Breast Cancer Cells Induce a Pro-inflammatory Response to Mitigate Immune Mediation in a 3D Culture Model.

BACKGROUND: Growth factors and cytokines mediate complex interactions between cells within the breast tumour microenvironment. In advanced cancer, an excess of regulatory T (TREG) lymphocytes and lack of natural killer (NK) cells in tumour-infiltrating lymphocyte populations may reflect a shift to pro-tumorigenic adaptive immune mechanisms. To facilitate targeted assessment of the interactions between tumour and immune cells ex vivo, three-dimensional (3D) culture systems are able to better recapitulate the in vivo microenvironment, recreating the anatomy of tumours. MATERIALS AND METHODS: We used 3D breast tumour models to determine morphological alterations, and the levels of secreted transforming growth factor-ß (TGFß) and induced cytokines. 3D luminal phenotype models and basal phenotype models were generated by culturing NK cells and CD4+CD25+ TREG cells with MCF-7 cells and MDA-MB-231 cells respectively, in growth factor-reduced Matrigel. TGFß was qualitatively assessed by immunolocalisation and cytokine data from culture supernatant was acquired with a multiplex cytokine assay. Traditional statistical analysis and principal component analysis were employed to unravel the cytokine response. RESULTS AND CONCLUSION: We identified that an interleukin-6 (IL6)-chemokine axis associated with TGFß is primarily responsible for differences detected between breast cancer models, with luminal and basal phenotype tumours responding differentially to immune mediation. Identified cytokines are implicated in facilitating tumour cell subversion of immune cell function to promote an invasive phenotype. Moreover, the disruption of the extracellular matrix and failure to form well-differentiated tumour masses/networks is indicative of enhanced malignancy. Tumour cells are implicated in promoting a pro-inflammatory microenvironment to attenuate NK cell function and in inducing a pro-tumorigenic profile that is facilitated by TREG lymphocytes.

Cystatin C- and creatinine-based equations in the assessment of renal function in HIV-positive patients prior to commencing Highly Active Antiretroviral Therapy.

BACKGROUND: We evaluated the accuracy and precision of creatinine- and cystatin C-based prediction equations for estimating glomerular filtration rate compared to measured glomerular filtration rate in an antiretroviral-naive human immunodeficiency virus population. METHODS: The study population consisted of 100 treatment-naive HIV patients. Glomerular filtration rate was estimated using the Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations, as well as cystatin C-based equations (CKD-EPIcystatin C, cystatin Cvan Deventer and CKD-EPIcombined)) compared to (51)Cr-EDTA plasma clearance-measured glomerular filtration rate. We calculated percentage bias, standard deviation of the differences, accuracy within 15 and 30% of measured glomerular filtration rate and sensitivity and specificity for predicting measured glomerular filtration rate <60 mL/min/1.73 m(2). RESULTS: Bias for all estimating glomerular filtration rate equations ranged from -9.4% to 38.4%. The CKD-EPIcombined without ethnicity correction factor equation had the least bias, 2.9% (-2.9 to 8.8). Bias was higher for the Modification of Diet in Renal Disease and CKD-EPI equation with the African-American ethnicity factor (38.4 and 33.7%) than without (14.2 and 15.3%). Standard deviation of the differences ranged from 29.2% (CKD-EPIcombined without ethnicity factor) to 54.0% (Modification of Diet in Renal Disease with ethnicity factor). Accuracy within 30% of measured glomerular filtration rate ranged from 78% for CKD-EPIcombined without ethnicity factor to 56.7% for the Cockcroft-Gault equation. Sensitivity for creatinine-based equations was less than 50% and for the CKD-EPIcystatin C equation was 75%. CONCLUSION: Sensitivity of creatinine-based equations for predicting glomerular filtration rate was poor in this group of patients. The CKD-EPIcombined equation performed better than creatinine-based equations.

Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer.

Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell-cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.

Lavage drain extension for local anaesthetic instillation into abdominal wounds.

BACKGROUND: A new device made by ThebeMedicare allows efficient local anaesthetic washout of wound areas, by utilising an attachment to an existing drain. The aim of this trial was to explore 'proof of concept' in patients undergoing abdominoplasty procedures. PATIENTS AND METHODS: Thirty-one patients who had undergone abdominoplasty procedures were selected for instillation of a local anaesthetic preparation, ropivacaine (Naropin, AstraZeneca) into the wound site on day 1 and 2 after surgery, followed by early mobilisation. Efficacy of the system, patient comfort and mobilisation were documented. RESULTS: The abdominoplasty patients experienced no discomfort from the procedure and claimed effective relief of pain for an average of 12 hours following instillation of local anaesthetic. All mobilised effectively. The device worked well, with no technical problems. CONCLUSION: The lavage drain extension has proved to be a cost-effective and efficient way of providing postoperative pain control and promoting early mobilisation in this patient group.

Lumbar disc replacement for junctional decompensation after fusion surgery: clinical and radiological outcome at an average follow-up of 33 months.

BACKGROUND: Failed fusion surgery remains difficult to treat. Few published data on disc replacement surgery after failed fusion procedures exist. Our objective was to evaluate outcomes of junctional lumbar disc replacement after previous fusion surgery and to correlate outcome with radiological changes to parameters of sagittal balance. METHODS: Out of a single-center prospective registry of 290 patients with 404 lumbar disc replacements, 27 patients had had a previous lumbar fusion operation on 1 to 4 lumbar segments and had completed a mean follow- up of 33 months (range: 18-56). We correlated the clinical outcome measures (patient satisfaction, 10-point pain score, and Oswestry Disability Index [ODI] score) to parameters of spinal sagittal alignment (sacral tilt, pelvic tilt, pelvic incidence, and lumbar lordosis). RESULTS: Postoperative hospital stay averaged 3.3 days (range: 2-8). Previously-employed patients went back to their jobs with a mean of 32 days (range: 21-42) after the procedure. At the latest follow-up, 1 of the patients considered the outcome to be poor, 3 fair, 8 good, and 15 excellent. Twenty-four patients "would undergo the operation again." Average pain score decreased from 9.1 ± 1.0 (SD) to 3.2 ± 2.1 (P < .01). Average ODI decreased from 50.2 ± 9.9 preoperatively to 21.7 ± 14.2 (P ≤ .01). We found the change in pelvic tilt to be an independent predictor of better clinical outcome by multivariate analysis (P < .05). CONCLUSIONS: In patients with junctional failure adjacent to a previous posterolateral fusion, disc replacement at the junctional level(s), compared with osteotomy and fusion surgery, offers the advantage of maintaining segmental mobility and correcting the flat-back deformity through a single approach with less operative time and blood loss. Early- to intermediate-term results are promising. The influence of changes in spinal sagittal alignment on clinical outcome needs to be addressed in future research. CLINICAL RELEVANCE: This is the first study on "junctional disc replacement patients" correlating clinical outcome to changes in spinal/pelvic alignment.