contemporary randomized controlled trials.

OBJECTIVE: Current randomized controlled trials (RCTs) comparing percutaneous coronary intervention with drug eluting stent (DES-PCI) with coronary artery bypass grafting (CABG) in multivessel disease are underpowered to detect a difference in hard clinical end-points such as mortality, myocardial infarction and stroke. We aimed to overcome this limitation by conducting a meta-analysis of contemporary RCTs. METHODS: A systematic literature search was conducted for all RCTs comparing DES-PCI versus CABG in multivessel disease published through May 2015. Inverse variance weighting was used to pool data from individual studies (<1 favouring DES-PCI and >1 CABG favouring surgery). RESULTS: A total of five randomized trials including 4563 subjects were analysed. After an average follow-up of 3.4 years, DES-PCI was associated with a significantly increased risk of overall mortality (HR 1.51; 95%CI 1.23-1.84; P<0.001), MI (HR 2.02; 95%CI 1.57-2.58; P<0.001) and repeat revascularization (HR 2.54; 95%CI 2.07-3.11; P=<0.001). CABG marginally increased the risk of stroke (HR 0.70; 95%CI 0.50-0.98; P=0.04). The absolute risk reduction for all-cause mortality (3.3%) and myocardial infarction (4.3%) with CABG was larger than the absolute risk reduction for stroke (0.9%) with DES-PCI. CONCLUSION: In patients with multivessel coronary disease, CABG was found to be superior to DES-PCI by reducing the risk of mortality and subsequent myocardial infarction at the expense of a marginally increased risk of stroke.

Right internal thoracic artery versus radial artery as the second best arterial conduit: Insights from a meta-analysis of propensity-matched data on long-term survival.

OBJECTIVE(S): We conducted a meta-analysis of propensity score-matching (PSM) studies comparing long-term survival of patients receiving right internal thoracic artery (RITA) versus radial artery (RA) as a second arterial conduit for coronary artery bypass grafting. METHODS: A literature search was conducted using MEDLINE, EMBASE, and Web of Science to identify relevant articles. Primary endpoint was long-term mortality. Secondary endpoints were operative mortality, incidence of sternal wound infection, and repeat revascularization. Binary events were pooled using the DerSimonian and Laird method. For time-to-event outcomes, estimates of log hazard ratio (HR) and standard errors obtained were combined using the generic inverse-variance method. RESULTS: A total of 8 PSM studies were finally selected including 15,374 patients (RITA, 6739; RA, 8635) with 2992 matched pairs for final comparison. Mean follow-up time ranged from 45 to 168 months. When compared with RA, RITA was associated with a lower risk reduction of late death (HR, 0.75; 95% confidence interval [CI], 0.58-0.97; P = .028) and repeat revascularization (HR, 0.37; 95% CI, 0.16-0.85; P = .03). On the other hand, RITA did not increase operative mortality (odds ratio [OR], 1.53; 95% CI, 0.97-2.39; P = .07). RITA was associated with an increased risk of sternal wound complication when pedicled harvesting was used (OR, 3.18; 95% CI, 1.34-7.57), but not with skeletonized harvesting (OR, 1.07; 95% CI, 0.67-1.71). CONCLUSIONS: The present PSM data meta-analysis suggests that the use of RITA compared with RA was associated with superior long-term survival and freedom from repeat revascularization, with similar operative mortality and incidence of sternal wound complication when the skeletonized harvesting technique was used.

Antecubital Fossa Solitary Osteochondroma with Associated Bicipitoradial Bursitis.

Antecubital fossa lesions are uncommon conditions that present to the orthopaedic clinic. Furthermore, the radius bone is an uncommonly reported location for an osteochondroma, especially when presenting with a concurrent reactive bicipitoradial bursitis. Osteochondromas are a type of developmental lesion rather than a true neoplasm. They constitute up to 15% of all bone tumours and up to 50% of benign bone tumours. They may occur as solitary or multiple lesions. Multiple lesions are usually associated with a syndrome known as hereditary multiple exostoses (HME). Malignant transformation is known to occur but is rare. Bicipitoradial bursitis is a condition which can occur as primary or secondary (reactive) pathology. In our case, the radius bone osteochondroma caused reactive bicipitoradial bursitis. The differential diagnosis of such antecubital fossa masses is vast but may be narrowed down through a targeted history, stepwise radiological investigations, and histological confirmation. Our aim is to ensure that orthopaedic clinicians keep a wide differential in mind when dealing with antecubital fossa mass lesions.

The Libyan civil conflict: selected case series of orthopaedic trauma managed in Malta in 2014.

AIM: The purpose of this series of cases was to analyse our management of orthopaedic trauma casualties in the Libyan civil war crisis in the European summer of 2014. We looked at both damage control orthopaedics and for case variety of war trauma at a civilian hospital. Due to our geographical proximity to Libya, Malta was the closest European tertiary referral centre. Having only one Level 1 trauma care hospital in our country, our Trauma and Orthopaedics department played a pivotal role in the management of Libyan battlefield injuries. Our aims were to assess acute outcomes and short term mortality of surgery within the perspective of a damage control orthopaedic strategy whereby aggressive wound management, early fixation using relative stability principles, antibiotic cover with adequate soft tissue cover are paramount. We also aim to describe the variety of war injuries we came across, with a goal for future improvement in regards to service providing. METHODS: Prospective collection of six interesting cases with severe limb and spinal injuries sustained in Libya during the Libyan civil war between June and November 2014. CONCLUSIONS: We applied current trends in the treatment of war injuries, specifically in damage control orthopaedic strategy and converting to definitive treatment where permissible. The majority of our cases were classified as most severe (Type IIIB/C) according to the Gustilo-Anderson classification of open fractures. The injuries treated reflected the type of standard and improved weaponry available in modern warfare affecting both militants and civilians alike with increasing severity and extent of damage. Due to this fact, multidisciplinary team approach to patient centred care was utilised with an ultimate aim of swift recovery and early mobilisation. It also highlighted the difficulties and complex issues required on a hospital management level as a neighbouring country to war zone countries in transforming care of civil trauma to military trauma.

Miniaturized extracorporeal circulation versus off-pump coronary artery bypass grafting: a meta-analysis of randomized controlled trials.

BACKGROUND: Controversies exist whether off-pump coronary artery bypass (OPCAB) is superior to miniaturized extracorporeal circulation (MECC) in reducing deleterious effects of cardiopulmonary bypass as only a number of smaller randomized controlled trials (RCT) currently provide a limited evidence base. The main purpose of conducting the present meta-analysis was to overcome the expected low power in RCTs in an attempt to establish whether MECC is comparable to OPCAB. METHODS: A MEDLINE/PubMed search was conducted to identify eligible RCTs. A pooled summary effect estimate was calculated by means of Mantel-Haenszel method. RESULTS: The search yielded 7 RCTs included in this meta-analysis enrolling 271 patients in the OPCAB group and 279 in the MECC group. The OPCAB and MECC groups were comparable in terms of incidence of in-hospital mortality (Risk Difference [RD] 0.01; 95%CI -0.02, 0.03; P = 0.55; I(2) = 0%), stroke (RD -0.01; 95%CI -0.05, 0.04; P = 0.69; I(2) = 0%), need for renal replacement therapy (RD 0.00; -0.06, 0.06; P = 1; I(2) = 0%), postoperative atrial fibrillation (RD -0.03; -0.17, 0.10; P = 0.64; I(2) = 0%), re-exploration for bleeding (RD -0.01; 95%CI -0.03, 0.02; P = 0.65; I(2) = 0%), transfusion rate (RD -0.01; 95%CI -0.03, 0.02; P = 0.65; I(2) = 0%) and the amount of blood loss (weighted mean difference -25 mL; 95%CI -71, 21; P = 0.28; I(2) = 0%). CONCLUSIONS: Using a meta-analytic approach, MECC achieves clinical results comparable to OPCAB including postoperative blood loss and blood transfusion requirement. On the basis of our findings, MECC should be considered as a valid alternative to OPCAB in order to reduce surgical morbidity of conventional cardiopulmonary bypass.

Culture of low passage colorectal cancer cells and demonstration of variation in selected tumour marker expression.

There is increasing evidence that a tumour comprises of heterogeneous population of cells. Thus, studying homogenous cell lines in vitro may yield results that are not reflective of the true situation in a tumour and studying low passage cell lines maintained in a heterogeneous population before they transform away from the original state may provide a more complete picture of colorectal cancer. A method was developed to isolate and establish low passage colorectal cancer cell lines from tumour biopsies. The media contents, combination of antimicrobials and specimen collection and transport conditions employed, successfully eliminated microbial contamination which is frequently present in samples obtained from the gastrointestinal tract. A variety of growth forms indicating a heterogeneous mixture of cells was seen in the initial cultures. Using fluorescence immunocytochemistry, primary tumour cultures were shown to variably express selected tumour markers, carcinoembryonic antigen and C2 antigen. These low passage cell lines growing in a heterogeneous environment would more closely reflect the characteristics of the cells of the original tumour.

Shape-dependent cell migration and focal adhesion organization on suspended and aligned nanofiber scaffolds.

In the body, cells dynamically respond to chemical and mechanical cues from the extracellular matrix (ECM), yet precise mechanisms by which biophysical parameters (stiffness, topography and alignment) affect cell behavior remain unclear. Here, highly aligned and suspended multilayer polystyrene (PS) nanofiber scaffolds are used to study biophysical influences on focal adhesion complex (FAC) arrangement and associated migration behavior of mouse C2C12 cells arranged in specific shapes: spindle, parallel and polygonal. Furthermore, the role of cytoskeletal-altering drugs including blebbistatin, nocodazole and cytochalasin-D on FAC formation and migratory behavior is investigated. For the first time, this work reports that cells on suspended fiber networks, including cells with administered drugs, elongated along the fiber axes and developed longer (∼ 4×) and more concentrated FAC clusters compared to cells on flat PS control substrates. Additionally, substrate designs which topographically restrict sites of cell attachment and align adhesions were found to promote higher migration speeds (spindle: 52µmh(-1), parallel: 39µmh(-1), polygonal: 25µmh(-1), flat: 32µmh(-1)). This work demonstrates that suspended fiber topography-induced concentration of FACs along fiber axes generates increased migration potential as opposed to flat surfaces, which diffuse and randomly orient adhesions.

Intraoperative methylene blue sentinel lymph node mapping in colorectal cancer.

BACKGROUND: Isosulfan blue is not available for clinical use in Malaysia. This study describes the use of methylene blue as an alternative to isosulfan blue in colorectal sentinel node mapping. METHODS: Methylene blue dye was injected around colonic and rectal tumours and the first blue-stained nodes were suture tagged and harvested after standard colorectal resection. Standard histopathological examination was then carried out to detect nodal metastasis. All negative sentinel lymph nodes (SLN) were subjected to 10 further step sectioning and immunoperoxidase staining for cytokeratin 20 to detect tumour deposits. RESULTS: Thirty-one patients were enrolled from August 2005 to July 2006. Twenty-five attempts at identifying the SLN were successful (80.7%). Of the 18 (58.1%) who had nodal metastases (stage III), 3 had negative SLN but positive other lymph nodes (false-negative rate of 21.4%). In one (4%), the SLN was the exclusive site of metastasis. CONCLUSION: Methylene blue can be used as an alternative sentinel node marker for rectal cancer (above the peritoneal reflection) and colonic cancer.