Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector(® )electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/10(6 )cells (range of 107.6–1079.3 pg /10(6 )cells/24 h) compared to mock transfected CIK cells (31 pg/10(6 )cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 ± 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 ± 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line

Towards segmentation and spatial alignment of the human embryonic brain using deep learning for atlas-based registration

We propose an unsupervised deep learning method for atlas based registration to achieve segmentation and spatial alignment of the embryonic brain in a single framework. Our approach consists of two sequential networks with a specifically designed loss function to address the challenges in 3D first trimester ultrasound. The first part learns the affine transformation and the second part learns the voxelwise nonrigid deformation between the target image and the atlas. We trained this network end-to-end and validated it against a ground truth on synthetic datasets designed to resemble the challenges present in 3D first trimester ultrasound. The method was tested on a dataset of human embryonic ultrasound volumes acquired at 9 weeks gestational age, which showed alignment of the brain in some cases and gave insight in open challenges for the proposed method. We conclude that our method is a promising approach towards fully automated spatial alignment and segmentation of embryonic brains in 3D ultrasound

Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC

BACKGROUND: Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet. METHODS: Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells. RESULTS: We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone. CONCLUSION: This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future

Novel non-viral method for transfection of primary leukemia cells and cell lines

BACKGROUND: Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods. METHODS: The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. RESULTS: Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP. CONCLUSION: The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy

Lattice worldline representation of correlators in a background field

We use a discrete worldline representation in order to study the continuum limit of the one-loop expectation value of dimension two and four local operators in a background field. We illustrate this technique in the case of a scalar field coupled to a non-Abelian background gauge field. The first two coefficients of the expansion in powers of the lattice spacing can be expressed as sums over random walks on a d-dimensional cubic lattice. Using combinatorial identities for the distribution of the areas of closed random walks on a lattice, these coefficients can be turned into simple integrals. Our results are valid for an anisotropic lattice, with arbitrary lattice spacings in each direction.Comment: 54 pages, 14 figure

Comparison of knowledge scores of medical students in problem-based learning and traditional curriculum on public health topics

BACKGROUND: The purpose of the study was to compare the knowledge scores of medical students in Problem-based Learning and traditional curriculum on public health topics. METHODS: We planned a cross-sectional study including the fifth and sixth year medical students of Dokuz Eylul University in Turkey. The fifth year students (PBL group, n = 56) were the pioneers educated with PBL curriculum since the 1997–1998 academic year. The sixth year students (traditional education group, n = 78) were the last students educated with traditional education methods. We prepared 25 multiple-choice questions in order to assess knowledge scores of students on selected subjects of Public Health. Our data were collected in year 2002. RESULTS: Mean test scores achieved in PBL and traditional groups were 65.0 and 60.5 respectively. PBL students were significantly more successful in the knowledge test (p = 0.01). The knowledge scores of two topics were statistically higher among PBL students. These topics were health management and chronic diseases. CONCLUSION: We found that mean total evaluation score in the PBL group was 4.5 points higher than in the traditional group in our study. Focusing only on the knowledge scores of students is the main limitation of our study. Upon the graduation of the first PBL students in the 2002–2003 academic year, we are planning additional studies regarding the other functions of a physician such as skill, behaviour and attitude

Distance Optimization and the Extremal Variety of the Grassmann Variety

The approximation of a multivector by a decomposable one is a distance-optimization problem between the multivector and the Grassmann variety of lines in a projective space. When the multivector diverges from the Grassmann variety, then the approximate solution sought is the worst possible. In this paper, it is shown that the worst solution of this problem is achieved, when the eigenvalues of the matrix representation of a related two-vector are all equal. Then, all these pathological points form a projective variety. We derive the equation describing this projective variety, as well as its maximum distance from the corresponding Grassmann variety. Several geometric and algebraic properties of this extremal variety are examined, providing a new aspect for the Grassmann varieties and the respective projective spaces

The intestinal expulsion of the roundworm Ascaris suum is associated with eosinophils, intra-epithelial T cells and decreased intestinal transit time

Ascaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections