Standard Cytotoxicity Test Research Articles

The Study of Nanosized Silicate-Substituted Hydroxyapatites Co-Doped with Sr2+ and Zn2+ Ions Related to Their Influence on Biological Activities.

Nanosized silicate-substituted hydroxyapatites, characterized by the general formula Ca9.8-x-nSrnZnx(PO4)6-y(SiO4)y(OH)2 (where: n = 0.2 [mol%]; x = 0.5-3.5 [mol%]; y = 4-5 [mol%]), co-doped with Zn2+ and Sr2+ ions, were synthesized with the help of a microwave-assisted hydrothermal technique. The structural properties were determined using XRD (X-ray powder diffraction) and Fourier-transformed infrared spectroscopy (FT-IR). The morphology, size and shape of biomaterials were detected using scanning electron microscopy techniques (SEM). The reference strains of Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa were used to assess bacterial survivability and the impact on biofilm formation in the presence of nanosilicate-substituted strontium-hydroxyapatites. Safety evaluation was also performed using the standard cytotoxicity test (MTT) and hemolysis assay. Moreover, the mutagenic potential of the materials was assessed (Ames test). The obtained results suggest the dose-dependent antibacterial activity of nanomaterials, especially observed for samples doped with 3.5 mol% Zn2+ ions. Moreover, the modification with five SiO4 groups enhanced the antibacterial effect; however, a rise in the toxicity was observed as well. No harmful activity was detected in the hemolysis assay as well as in the mutagenic assay (Ames test).

The Preliminary Assessment of New Biomaterials Necessitates a Comparison of Direct and Indirect Cytotoxicity Methodological Approaches.

Cytotoxicity testing is a primary method to establish the safety of biomaterials, e.g., biocomposites. Biomaterials involve a wide range of medical materials, which are usually solid materials and are used in bone regeneration, cardiology, or dermatology. Current advancements in science and technology provide several standard cytotoxicity testing methods that are sufficiently sensitive to detect various levels of cellular toxicity, i.e., from low to high. The aim was to compare the direct and indirect methodology described in the ISO guidelines UNE-EN ISO 10993-5:2009 Part 5. Cell proliferation was measured using WST-1 assay, and cytotoxicity was measured using LDH test kit. The results indicate that the molecular surface of biomaterials have impact on the cytotoxicity and proliferation profile. Based on these results, we confirm that the indirect method does not provide a clear picture of the cell condition after the exposure to the surface, and moreover, cannot provide complete results about the effects of the material. Comparison of both methods shows that it is pivotal to investigate biomaterials at the very early stages using both indirect and direct methods to access the influence of the released toxins and surface of the material on the cell condition.

In Vitro Direct and Indirect Cytotoxicity Comparative Analysis of One Pre-Hydrated versus One Dried Acellular Porcine Dermal Matrix.

Aim: The aim of the present study was to compare the direct and indirect cytotoxicity of a porcine dried acellular dermal matrix (PDADM) versus a porcine hydrated acellular dermal matrix (PHADM) in vitro. Both are used for periodontal and peri-implant soft tissue regeneration. Materials and methods: Two standard direct cytotoxicity tests—namely, the Trypan exclusion method (TEM) and the reagent WST-1 test (4-3-[4-iodophenyl]-2-[4-nitrophenyl]-2H-[5-tetrazolio]-1,3-benzol-desulphonated)—were performed using human primary mesenchymal stem cells (HPMSCs) seeded directly onto a PDADM and PHADM after seven days. Two standard indirect cytotoxicity tests—namely, lactate dehydrogenase (LTT) and MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazoliumbromide)—were performed using HPMSCs cultivated in eluates from the matrices incubated for 0.16 h (10 min), 1 h, and 24 h in a serum-free cell culture medium. Results: The WST and the TEM tests revealed significantly lower direct cytotoxicity values of HPMSCs on the PHADM compared with the PDADM. The indirect cytotoxicity levels were low for both the PHADM and PDADM, peaking in short-term eluates and decreasing with longer incubation times. However, they were lower for the PHADM with a statistically significant difference (p < 0.005). Conclusions: The results of the current study demonstrated a different biologic behavior between the PHADM and the PDADM, with the hydrated form showing a lower direct and indirect cytotoxicity.

Substituted Furanocoumarins as Novel Class of Antibacterial Translation Inhibitors.

A variety of organic compounds has been reported to have antibacterial activity. However, antimicrobial resistance is one of the main problems of current anti-infective therapy, and the development of novel antibacterials is one of the main challenges of current drug discovery. Using our previously developed dual-reporter High-Throughput Screening (HTS) platform, we identified a series of furanocoumarins as having high antibacterial activity. The construction of the reporter system allows us to differentiate three mechanisms of action for the active compounds: inhibition of protein synthesis (induction of Katushka2S), DNA damaging (induction of RFP) or other (inhibition of bacterial growth without reporter induction). Two primary hit-molecules of furanocoumarin series demonstrated relatively low MIC values comparable to that observed for Erythromycin (Ery) against E. coli and weakly induced both reporters. Dose-dependent translation inhibition was shown using in vitro luciferase assay, however it was not confirmed using C14-test. A series of close structure analogs of the identified hits was obtained and investigated using the same screening platform. Compound 19 was found to have slightly lower MIC value (15.18 µM) and higher induction of Katushka2S reporter in contrast to the parent structures. Moreover, translation blockage was clearly identified using both in vitro luciferase assay and C14 test. The standard cytotoxicity test revealed a relatively low cytotoxicity of the most active molecules. High antibacterial activity in combination with low cytotoxicity was demonstrated for a series of furanocoumarins. Further optimization of the described structures may result in novel and attractive lead compounds with promising antibacterial efficiency.

Optimization of photocrosslinkable resin components and 3D printing process parameters

The role of 3D printing in the biomedical field is growing. In this context, photocrosslink-based 3D printing procedures for resorbable polymers stand out. Despite much work, more studies are needed on photocuring stereochemistry, new resin additives, new polymers and resin components. As part of these studies it is vital to present the logic used to optimize the amount of each resin constituent and how that effects printing process parameters.The present manuscript aims to analyze the effects of poly(propylene fumarate) (PPF) resin components and their effect on 3D printing process parameters. Diethyl fumarate (DEF), bisacylphosphine oxide (BAPO), Irgacure 784, 2-hydroxy-4-methoxybenzophenone (HMB) and, for the first time, in biomedical 3D printing, ethyl acetate (EA), were the resin components under investigation in this study. Regarding printing process parameters, Exposure Time, Voxel Depth, and Overcuring Depth were the parameters studied. Taguchi Design of Experiments was used to search for the effect of varying these resin constituent concentrations and 3D printing parameters on the curing behavior of 3D printable PPF resins.Our results indicate that resins with higher polymer cross-link density, especially those with a higher content of PPF, are able to be printed at higher voxel depth and with greater success (i.e., high yield). High voxel depth, as long as it does not sacrifice required resolution, is desirable as it speeds printing. Nevertheless, the overall process is governed by the correct setup of the voxel depth in relation to overcuring depth. In regards to resin biocompatibility, it was observed that EA is more effective than DEF, the material we had previously relied on. Our preliminary in vitro cytotoxicity tests indicate that the use of EA does not reduce scaffold biocompatibility as measured by standard cytotoxicity testing (i.e., ISO 10993-5). We demonstrate a workpath for resin constituent concentration optimization through thin film tests and photocrosslinkable process optimization. Statement of SignificanceWe report here the results of a study of photo-crosslinkable polymer resin component optimization for the 3D printing of resorbable poly(propylene fumarate) (PPF) scaffolds. Resin additives are initially optimized for PPF thin film printing. Once those parameters have been optimized the 3D printing process parameters for PPF objects with complex, porous shapes can be optimized. The design of experiments to optimize both polymer thin films and complex porous resorbable polymer scaffolds is important as a guess and check, or in some cases a systematic method, are very likely to be too time consuming to accomplish. Previously unstudied resin components and process parameters are reported.

Optimization of Photocrosslinkable Resin Components and 3D Printing Process Parameters

The role of 3D printing in the biomedical field is growing. In this context, photocrosslink-based 3Dprinting procedures for resorbable polymers stand out. Despite much work, more studies are needed on photocuring stereochemistry, new resin additives, new polymers and resin components. As par tof these studies it is vital to present the logic used to optimize the amount of each resin constituent and how that effects printing process parameters.The present manuscript aims to analyze the effects the poly(propylene fumarate) (PPF) resin components and their effect on 3D printing process parameters. Diethyl fumarate (DEF),bisacylphosphine oxide (BAPO), Irgacure 784, 2-hydroxy-4-methoxybenzophenone (HMB) and,for the first time, in biomedical 3D printing, ethyl acetate (EA), were the resin components under investigation in this study. Regarding printing process parameters, Exposure Time, Voxel Depth,and Overcuring Depth were the parameters studied. Taguchi Design of Experiments was used tosearch for the effect of varying these resin constituent concentrations and 3D printing parameters onthe curing behavior of 3D printable PPF resins.Our results indicate that resins with higher polymer cross-link density, especially those with a higher content of PPF, are able to be printed at higher voxel depth and with greater success (i.e.,high yield). High voxel depth, as long as it does not sacrifice required resolution, is desirable as it speeds printing. Nevertheless, the overall process is governed by the correct setup of the voxeldepth in relation to overcuring depth. In regards to resin biocompatibility, it was observed that EAis more effective than DEF, the material we had previously relied on. Our preliminary in vitrocytotoxicity tests indicate that the use of EA does not reduce scaffold biocompatibility as measured by standard cytotoxicity testing (i.e., ISO 10993-5). We demonstrate a workpath for resin constituent concentration optimization through thin film tests and photocrosslinkable process optimization.

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO2) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Development and characterisation of a large diameter decellularised vascular allograft

The aims of this study were to develop a biological large diameter vascular graft by decellularisation of native human aorta to remove the immunogenic cells whilst retaining the essential biomechanical, and biochemical properties for the ultimate benefit of patients with infected synthetic grafts. Donor aortas (n = 6) were subjected to an adaptation of a propriety decellularisation process to remove the cells and acellularity assessed by histological analysis and extraction and quantification of total DNA. The biocompatibility of the acellular aortas was determined using standard contact cytotoxicity tests. Collagen and denatured collagen content of aortas was determined and immunohistochemistry was used to determine the presence of specific extracellular matrix proteins. Donor aortas (n = 6) were divided into two, with one half subject to decellularisation and the other half retained as native tissue. The native and decellularised aorta sections were then subject to uniaxial tensile testing to failure [axial and circumferential directions] and suture retention testing. The data was compared using a paired t-test. Histological evaluation showed an absence of cells in the treated aortas and retention of histoarchitecture including elastin content. The decellularised aortas had less than 15 ng mg−1 total DNA per dry weight (mean 94% reduction) and were biocompatible as determined by in vitro contact cytotoxicity tests. There were no gross changes in the histoarchitecture [elastin and collagen matrix] of the acellular aortas compared to native controls. The decellularisation process also reduced calcium deposits within the tissue. The uniaxial tensile and suture retention testing revealed no significant differences in the material properties (p > 0.05) of decellularised aorta. The decellularisation procedure resulted in minimal changes to the biological and biomechanical properties of the donor aortas. Acellular donor aorta has excellent potential for use as a large diameter vascular graft.

Artificial urine and FBS supplemented media in cytocompatibility assays for PLGA-PEG-based intravesical devices using the urothelium cell line UROtsa.

European and German directives for approval of new medical devices require tests for cytotoxicity in relevant media, since urine can influence cytotoxicity of biodegradable devices. The aim of this study was to determine the long-term cytotoxicity of PLGA-b-mPEG (PLGA-PEG) polymer carriers and artificial urine (AU) to human UROtsa cells. Benign urothelial UROtsa cells were incubated in fetal bovine serum-containing RPMI 1640 medium supplemented with a range of concentrations of AU for 24 h and 7 days. Cell viability was determined by the XTT assay and by live/dead staining. The cytotoxicity of medium containing degradation products from PLGA-PEG carriers was also tested on the UROtsa cells in AU-containing and control medium. PLGA-PEG carriers exhibited no cytotoxicity to UROtsa cells after 24 h of incubation. However, after 7 days, cytotoxicity was observed, but this was largely attributable to the effects of 30% AU on the cells. Compared to phosphate buffer saline (PBS) and normalized to RPMI 1640 medium, significant cytotoxicity was observed by 24 h in medium containing 50% AU and by 7 days in medium containing 30% AU. Live/Dead staining confirmed proliferation results and no pH-changes could be observed. Here we demonstrate for the first time the impact of AU on standard cytotoxicity tests related to biomaterials for urinary-tract applications. Our study showed cytotoxic effects of high concentrations of 50% AU by 24 h and by physiological concentrations of AU (i.e., 30%) by 7 days. We have also demonstrated that PLGA-PEG has no cytotoxic effects in the appropriate AU-containing test environment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2140-2147, 2018.

Differences in cytocompatibility, dynamics of the oxide layers' formation, and nickel release between superelastic and thermo-activated nickel-titanium archwires.

Superelastic (SE) and thermo-activated (TA) nickel-titanium (NiTi) archwires are used in everyday orthodontic practice, based on their acceptable biocompatibility and well-defined shape memory properties. However, the differences in their surface microstructure and cytotoxicity have not been clearly defined, and the standard cytotoxicity tests are too robust to detect small differences in the cytotoxicity of these alloys, all of which can lead to unexpected adverse reactions in some patients. Therefore, we tested the hypothesis that the differences in manufacture and microstructure of commercially available SE and TA archwires may influence their biocompatibility. The archwires were studied as-received and after conditioning for 24h or 35days in a cell culture medium under static conditions. All of the tested archwires, including their conditioned medium (CM), were non-cytotoxic for L929 cells, but Rematitan SE (both as received and conditioned) induced the apoptosis of rat thymocytes in a direct contact. In contrast, TruFlex SE and Equire TA increased the proliferation of thymocytes. The cytotoxic effect of Rematitan SE correlated with the higher release of Ni ions in CM, higher concentration of surface Ni and an increased oxygen layer thickness after the conditioning. In conclusion, the apoptosis assay on rat thymocytes, in contrast to the less sensitive standard assay on L929 cells, revealed that Rematitan SE was less cytocompatible compared to other archwires and the effect was most probably associated with a higher exposition of the cells to Ni on the surface of the archwire, due to the formation of unstable oxide layer.

Comparison of different in vitro tests for biocompatibility screening of Mg alloys

Standard cell culture tests according to ISO 10993 have only limited value for the biocompatibility screening of degradable biomaterials such as Mg alloys. The correlation between in vitro and in vivo results is poor. Standard cytotoxicity tests mimic the clinical situation to only a limited extent, since in vivo proteins and macromolecules in the blood and interstitial liquid will influence the corrosion behaviour and, hence, biocompatibility of Mg alloys to a significant extent. We therefore developed a modified cytotoxicity test simulating the in vivo conditions by use of bovine serum as the extraction vehicle instead of the cell culture medium routinely used in standard cytotoxicity testing according to ISO 10993-5. The modified extraction test was applied to eight experimental Mg alloys. Cytotoxicity was assayed by inhibition of cell metabolic activity (XTT test). When extraction of the alloy samples was performed in serum instead of cell culture medium the metabolic activity was significantly less inhibited for six of the eight alloys. The reduction in apparent cytotoxicity under serum extraction conditions was most pronounced for MgZn1 (109% relative metabolic activity with serum extracts vs. 26% in Dulbecco’s modified Eagle’s medium (DMEM)), for MgY4 (103% in serum vs. 32% in DMEM) and for MgAl3Zn1 (84% vs. 17%), resulting in a completely different cytotoxicity ranking of the tested materials when serum extraction was used. We suppose that this test system has the potential to enhance the predictability of in vivo corrosion behaviour and biocompatibility of Mg-based materials for biodegradable medical devices.

Immortalized gingival fibroblasts as a cytotoxicity test model for dental materials

In vitro cytotoxicity test is an initial step to identify the harmful effects of new dental materials. Aim of this study was to develop a stable human cell line derived from normal gingival fibroblasts (hNOF) and to assess its feasibility in in vitro cytotoxicity testing. Immortalized human gingival fibroblasts (hTERT-hNOF) were successfully established with human telomerase reverse transcriptase gene transfection, preserving its phenotypical characteristics, replicative potential and biological properties. Utilizing standard cytotoxicity test modeling and dental materials, hTERT-hNOF were evaluated for their feasibility in cytotoxicity testing, compared with hNOF and L929 cells. Similar pattern of cytotoxic response was observed among hNOF, hTERT-hNOF and L929 cells. Cytotoxicity response of hTERT-hNOF was significantly similar to hNOF, moreover hTERT-hNOF and hNOF were found to be more sensitive towards the tested dental materials compared to L929 cells. This study suggested that hTERT-hNOF is an effective cytotoxic test model for dental materials.

The study on the induction of specific immune cytotoxic T lymphocyte responses against pancreatic cancer by transfected dendritic cells with common tumor antigen survivin mRNAs in vitro

Objective To investigate the induction of specific anti-tumor immune response by transfected dendritic cells (DCs) with survivin mRNA of human pancreatic cancer, and to provide the experimental evidences for the treatment of human pancreatic cancer with DCs vaccine. Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs). After being transcripted and amplified, survivin mRNA was transfected into DCs by electroporation. The expression of survivin in DCs at different time points was detected by quantitative real-time PCR. The survival rate of DCs before and after transfection was determined by MTT method. The induction of specific cytotoxic T lymphocyte (CTL) response by survivin mRNA transfected DCs was measured by 51Cr standard cytotoxicity test. The induction of specific CTL activation by survivin mRNA transfected DCs was evaluated through testing released IFN-γ by ELISA method. Results After survivin mRNA transfection for 48h, the expression of survivin mRNA in DCs reached the highest point (46.09±6.57). After transfection, the survival rate of DCs was stabilized around 80%. The DCs transfected with survivin mRNA could effectively induce HLA-A2+ / survivin+ specific CTL immune responses. Stimulated with pancreatic cancer cell line Capan-2 cells or SCL-1 cells as control group, the IFN-γ released in 24 hours by survivin specific CTL were (28.79±5.70) U/ml and (25.12±2.13) U/ml respectively, there was no significant difference (P=0.761). Conclusion The induction of CTLs by DCs transfected with human pancreatic cancer survivin mRNA could produce specific anti-tumor immunity. Key words: Dendritic cells; Microtubule-associated proteins; RNA, messenger; Transfection; Pancreatic neoplasms; T-lymphocytes, cytotoxic

C1 Domain-Targeted Isophthalate Derivatives Induce Cell Elongation and Cell Cycle Arrest in HeLa Cells

Diacylglycerol (DAG)-mediated signaling pathways, such as those mediated by protein kinase C (PKC), are central in regulating cell proliferation and apoptosis. DAG-responsive C1 domains are therefore considered attractive drug targets. Our group has designed a novel class of compounds targeted to the DAG binding site within the C1 domain of PKC. We have previously shown that these 5-(hydroxymethyl)isophthalates modulate PKC activation in living cells. In this study we investigated their effects on HeLa human cervical cancer cell viability and proliferation by using standard cytotoxicity tests and an automated imaging platform with machine vision technology. Cellular effects and their mechanisms were further characterized with the most potent compound, HMI-1a3. Isophthalate derivatives with high affinity to the PKC C1 domain exhibited antiproliferative and non-necrotic cytotoxic effects on HeLa cells. The anti-proliferative effect was irreversible and accompanied by cell elongation. HMI-1a3 induced down-regulation of retinoblastoma protein and cyclins A, B1, D1, and E. Effects of isophthalates on cell morphology, cell proliferation and expression of cell cycle-related proteins were different from those induced by phorbol 12-myristate-13-acetate (PMA) or bryostatin 1, but correlated closely to binding affinities. Therefore, the results strongly indicate that the effect is C1 domain-mediated.

Issues surrounding standard cytotoxicity testing for assessing activity of non-covalent DNA-binding metallo-drugs

Investigating the methods commonly used to evaluate in vitro cytotoxicity of novel compounds, specifically non-covalent DNA binders, identifies that these methods may not be appropriate. The level of anticancer activity depends not only on the incubation time but also on the absolute amount (number of moles) of drug compound applied, rather than the concentration.

Gellan gum: A new biomaterial for cartilage tissue engineering applications

Gellan gum is a polysaccharide manufactured by microbial fermentation of the Sphingomonas paucimobilis microorganism, being commonly used in the food and pharmaceutical industry. It can be dissolved in water, and when heated and mixed with mono or divalent cations, forms a gel upon lowering the temperature under mild conditions. In this work, gellan gum hydrogels were analyzed as cells supports in the context of cartilage regeneration. Gellan gum hydrogel discs were characterized in terms of mechanical and structural properties. Transmissionelectron microscopy revealed a quite homogeneous chain arrangement within the hydrogels matrix, and dynamic mechanical analysis allowed to characterize the hydrogels discs viscoelastic properties upon compression solicitation, being the compressive storage and loss modulus of approximately 40 kPa and 3 kPa, respectively, at a frequency of 1 Hz. Rheological measurements determined the sol-gel transition started to occur at approximately 36 degrees C, exhibiting a gelation time of approximately 11 s. Evaluation of the gellan gum hydrogels biological performance was performed using a standard MTS cytotoxicity test, which showed that the leachables released are not deleterious to the cells and hence were noncytotoxic. Gellan gum hydrogels were afterwards used to encapsulate human nasal chondrocytes (1 x 10(6) cells/mL) and culture them for total periods of 2 weeks. Cells viability was confirmed using confocal calcein AM staining. Histological observations revealed normal chondrocytes morphology and the obtained data supports the claim that this new biomaterial has the potential to serve as a cell support in the field of cartilage regeneration.

Measuring Human Complement Activation by HLA Antibodies

The clinical significance of HLA antibodies in transplantation depends on their ability to activate complement, which is measured by the standard complement-dependent cytotoxicity test. Recent reports indicate that C3b measurement on target cells may be a better indicator of human complement activation than standard cytotoxicity. To determine the characteristics of the test, the role of other complement components, and the potential influence of the patient's own complement activity. The T-cell deposition of multiple complement components triggered by HLA alloantibodies was evaluated by flow cytometry using normal human serum as the source of complement. Complement activity in patients' sera was measured after activation with a standard antibody. When HLA antibodies activate complement, C3b deposition is usually highest, followed by that of C4b and C5b. Deposition of C1q, C3d, iC3b, or C5b-9 was low or undetectable in this study. The C5 was activated only when relatively high levels of C3b were present. There was a critical concentration of antibody, unique for each patient, below which activation of C3 declined abruptly. The complement activity in the serum of candidates for liver, heart, and lung transplantation that was tested with a standard antibody was similar to normal serum. In contrast, kidney transplant candidates exhibited higher complement activity. Unexpectedly, serum C3 activity was retained for at least 72 hours after collection. Measurement of C3b, and in some instances C4b or C5b, offers a better definition of the ability of HLA antibodies to activate human complement than tests that are in current use. The results provide a necessary baseline to conduct clinical correlation studies.

A mutated human tumor necrosis factor-alpha improves the therapeutic index in vitro and in vivo

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has cytotoxic, cytostatic and immunomodulatory effects on malignant tumors. However, clinical trials have revealed high systemic toxicity and this has hampered its utilization as an anti-cancer agent. In this study, a human TNF-alpha mutant was created and tested for its anti-tumor effects. The TNF mutant (recombinant mutated human TNF; rmhTNF) was prepared by protein engineering in which amino acids Pro, Ser and Asp at positions 8, 9 and 10 of TNF-alpha were substituted by Arg, Lys and Arg, and C terminal Leu157 was substituted by Phe, along with deletion of the first seven N-terminal amino acids. Prokaryotic expression recombinant vector pBV-mhTNF containing the PLPR promotor was constructed and transformed into E. coli DH5alpha. The rmhTNF was expressed in a partially soluble form in DH5alpha, purified from the supernatant of cell lysate by ammonia sulfate precipitation and two sequential chromatographic steps. The purified rmhTNF was >95% pure by SDS-PAGE stained with silver and high-pressure size exclusion chromatography (SEC-HPLC). Its yield was about 1.22 mg/g wet cell paste. The mutant rmhTNF exhibited an approximately 50-fold increase in cytotoxicity relative to the wild-type rhTNF on the mouse fibroblast cell line L929 in a standard cytotoxicity test, and at least and at least 50 times higher LD50 as wild type rhTNF in mice. In vivo biological activity studies carried out on tumor cell transplanted mice and nude mice also showed a more effective cytotoxicity of rmhTNF than rhTNF. These results suggest that rmhTNF has potential for developing an effective anti-tumor reagent for some tumors.

Tetragametic chimerism detected in a healthy woman with mixed‐field agglutination reactions in ABO blood grouping

The case of a healthy woman with serologic blood group AB and her biologic father showing blood group O was investigated. Further analysis, including blood, buccal swabs, and nail clippings, revealed a tetragametic chimerism. Blood grouping was performed with standard gel centrifugation test cards, ABO genotyping by sequence-specific primers (SSPs) and sequence-based typing, and HLA Class I and II typing by standard NIH cytotoxicity testing and SSP. Additionally, short-tandem-repeat (STR) and variable-number tandem-repeat (VNTR) typing was performed on blood, nail clippings, and buccal swab samples. The karyotype was analyzed by G-banded chromosomes. The proposita's RBCs were typed AB with a mixed-field agglutination whereas in molecular typing A, B, and O alleles were found. One paternal and two maternal haplotypes were determined by use of HLA typing. Interestingly, both paternal alleles were detected in 4 of 23 tested STR and VNTR loci only, with whole blood, nail clippings, and buccal swabs. The karyotype was identified as 46XX. The family members including the proposita's healthy twin children displayed no abnormal findings in tests performed. By investigation of DNA polymorphisms, it was possible to determine a rare case of tetragametic chimerism being the result of double parental contribution of nuclei.

Clinical relevance of antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test

Clinical relevance of antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test