Safety and immunological effects of recombinant canine IL-15 in dogs.

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays pivotal roles in innate and adaptive immunity. It is also a promising cytokine for treating cancer. Despite growing interest in its use as an immunotherapeutic, its safety and immunological effects in dogs have not been reported. In this study, healthy dogs were given recombinant canine IL-15 (rcIL-15) intravenously at a daily dose of 20 µg/kg for 8 days and monitored for 32 days to determine the safety and immunological effects of rcIL-15. The repeated administration of rcIL-15 was well tolerated, did not cause any serious side effects, and promoted the selective proliferation and activation of canine anti-cancer effector cells, including CD3+CD8+ cytotoxic T lymphocytes, CD3+CD5dimCD21-, and non-B/non-T NK cell populations, without stimulating Treg lymphocytes. The rcIL-15 injections also stimulated the expression of molecules and transcription factors associated with the activation and effector functions of NK cells, including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, granzyme B, Ly49, T-bet, and Eomes. These results suggest that rcIL-15 might be a valuable therapeutic adjuvant to improve immunity against cancer in dogs.

Canine non-B, non-T NK lymphocytes have a potential antibody-dependent cellular cytotoxicity function against antibody-coated tumor cells.

BACKGROUND: The antibody-dependent cellular cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector immune cells actively lyse antibody-coated target cells. The ADCC of tumor cells is employed in the treatment of various cancers overexpressing unique antigens, and only natural killer (NK) cells are known to be major effectors of antibody mediated ADCC activity. Canine NK cells are still defined as non-B, non-T large granular lymphocytes because of the lack of information regarding the NK cell-restricted specific marker in dogs, and it has never been demonstrated that canine NK cells have ADCC ability against tumor cells. In the present study, we investigated whether canine non-B, non-T NK cells have ADCC ability against target antibody-coated tumor cells, using cetuximab and trastuzumab, the only human antibodies reported binding to canine cancer cells. RESULTS: Activated canine non-B, non-T NK cells (CD3-CD21-CD5-TCRαß-TCRγδ-) for 13~17 days ex vivo showed ADCC ability against trastuzumab- or cetuximab-coated target tumor cells expressing various levels of human epidermal growth factor receptor 2 (HER-2) and epidermal growth factor receptor (EGFR). Trastuzumab and cetuximab induced significant ADCC responses of canine NK cells even in CMT-U334 and CF41.Mg cells expressing low levels of HER-2 and/or EGFR, as well as in SKBR3 and DU145 cells overexpressing HER-2 and/or EGFR. The trastuzumab-mediated ADCC activity of NK cells was significantly enhanced by treatment with rcIL-21. CONCLUSIONS: The results of this study suggest that canine non-B, non-T NK lymphocytes have a potential ADCC function and that combinational strategies of monoclonal antibodies with either cytokines, which activate NK cells in vivo, or adoptive transfer of NK cells may be a feasible method for amplifying the efficacy of immunotherapy against malignant cancers even with very low expression of target molecules in dogs.

Effect of irradiation-induced intercellular adhesion molecule-1 expression on natural killer cell-mediated cytotoxicity toward human cancer cells.

BACKGROUND AIMS: Irradiation enhances the adhesion between natural killer (NK) cells and target cells by up-regulating intercellular adhesion molecule-1 (ICAM-1) on target cells. Therefore, we investigated the effect of irradiation-induced ICAM-1 expression on human cancer cells on NK cell-mediated cytotoxicity. METHODS: Expression levels of ICAM-1 on the target cell surface before and after irradiation of six human cancer cell lines (HL60, SKBR-3, T47D, HCT-116, U937 and U251) were analyzed by flow cytometry. Ex vivo expansion of NK cells from human peripheral blood mononuclear cells was performed by co-culture with irradiated K562 cells. The related adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) on NK cells was analyzed by flow cytometry. An enzyme-linked immunosorbent assay was used to detect interferon-γ (IFN-γ), and WST-8 assays were performed to check NK cell cytotoxicity. Finally, blocking assays were performed using monoclonal antibodies against ICAM-1 or LFA-1. RESULTS: LFA-1 expression increased on NK cells after expansion (P <0.001). The expression of ICAM-1 was significantly upregulated by irradiation after 24 h in various cell lines, including HL60 (P <0.001), SKBR-3 (P <0.001), T47D (P <0.001) and U937 (P <0.001), although the level of expression depended on the cell line. ICAM-1 expression was extremely low before and after irradiation in U251 cells. NK cell-mediated cytotoxicity increased after irradiation of HL60 (P <0.001), SKBR-3 (P <0.001), T47D (P = 0.003), and U937 (P = 0.004) cells, in which ICAM-1 expression was significantly increased after irradiation. IFN-γ production by NK cells in response to HL60 (P <0.001) and T47D (P = 0.011) cells significantly increased after irradiation. NK cell-mediated cytotoxicity against irradiated SKBR-3 (P <0.001) and irradiated T47D cells (P = 0.035) significantly decreased after blocking of ICAM-1. Blocking of LFA-1 on NK cells resulted in reduced cytotoxicity against irradiated HL60 (P <0.001) and irradiated SKBR-3 (P <0.001). CONCLUSIONS: Irradiation upregulates ICAM-1 expression on the surface of human cancer cells and enhances activated NK cell-mediated cytotoxicity. Therefore, irradiation combined with NK cell therapy may improve the antitumor effects of NK cells.

The Development of an IMU Integrated Clothes for Postural Monitoring Using Conductive Yarn and Interconnecting Technology.

Spinal disease is a common yet important condition that occurs because of inappropriate posture. Prevention could be achieved by continuous posture monitoring, but most measurement systems cannot be used in daily life due to factors such as burdensome wires and large sensing modules. To improve upon these weaknesses, we developed comfortable "smart wear" for posture measurement using conductive yarn for circuit patterning and a flexible printed circuit board (FPCB) for interconnections. The conductive yarn was made by twisting polyester yarn and metal filaments, and the resistance per unit length was about 0.05 Ω/cm. An embroidered circuit was made using the conductive yarn, which showed increased yield strength and uniform electrical resistance per unit length. Circuit networks of sensors and FPCBs for interconnection were integrated into clothes using a computer numerical control (CNC) embroidery process. The system was calibrated and verified by comparing the values measured by the smart wear with those measured by a motion capture camera system. Six subjects performed fixed movements and free computer work, and, with this system, we were able to measure the anterior/posterior direction tilt angle with an error of less than 4°. The smart wear does not have excessive wires, and its structure will be optimized for better posture estimation in a later study.

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor.

BACKGROUND AIMS: Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells. METHODS: The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays. RESULTS: The activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R2 = 0.91; P <0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines. CONCLUSIONS: Our data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer.

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture.

BACKGROUND AIMS: Interleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture. METHODS: IL-2/IL-15-activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry. RESULTS: Compared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the "first week" group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the "first week" group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of "first week" NK cells that received IL-21 treatment for an additional 2 days compared with the "first week" NK cells (P < 0.05). CONCLUSIONS: These data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.

The p.R168Q mutation is associated with the Bw phenotype and a predicted decrease in the stability of the resulting ABO glycosyltransferase.

BACKGROUND: Mutation of ABO glycosyltransferase (GT) can cause protein stability changes that can result in a weak ABO phenotype. To explain the Bw phenotype of a novel ABO*Bw allele, a protein stability of the mutant GT, which enhances the information of the three-dimensional (3D) structural analysis, was calculated. STUDY DESIGN AND METHODS: ABO serology and genotyping were performed on a neonate and her five family members. A 3D structural analysis of the wild-type GTB and enzymes with a variety of mutations at Residue 168, along with predicted protein stability changes (ΔΔG) and flow cytometric analysis of ABO antigen expression on HeLa cells transfected with plasmids containing R168Q, R168L, and R168P mutants was also performed. RESULTS: A novel ABO*Bw allele (c.503G>A, p.R168Q) was discovered. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTB, and the ΔΔG values, which inversely correlated with the mean relative fluorescence intensity of ABO antigen expression, quantitatively explained the reduced ABO antigen expression. CONCLUSIONS: The predicted protein stability change of a mutant GT enzyme might be a useful and convenient approach to objectively and quantitatively explain the reduced ABO antigen expression.

The postnatal injection of AAV9-FOXG1 rescues corpus callosum agenesis and other brain deficits in the mouse model of FOXG1 syndrome.

Heterozygous mutations in the FOXG1 gene manifest as FOXG1 syndrome, a severe neurodevelopmental disorder characterized by structural brain anomalies, including agenesis of the corpus callosum, hippocampal reduction, and myelination delays. Despite the well-defined genetic basis of FOXG1 syndrome, therapeutic interventions targeting the underlying cause of the disorder are nonexistent. In this study, we explore the therapeutic potential of adeno-associated virus 9 (AAV9)-mediated delivery of the FOXG1 gene. Remarkably, intracerebroventricular injection of AAV9-FOXG1 to Foxg1 heterozygous mouse model at the postnatal stage rescues a wide range of brain pathologies. This includes the amelioration of corpus callosum deficiencies, the restoration of dentate gyrus morphology in the hippocampus, the normalization of oligodendrocyte lineage cell numbers, and the rectification of myelination anomalies. Our findings highlight the efficacy of AAV9-based gene therapy as a viable treatment strategy for FOXG1 syndrome and potentially other neurodevelopmental disorders with similar brain malformations, asserting its therapeutic relevance in postnatal stages.

The patient-specific mouse model with Foxg1 frameshift mutation uncovers the pathophysiology of FOXG1 syndrome.

Single allelic mutations in the gene encoding the forebrain-specific transcription factor FOXG1 lead to FOXG1 syndrome (FS). Patient-specific animal models are needed to understand the etiology of FS, as FS patients show a wide spectrum of symptoms correlated with location and mutation type in the FOXG1 gene. Here we report the first patient-specific FS mouse model, Q84Pfs heterozygous (Q84Pfs-Het) mice, mimicking one of the most predominant single nucleotide variants in FS. Intriguingly, we found that Q84Pfs-Het mice faithfully recapitulate human FS phenotypes at the cellular, brain structural, and behavioral levels. Importantly, Q84Pfs-Het mice exhibited myelination deficits like FS patients. Further, our transcriptome analysis of Q84Pfs-Het cortex revealed a new role for FOXG1 in synapse and oligodendrocyte development. The dysregulated genes in Q84Pfs-Het brains also predicted motor dysfunction and autism-like phenotypes. Correspondingly, Q84Pfs-Het mice showed movement deficits, repetitive behaviors, increased anxiety, and prolonged behavior arrest. Together, our study revealed the crucial postnatal role of FOXG1 in neuronal maturation and myelination and elucidated the essential pathophysiology mechanisms of FS.

Safety analysis of ex vivo-expanded canine natural killer cells in a xenogeneic mouse model of graft-versus-host disease.

Canine natural killer (NK) cells are large, granular lymphocytes that are neither B lymphocytes nor T lymphocytes. However, it has been reported that canine NK cells share some of the phenotypic characteristics of T lymphocytes, such as CD3 and CD5. Studies are needed to assess the safety of canine NK cells for immunotherapy, especially because the safety of using allogeneic NK cells as an immunotherapy for dogs has yet to be shown. In this study, the safety of cultured canine NK cells was assessed using a xenogeneic mouse model of graft-versus-host disease (GVHD). Mice were injected with either canine peripheral blood mononuclear cells (PBMCs) or cultured NK cells for 2 or 3 weeks. Data were then collected on changes in mice body weights, disease severity scores, and survival rates. Histopathological and immunohistochemical evaluations were also performed. All mice injected with canine PBMCs died within 45 days after injection. Severe clinical signs were caused by GVHD. The histopathological and immunohistochemical evaluations showed that mice injected with canine PBMCs had multiple lesions, including necrosis in their lungs, livers, kidneys, and stomachs, and the injected cells were present around the lesions. By contrast, no mice injected with cultured NK cells without removing the CD3+ TCR- cells exhibited any clinical abnormalities. Moreover, they all survived the 90-day experimental period without exhibiting any histopathological changes. Accordingly, the results of this study suggest that canine NK cells do not cause significant side effects such as GVHD and allogeneic NK cells can safely be used for cancer immunotherapy in dogs.

An Air Recirculation System Based on Bioinspired Soft Re-Air Valve for Highly Efficient Pneumatic Actuation.

Owing to their compliance, lightweight, and high force density characteristics, pneumatic actuation systems have been widely implemented in various soft robots. However, pneumatic actuation systems exhibit low efficiency, poor control performance, and high noise; these make it extremely challenging to widely employ a pneumatic actuation system in mobile robots. To overcome these limitations, many researches were conducted on recycling the compressed air within such systems. However, the proposed approaches do not consider the system efficiency and exhaust performance of pneumatic systems. Therefore, this article proposes a recirculation system using a novel soft re-air valve based on the cardiac structure of fish. In particular, the proposed recirculation system recycles the compressed air to improve the system efficiency and pressurizing performance, and the soft re-air valve simultaneously prevents a decrease in the depressurizing performance. For the validation of the proposed scheme, experiments were conducted to evaluate the system efficiency, control performance, and exhaust noise. In contrast to conventional pneumatic systems, the experimental results revealed that the proposed system increased the overall system efficiency by 47.58%, reduced the position root mean square error by 8.16%, and reduced the exhaust noise by 47.52%.

Detection and molecular characterization of porcine toroviruses in Korea.

This study examined the prevalence and genetic diversity of the porcine torovirus (PToV) in Korea. Of 295 samples, 19 (6.4%) samples tested positive for PToVs by RT-PCR. A low nucleotide sequence identity of the partial S gene was detected among the Korean PToVs (73.5%) and between the Korean and European PToVs (74.0%). Phylogenetic analysis of the spike and nucleocapsid genes showed that the Korean PToVs form distinct branches with clusters corresponding to the farm of origin, which were separate from the other known foreign PToVs. These findings suggest that genetically diverse Korean PToV strains cause sporadic infections in Korea.

Development of one-step real-time reverse transcription polymerase chain reaction assays for rapid detection of porcine group C rotaviruses.

Although the widespread occurrence of porcine group C rotaviruses (GCRV) is assumed, precise prevalence remains largely unknown because of the absence of reliable, specific, and rapid diagnostic methods. To detect and quantify porcine GCRV, the authors evaluated and optimized SYBR Green and TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR) assays and applied them to 108 piglet fecal samples. Using serially diluted standard RNA transcripts of porcine GCRV VP6 gene, both SYBR Green and TaqMan real-time RT-PCR assays detected as few as 1 x 10(1) genome copies/microl (correlation coefficiency >0.99), whereas conventional RT-PCR detected 1.0 x 10(3) copies/microl. In addition, the conventional assay detected porcine GCRV in 24% (26/108) of fecal samples, whereas the detection rates of both SYBR Green and TaqMan assays were 72% (78 of 108) and 64% (70 of 108), respectively. The current study indicated that both real-time RT-PCR assays were reliable, specific, and rapid methods for the detection of porcine GCRV in porcine fecal samples.

Laser Pyrolysis of Imprinted Furan Pattern for the Precise Fabrication of Microsupercapacitor Electrodes.

The design or dimension of micro-supercapacitor electrodes is an important factor that determines their performance. In this study, a microsupercapacitor was precisely fabricated on a silicon substrate by irradiating an imprinted furan micropattern with a CO2 laser beam under ambient conditions. Since furan is a carbon-abundant polymer, electrically conductive and porous carbon structures were produced by laser-induced pyrolysis. While the pyrolysis of a furan film in a general electric furnace resulted in severe cracks and delamination, the laser pyrolysis method proposed herein yielded porous carbon films without cracks or delamination. Moreover, as the imprinting process already designated the furan area for laser pyrolysis, high-precision patterning was achieved in the subsequent laser pyrolysis step. This two-step process exploited the superior resolution of imprinting for the fabrication of a laser-pyrolyzed carbon micropattern. As a result, the technical limitations of conventional laser direct writing could be overcome. The laser-pyrolyzed carbon structure was employed for microsupercapacitor electrodes. The microsupercapacitor showed a specific capacitance of 0.92 mF/cm2 at 1 mA/cm2 with a PVA-H2SO4 gel electrolyte, and retained an up to 88% capacitance after 10,000 charging/discharging cycles.

Genetic diversity of porcine circovirus type 2 in Korean pigs with postweaning multisystemic wasting syndrome during 2005-2007.

The comparison of nucleotide and deduced amino acid sequence was conducted with 9 porcine circovirus type 2 (PCV2) strains isolated from PCV2-infected pigs with postweaning multisystemic wasting syndrome (PMWS) and 50 tissue samples obtained from PCV2-infected 50 pigs with PMWS during 2005-2007. At amino acid positions 88-89 of the ORF2 gene, 50 Korean PCV2 had amino acids PR/L consistent with group 1 PCV2, whereas 9 Korean PCV2 contained amino acids KI, characteristic of group 2 PCV2. Phylogenetically, 47, 3, 2 and 7 Korean PCV2 belonged to subgroups 1A/B (79.7%), 1C (5.1%), 2D (3.4%) and 2E (11.9%), respectively. Although the predominant Korean PCV2 was subgroup 1A/B, subgroups 1C and 2E were still circulating and subgroup 2D PCV2 were found to be newly emerged in Korea.

A Novel Soft Pneumatic Artificial Muscle with High-Contraction Ratio.

There is a growing interest in soft actuators for human-friendly robotic applications. However, it is very challenging for conventional soft actuators to achieve both a large working distance and high force. To address this problem, we present a high-contraction ratio pneumatic artificial muscle (HCRPAM), which has a novel actuation concept. The HCRPAM can contract substantially while generating a large force suitable for a wide range of robotic applications. Our proposed prototyping method allows for an easy and quick fabrication, considering various design variables. We derived a mathematical model using a virtual work principle, and validated the model experimentally. We conducted simulations for the design optimization using this model. Our experimental results show that the HCRPAM has a 183.3% larger contraction ratio and 37.1% higher force output than the conventional pneumatic artificial muscle (McKibben muscle). Furthermore, the actuator has a compatible position tracking performance of 1.0 Hz and relatively low hysteresis error of 4.8%. Finally, we discussed the controllable bending characteristics of the HCRPAM, which uses heterogeneous materials and has an asymmetrical structure to make it comfortable for a human to wear.

Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes.

Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3-CD21-) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3+CD5dimCD21- lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3+CD5dimCD21- (cytotoxic large granular lymphocytes) and CD3-CD5-CD21- NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3+CD5dimCD21- lymphocytes can be differentiated into non-B, non-T NK (CD3-CD5-CD21-TCRαß-TCRγδ-GranzymeB+) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3+CD5dimCD21- cells showed that CD3-CD5-CD21- cells are derived from CD3+CD5dimCD21- cells through phenotypic modulation. CD3+CD5dimCD21- cells share more NK cell functional characteristics compared with CD3-CD5-CD21- cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3+CD5dimCD21- and CD3-CD5-CD21- cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.

Identification of genes differentially expressed in RAW264.7 cells infected by Salmonella typhimurium using PCR method.

Salmonella typhimurium, causing mouse typhoid, infects hosts such as macrophage cells, and proliferates in intracellular vacuoles causing infected cells to trigger numerous genes to respond against the infection. In this study, we tried to identify such genes in RAW264.7 cells by using the PCR screening method with degenerate primers. Fourteen genes were found to be differentially expressed after a 4 h infection in which the expression of 8 genes increased while expression of the others decreased. Most of the genes were involved in proinflammatory responses such as cytokines production and cell death. The mutation in msbB gene encoding the myristoyl transferase in lipid A of lipopolysaccharide (LPS) resulted in much lower toxicity to the inoculated animals. We compared the expression of the identified genes in wild-type and msbB-mutated S. typhimurium infections and found that Lyzs encoding lysozyme type M was differentially expressed. This gene is quite likely to be related to bacterial survival in the host cells.

The A312 Allele (c.280A>T) Is Responsible for the Weak A Phenotype.

The ABO*A312 allele was found in a 71-year-old Korean male with ABO discrepancy and in his two sons. Although the ABO*A312 allele (c.280A>T, I94F) in an AwB case was registered in GenBank, the impact of the I94F mutation of the ABO gene on the activity of A transferase has not been studied. Transient transfection experiments were performed in HeLa cells using A101, A102, and A312 alleles synthesized by site-directed mutagenesis, and the functional expression level of A antigen was assessed by flow cytometry. The results showed that the A102 and A312 alleles expressed A antigen levels that were 80.28% and 19.32%, respectively, of that of the A101 allele. Our study results demonstrate that the c.280A>T variant is responsible for the weakened expression of A antigen.

Effects of ADAM10 and ADAM17 Inhibitors on Natural Killer Cell Expansion and Antibody-dependent Cellular Cytotoxicity Against Breast Cancer Cells In Vitro.

BACKGROUND/AIM: The inhibition of a disintegrin and metalloproteinase (ADAM) has the potential to become a novel approach for natural killer (NK) cell-based cancer immunotherapy. Thus, the aim of this study was to investigate the influence of ADAM10 and ADAM17 inhibitors on expanded NK cell to enhance antibody-dependent cellular cytotoxicity (ADCC) in breast cancer cell lines. MATERIALS AND METHODS: NK cells were expanded in medium supplemented with an ADAM10 or ADAM17 inhibitor to prevent the shedding of soluble CD16/FcγRIII. The expression level of CD16 and production of interferon-gamma (IFN-γ) was detected by flow cytometry using specific antibodies. ADCC activity of expanded NK cells was estimated in trastuzumab treated breast cancer cell lines such as MCF-7, MDA-MB-231, SKBR3, and BT-474 cells. RESULTS: The ADAM17 inhibitor increased the purity of expanded NK cells to 90% after 14 days at 5 and 10 µM in vitro (p=0.043). However, the expansion rate of NK cells was decreased at 10 µM of the ADAM 17 inhibitor (p=0.043). Inhibition of ADAM10 suppressed the expansion of NK cells, although the NK purity was increased at 1 µM of the inhibitor. The expression of CD16 was significantly increased at 1 and 5 µM of the ADAM17 inhibitor (p=0.046, 0.028, respectively) during the culturing period. Inhibition of ADAM10 reduced the expression of CD16 on NK cells. The cytotoxic activity of the ADAM17 inhibitor treated NK cells against MCF-7 (p=0.039) and BT-474 (p=0.027) cells was significantly elevated. The ADCC activity of NK cells treated with 5 µM of ADAM17 inhibitor was significantly increased against SKBR-3 and BT-474 (p=0.027). Inhibition of ADAM17 increased the production of IFN-γ in expanded NK cells. CONCLUSION: The inhibition of ADAM17 enhanced the purity of expanded NK cells and the ADCC activity of these cells against trastuzumab treated breast cancer cell lines.