Role of tumor architecture in elicitation of effector functions of human cytotoxic T-lymphocytes recognizing melanoma associated antigens

Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. 3D architectures might also play a role in impairing immunorecognition of cancer cells by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens. Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA-) melanoma cell lines on poly-Hydroxyethylmethacrylate-coated plates, resulted in generation of 3D multicellular tumor spheroids (MCTS). Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10-15 days) MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. To obtain an insight into the role played by tumor architecture in the elicitation of specific gene expression patterns, we addressed gene expression profiles of NA8 melanoma cells cultured in two-dimensional monolayers (2D) or in 3D (MCTS). Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with more than threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, more than threefold, upregulation of the expression of >100 transcripts, while 73 transcripts were more than threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-α, -β, and γ), IL-8, CCL20 (MIP-3α), and Angiopoietin-like 4 were significantly upregulated, whereas basic-FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process. Then we investigated the effects of 3D culture on the recognition of melanoma cells by antigenspecific HLA class I-restricted Cytotoxic T-Lymphocytes (CTL). IFN-γ production can be used as a surrogate marker for tumor cell immunorecognition. Co-culture of melanoma spheroids with HLA-A0201 restricted Melan-A/MART-127-35-specific CTL clones resulted in significantly defective TAA recognition by CTL as compared to 2D, as witnessed by decreased IFN-γ production and decreased Fas Ligand, perforin and granzyme B gene expression. Indeed, Melan- A/MART-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D tumor cell cultures. Concomitantly, a parallel decrease of HLA class I molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures. We further identified a multiplicity of mechanisms potentially involved. In particular : 1) MCTS per se limit CTL capacity of recognizing HLA class I restricted antigens by reducing exposed cell surfaces. 2) Expression of melanoma differentiation antigens is down-regulated in tumor cell spheroids as compared to 2D unrelated to hypoxia or increased Oncostatin M gene expression but rather to decreased MITF gene expression. 3) Expression of HLA class I molecules is frequently down-regulated in melanoma MCTS, as compared to 2D, possibly due to decreased IRF-1 gene expression. 4) Lactate production by melanoma cells is increased in MCTS, as compared to 2D and lactate significantly inhibits TAA triggered IFN-γ production by CTL. Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL and a constellation of mechanisms are involved in causing this impairment of immunorecognition

Where do free-ranging dogs rest? A population level study reveals hidden patterns in resting site choice

Free-ranging dogs (FRDs) in human-dominated areas encounter obstacles such as noise, pollution, limited food sources, and anthropogenic disturbance while resting. Since FRDs have survived as a population in India, as in many other parts of the Global South for centuries, they provide a unique opportunity to study adaptation of animals to the human-dominated urban landscape. We documented factors impacting resting behaviour and site preferences in three states of India, for 284 dogs, leading to 6047 observations over 3 years. 7 physical parameters of the resting sites, along with the biological factors like mating and pup-rearing and time of day affected their choice of resting sites. The frequency-rank distribution of the unique combinations in which the parameters were selected followed a Power law distribution, which suggests underlying biological reasons for the observed preferences. Further, 3 of these parameters showed maximum consistency of choice in terms of the sub-parameters selected, explaining 30% of the observations. FRDs prefer to rest close to their resource sites within the territory, at a place that enabled maximum visibility of the surroundings. They chose such sites in the core of the territory for sleeping. At other times, they chose such sites away from the core, and were less restive, thus allowing for immediate response in case of intrusion or threat. They generally avoided anthropogenic disturbance for sleeping, and preferred areas with shade.Incorporating these aspects into urban management plans can promote human-dog cooperation and reduce situations of conflict. We envisage more inclusive urban areas in the future, that can allow for co-existence of the humans and their oldest companions in the commensal relationship that has been maintained for hundreds of generations of dogs in this part of the world.Comment: 2 figures, 2 tables, ES

Bayesian Inference in Nonparametric Dynamic State-Space Models

We introduce state-space models where the functionals of the observational and the evolutionary equations are unknown, and treated as random functions evolving with time. Thus, our model is nonparametric and generalizes the traditional parametric state-space models. This random function approach also frees us from the restrictive assumption that the functional forms, although time-dependent, are of fixed forms. The traditional approach of assuming known, parametric functional forms is questionable, particularly in state-space models, since the validation of the assumptions require data on both the observed time series and the latent states; however, data on the latter are not available in state-space models. We specify Gaussian processes as priors of the random functions and exploit the "look-up table approach" of \ctn{Bhattacharya07} to efficiently handle the dynamic structure of the model. We consider both univariate and multivariate situations, using the Markov chain Monte Carlo (MCMC) approach for studying the posterior distributions of interest. In the case of challenging multivariate situations we demonstrate that the newly developed Transformation-based MCMC (TMCMC) of \ctn{Dutta11} provides interesting and efficient alternatives to the usual proposal distributions. We illustrate our methods with a challenging multivariate simulated data set, where the true observational and the evolutionary equations are highly non-linear, and treated as unknown. The results we obtain are quite encouraging. Moreover, using our Gaussian process approach we analysed a real data set, which has also been analysed by \ctn{Shumway82} and \ctn{Carlin92} using the linearity assumption. Our analyses show that towards the end of the time series, the linearity assumption of the previous authors breaks down.Comment: This version contains much greater clarification of the look-up table idea and a theorem regarding this is also proven and included in the supplement. Will appear in Statistical Methodolog

PLGA Nanoparticles for Peptide Receptor Radionuclide Therapy of Neuroendocrine Tumors: A Novel Approach towards Reduction of Renal Radiation Dose

BACKGROUND:Peptide receptor radionuclide therapy (PRRT), employed for treatment of neuroendocrine tumors (NETs) is based on over-expression of Somatostatin Receptors (SSTRs) on NETs. It is, however, limited by high uptake and retention of radiolabeled peptide in kidneys resulting in unnecessary radiation exposure thus causing nephrotoxicity. Employing a nanocarrier to deliver PRRT drugs specifically to the tumor can reduce the associated nephrotoxicity. Based on this, (177)Lu-DOTATATE loaded PLGA nanoparticles (NPs) were formulated in the present study, as a potential therapeutic model for NETs. METHODOLOGY AND FINDINGS:DOTATATE was labeled with Lutetium-177 ((177)Lu) (labeling efficiency 98%; R(f)∼0.8). Polyethylene Glycol (PEG) coated (177)Lu-DOTATATE-PLGA NPs (50:50 and 75:25) formulated, were spherical with mean size of 304.5±80.8 and 733.4±101.3 nm (uncoated) and 303.8±67.2 and 494.3±71.8 nm (coated) for PLGA(50:50) and PLGA(75:25) respectively. Encapsulation efficiency (EE) and In-vitro release kinetics for uncoated and coated NPs of PLGA (50:50 & 75:25) were assessed and compared. Mean EE was 77.375±4.98% & 67.885±5.12% (uncoated) and 65.385±5.67% & 58.495±5.35% (coated). NPs showed initial burst release between 16.64-21.65% with total 42.83-44.79% over 21 days. The release increased with coating to 20.4-23.95% initially and 60.97-69.12% over 21 days. In-vivo studies were done in rats injected with (177)Lu-DOTATATE and (177)Lu-DOTATATE-NP (uncoated and PEG-coated) by imaging and organ counting after sacrificing rats at different time points over 24 hr post-injection. With (177)Lu-DOTATATE, renal uptake of 37.89±10.2%ID/g was observed, which reduced to 4.6±1.97% and 5.27±1.66%ID/g with uncoated and coated (177)Lu-DOTATATE-NP. The high liver uptake with uncoated (177)Lu-DOTATATE-NP (13.68±3.08% ID/g), reduced to 7.20±2.04%ID/g (p = 0.02) with PEG coating. CONCLUSION:PLGA NPs were easily formulated and modified for desired release properties. PLGA 50:50 NPs were a more suitable delivery vehicle for (177)Lu-DOTATATE than PLGA 75:25 because of higher EE and slower release rate. Reduced renal retention of (177)Lu-DOTATATE and reduced opsonisation strongly advocate the potential of (177)Lu-DOTATATE-PLGA-PEG NPs to reduce radiation dose in PRRT

Study of the functional outcome of medial compartment osteoarthritis of the knee treated with proximal fibular osteotomy

Background: Knee osteoarthritis (OA) is a common painful and chronic condition that affects a large proportion of the population particularly older individuals. Increasing age and obesity are important predisposing factors for the development of knee OA. While in most of the cases, conservative approach is utilized, in patients not responding to conservative management surgical intervention is required. Proximal fibular osteotomy (PFO) is one of the important surgical procedures which is being increasingly used for managing knee OA not responding to conservative treatment. Aims and Objectives: The objectives of the study are as follows: To study the role of PFO in medial compartment OA of knee. To study the clinical and functional outcomes of OA of knee treated with PFO with respect to pain, disability, and range of movements. Materials and Methods: This was a prospective study conducted in the orthopedic division at Bharati Vidyapeeth Deemed College and Hospital, Sangli. Thirty patients with medial compartment knee OA and treated by PFO were included in this study on the basis of pre-defined inclusion and exclusion criteria. Body mass index (BMI) of all patients was determined. Pre-operative and post-operative visual analog scale index was analyzed to asses pain relief. Functional outcome was assessed by the Japanese Orthopaedic Association (JOA) score. P<0.05 was taken as statistically significant. Results: Out of 30 studied cases, there were 18 (60%) females with a M: F ratio of 1:1.5. Fourteen (46.7%) of the participants had right side affected whereas 16 (53.3%) of the participants had left knee OA. The mean age of affected cases was found to be 71.27±9.92 years. The median (IQR) of age (years) was 72.00 (64–80) and the age ranged from 52 to 90 years. Nine patients (30%) were obese (BMI ≥30) and 13 (43.33%) patients were overweight (BMI ≥25 but <30). There was a significant reduction in pain at the time of final follow-up (P<0.0001). Analysis of functional assessment showed that mean JOA scores at the time of final follow-up had significantly improved as compared to JOA scores at the time of presentation. Complications were seen in 4 (13.33%) patients. Conclusion: Proximal femoral osteotomy is effective in relieving pain and improving joint function in patients with medial compartment OA. It is an easy, safe, and cost-effective procedure with minimal complications