Real-time biomechanical modelling of the liver using LightGBM model.

BACKGROUND: Accurate and real-time biomechanical modelling of the liver is a major challenge in computer-assisted surgery. Finite element method is often used to predict the deformation of organs for its high modelling accuracy. However, its high computation cost hinders its application in real time, such as virtual surgery simulations. METHOD: A liver model with biomechanical properties similar to real one is created using finite element method and a data set of the liver deformation with different forces (whose magnitude ranges from 0.1 to 0.5 N in omni-direction) acting on different surface points is generated. The mechanical behaviour of liver is simulated in real time by a tree-based LightGBM regression model trained with the generated data set. RESULTS: In comparison with the Random Forest and XGBoost, the LightGBM model achieves the best accuracy with 0.0774 mm, 0.0786 mm, 0.0801 mm in the mean absolute error (MAE) and 0.0591 mm, 0.0609 and 0.0622 mm in the root mean square error (RMSE) along x, y and z axis, respectively. In addition, it only takes 33 ms for the LightGBM model to estimate the deformation of the liver, which is much faster than finite element model (29.91 s). CONCLUSION: These results lay a foundation for the future development of real-time virtual surgery systems of simulating liver deformation during minimally invasive surgeries using our method.

Target selection of CAR T cell therapy in accordance with the TME for solid tumors.

Chimeric antigen receptor-engineered T (CAR T) cell therapy has made great progress in hematological malignancies and resulted in two newly FDA-approved drugs specific for CD19, Kymriah and Yescarta. To some extent, this success is attributable to the appropriately selected antigen, CD19, a cell surface protein that is uniformly and strongly expressed on malignant B cells. This result indicates that a proper CAR target is of great importance to the success of this technique. Another key factor contributing to the success of hematological malignancies can be ascribed to the nonphysical tumor microenvironment (TME). The TME in solid tumors is complicated and has a specific niche favorable for tumor progression with physical barriers, multiple mechanisms of immunosuppression, and a variety of biochemical factors, thus resulting in limited efficacy of CAR T cell therapy in clinical trials with cancer patients. Therefore, the inhospitable solid TME becomes a major hurdle in translating the success of CAR T cell therapy in hematological malignancies to solid tumors. Here, we provide our perspective on how to improve the success of CAR T therapy in solid tumors by focusing on the aspects of target selection and the related TME in CAR T cell design, especially stressing the interplay between them. With four kinds of antigenic CAR targets as examples in this review, we anticipate that the overall consideration of both factors will further expand CAR T cell therapy in clinical trials.

Critical factors in chimeric antigen receptor-modified T-cell (CAR-T) therapy for solid tumors.

The success of chimeric antigen receptor-modified T-cell (CAR-T) therapy for B-cell lymphocyte malignancies targeting CD19 places it in a rapidly growing field in cancer immunotherapy for both hematological and solid tumors. However, the two types of tumor are quite different in the following respects. Solid tumors are characterized by complex vasculatures and matrix barriers that significantly affect T-cell functions and migration. Moreover, various immunosuppressive molecules expressed in the tumor microenvironment can impede T-cell activation, and the high metabolic rate of tumors competitively suppresses the metabolism of immune cells. All these factors will exert their influences on the development of a cancer, which is a dynamic balance between the host's immune system and the tumor. At present, solid tumors are treated primarily by surgical resection combined with radiotherapy and chemotherapy, a treatment process that is painful and not always effective. With advantages over traditional treatments, the recently developed CAR-T immunotherapy has been applied and has shown highly promising results. Nevertheless, the complexity of solid tumors presents a great challenge to this technique. This review focuses on elucidating the factors influencing the anti-tumor effects of CAR-T in the specific tumor environment, and hence exploring feasible approaches to overcome them.

Codon optimization significantly enhanced the expression of human 37-kDa iLRP in Escherichia coli.

37-kDa immature laminin receptor protein (iLRP), the precursor of 67-kDa laminin receptor protein (LRP), is overexpressed on the surface of most cancer cells and recognized as a universal tumor antigen. The role makes it a potential target for cancer immunotherapy, which has been well-studied. Our study aimed to produce high quality of human iLRP in bacteria so that the needs in research of its clinical application could be met. The powerful system for heterologous protein expression, pET system was used. Two types of DNA sequences encoding the same amino acid sequences were separately cloned into the vector pET30a(+). One of the resulting vectors includes the wild-type iLRP, and other one includes the codon-optimized iLRP. The expression by both genes was then compared in Escherichia coli BL21(DE3). Our results revealed that the performance of codon optimization was crucial for the expression of human iLRP in Escherichia coli. The yield was significantly enhanced up to 300 mg/L of bacterial culture by this approach.

Virtual-screening targeting Human Immunodeficiency Virus type 1 integrase-lens epithelium-derived growth factor/p75 interaction for drug development.

Three integrase (IN) inhibitors have been approved by FDA for clinical treatment of Human Immunodeficiency Virus (HIV) infection. This stimulates more researchers to focus their studies on this target for anti-HIV drug development. Three steps regarding of IN activity have been validated for inhibitor discovery: strand transfer, 3'-terminal processing, and IN-lens epithelium-derived growth factor (LEDGF)/p75 interaction. Among them, IN-LEDGF/p75 interaction is a new target validated in recent years. Emergence of drug-resistant virus strains makes this target appealing to pharmacologists. Compared with the traditional screening methods such as AlphaScreen and cell-based screening developed for IN inhibitor discovery, virtual screening is a powerful technique in modern drug discovery. Here we summarized the recent advances of virtual-screening targeting IN-LEDFG/p75 interaction. The combined application of virtual screening and experiments in drug discovery against IN-LEDFG/p75 interaction sheds light on anti-HIV research and drug discovery.

Transcriptional regulation of α1H T-type calcium channel under hypoxia.

The low-voltage-activated T-type Ca(2+) channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5'-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site -1,173cacgc-1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region.

Immunotherapy treatments of warm autoimmune hemolytic anemia.

Warm autoimmune hemolytic anemia (WAIHA) is one of four clinical types of autoimmune hemolytic anemia (AIHA), with the characteristics of autoantibodies maximally active at body temperature. It produces a variable anemia-sometimes mild and sometimes severe. With respect to the absence or presence of an underlying condition, WAIHA is either idiopathic (primary) or secondary, which determines the treatment strategies in practice. Conventional treatments include immune suppression with corticosteroids and, in some cases, splenectomy. In recent years, the number of clinical studies with monoclonal antibodies and immunosuppressants in the treatment of WAIHA increased as the knowledge of autoimmunity mechanisms extended. This thread of developing new tools of treating WAIHA is well exemplified with the success in using anti-CD20 monoclonal antibody, Rituximab. Following this success, other treatment methods based on the immune mechanisms of WAIHA have emerged. We reviewed these newly developed immunotherapy treatments here in order to provide the clinicians with more options in selecting the best therapy for patients with WAIHA, hoping to stimulate researchers to find more novel immunotherapy strategies.

Requirement of TPO/c-mpl for IL-17A-induced granulopoiesis and megakaryopoiesis.

IL-17A is a critical, proinflammatory cytokine essential to host defense and is induced in response to microbial invasion. It stimulates granulopoiesis, leading to neutrophilia, neutrophil activation, and mobilization. TPO synergizes with other cytokines in stimulating and expanding hematopoietic progenitors, also leading to granulopoiesis and megakaryopoiesis, and is required for thrombocytopoiesis. We investigated the effects of in vivo expression of IL-17A on granulopoiesis and megakaryopoiesis in TPO receptor c-mpl-/- mice. IL-17A expression expanded megakaryocytes by 2.5-fold in normal mice but had no such effect in c-mpl-/- mice. The megakaryocyte expansion did not result in increased peripheral platelet counts. IL-17A expression did not impact bone marrow precursors in c-mpl-/- mice; however, it expanded splenic precursors, although to a lesser extent compared with normal controls (CFU-HPP). No peripheral neutrophil expansion was observed in c-mpl-/- mice. Moreover, in c-mpl-/- mice, release of IL-17A downstream cytokines was reduced significantly (KC, MIP-2, GM-CSF). The data suggest that IL-17A requires the presence of functional TPO/c-mpl to exert its effects on granulopoiesis and megakaryopoiesis. Furthermore, IL-17A and its downstream cytokines are important regulators and synergistic factors for the physiologic function of TPO/c-mpl on hematopoiesis.

IL-17 is a potent synergistic factor with GM-CSF in mice in stimulating myelopoiesis, dendritic cell expansion, proliferation, and functional enhancement.

OBJECTIVE: Interleukin (IL)-17, which now defines the Th(17) immune response, is a critical cytokine expressed and required for stress granulopoiesis during microbial invasion. Dendritic cells (DC) can instigate this response by inducing IL-17 expression in CD4(+) T cells. Besides IL-17, microbial invasion also stimulates production of the DC growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The objective was the in vitro and in vivo investigation of IL-17 on DC proliferation and function in mice. MATERIALS AND METHODS: Murine IL-17 (mIL-7) or murine GM-CSF (mGM-CSF), or both, was expressed in C57BL6 mice using adenoviral technology to assess hematopoietic and DC changes. The E-22 tymoma tumor cell line using a previously described vaccinia virus ovalbumin/LacZ murine tumor model was employed to study effects on tumor rejection. RESULTS: The combination of mIL-17 and mGM-CSF increased peripheral neutrophila by 28-fold and splenic colonies by 11- and 14-fold over each individual factor in mice, respectively. The effect of mIL-17 by itself on murine DCs in vitro and in vivo was minimal; however, the combination greatly enhanced the stimulating effects of mGM-CSF, increasing the total numbers of CD14b/c(+) spleen DC by fourfold, as well as their function measured by enhanced endocytosis. Mixed lymphocyte reactions using mIL-17/mGM-CSF cultured DCs stimulator cells enhanced lymphocyte responses by twofold over mGM-CSF alone. Vaccination against LacZ in the C57BL6 E22 syngenic thymoma tumor model effectively delayed tumor growth in animals pretreated with the mIL-17/mGM-CSF combination prior to vaccination. CONCLUSIONS: mIL-17 effectively synergizes with mGM-CSF in stimulating granulopoiesis and DC expansion, as well as in functional enhancement of DCs.

Production, safety and antitumor efficacy of recombinant Oncofetal Antigen/immature laminin receptor protein.

We describe here for the first time an efficient high yield production method for clinical grade recombinant human Oncofetal Antigen/immature laminin receptor protein (OFA/iLRP). We also demonstrate significant antitumor activity for this protein when administered in liposomal delivery form in a murine model of syngeneic fibrosarcoma. OFA/iLRP is a therapeutically very promising universal tumor antigen that is expressed in all mammalian solid tumors tested so far. We have cloned the human OFA/iLRP cDNA in a bacterial expression plasmid which incorporates a 6x HIS-tag. Large scale cultures of the plasmid transformed Escherichia coli were performed and the crude HIS-tagged OFA/iLRP was isolated as inclusion bodies and solubilized in guanidine chloride. The protein was then purified by successive passage through three column chromatography steps of immobilized metal affinity, anion exchange, and gel filtration. The resulting protein was 94% pure and practically devoid of endotoxin and host cell protein. The purified OFA/iLRP was tested in mice for safety and efficacy in tumor rejection with satisfactory results. This protein will be used for loading onto autologous dendritic cells in an FDA approved phase I/II human cancer vaccine trial in OFA/iLRP-positive breast cancer patients.

IL-17F/IL-17R interaction stimulates granulopoiesis in mice.

OBJECTIVE: IL-17F, a member of the interleukin (IL)-17 cytokine family, most closely resembles IL-17A structurally. IL-17A is a potent stimulator of granulopoiesis; its expression is induced in response to microbial challenge. Although IL-17F is considered to be a weak IL-17A analog that is also mediating its effect via IL-17R, its exact role and in vivo functions are unknown. Our goal was to determine the in vivo activity of IL-17F on granulopoiesis as well as on release of granulopoiesis-stimulating downstream cytokines in mice and directly compare its effect to IL-17A. MATERIALS AND METHODS: Murine IL-17A (mIL-17A) or IL-17F (mIL-17F) was expressed in vivo in C57BL6 mice using adenoviral gene transfer technology. Peripheral cell counts were assessed as well as hematopoietic precursors using colony-forming assays at set time points. Downstream cytokines were measured using enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction. RESULTS: We found mIL-17F to have similar expression kinetics as mIL-17A in splenocytes in vitro and in vivo, following challenge with microbial agents. Overexpression of mIL-17F in vivo resulted in similar neutrophilia and only in slightly reduced myeloid progenitor expansion when compared to mIL-17A. In vivo, there was no difference in releases for granulocyte-macrophage colony-stimulating factor; regulated on activation, normal T expressed and secreted; interferon-inducible protein-10; IL-6; and monocyte chemotactic protein-1 between either cytokine. IL-1A, macrophage inflammatory protein -2 (MIP), KC, and granulocyte colony-stimulating factor expression was approximately half of that seen with mIL-17A. CONCLUSION: Both IL-17A and IL-17F are induced by similar stimuli, have similar expression kinetics and despite only minimal in vitro activity for IL-17F, surprisingly they exert similar in vivo bioactivity. IL-17F bioactivity appears to be augmented in vivo through mechanisms that require further investigation.

The expression of functional human parathyroid hormone in a gene therapy model for osteoporosis.

The pro-peptide sequence of human parathyroid hormone (hPTH) is essential for the efficient translocation of the nascent polypeptide and the precise cleavage at residue +1 from the cleavage site. If residue +1 is not a serine, the bioactivity of hPTH decreases dramatically. In order to express the functional hPTH properly in a gene therapy model, we constructed three sets of eukaryotic expression vectors: one combining hPTH cDNA with its pre-sequence or its prepro-sequence; one combining hPTH cDNA with the insulin pre-sequence or with the insulin pre-sequence and the hPTH pro-sequence; one combining pro-insulin cDNA with the hPTH pre-sequence or prepro-sequence. Radioimmunoassay and reverse transcription/polymerase chain reaction showed that transgenes were expressed in the animal model. The hPTH expressed by the vectors with the pro-sequence of PTH improved bone mechanical properties and bone mineral content, whereas the expressed insulin decreased blood glucose. Our results demonstrated that the pro-sequence of hPTH was required for skeletal muscle cells to secrete functional hPTH into the circulation of our gene therapy model. However, the function of expressed insulin was not affected by the fusion of the hPTH pro-sequence. We concluded that pre-sequence, pro-sequence and mature hPTH acted as an integrated unit in skeletal muscle to guarantee the proper processing and release of functional hPTH into the circulation.

Prevention of osteoporosis in ovariectomized mice by electroporational gene transfer of parathyroid hormone.

Non-viral vector, pCMV-Pth, with full-length human parathyroid hormone (prepro-hPTH) cDNA was constructed and delivered into the ovariectomized mouse quadriceps to explore the effects of electroporational gene transfer of PTH on the bone. The expression of hPTH in the transfected mice was detected by RT-PCR and radioimmunoassay. Mechanical testing and the analysis of bone mineral content demonstrated the improvement of bone properties. These results suggest that the electroporational gene taransfer of parathyroid hormone might be a promising method to prevent the bone loss in the postmenopausal women.