Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Matrix-material dependence on the elongation of embedded gold nanoparticles induced by 4 MeV C60 and 200 MeV Xe ion irradiation.

We report the elongation of embedded Au nanoparticles (NPs) in three different matrices, i.e. amorphous carbon (a-C), crystalline indium tin oxide (InxSn1-xOz; ITO) and crystalline calcium fluoride (CaF2), under irradiations of 4 MeV C60 + cluster ions and 200 MeV Xe14+ ions. Under 4 MeV C60 cluster irradiation, strong sputtering is induced in CaF2 layer so that the whole the layer was completely lost at a fluence of 5 × 1013 ions cm-2. Au NPs were partly observed in the SiO2, probably due to the recoil implantation. Amorphous carbon (a-C) layer exhibits low sputtering loss even under 4 MeV C60 irradiation. However, the elongation in a-C layer was low. While the ITO layer showed a certain decrease in thickness under 4 MeV C60 irradiation, large elongation of Au NPs was observed under both 4 MeV C60 and 200 MeV Xe irradiation. The ITO layer preserved the crystallinity even after large elongation was induced. This is the first report of the elongation of metal NPs in a crystalline matrix.

Characterization of secondary ion emission processes of sub-MeV C60 ion impacts via analysis of statistical distributions of the emitted ion number.

We report probability distributions of the number of secondary ions (SIs) emitted by sub-MeV C60 ion impacts on an organic polymer target and the characterization of their emission processes through the analysis of the distributions. The probability distributions were obtained by analyzing experimental SI counting data obtained by a time-of-flight SI mass spectrometer combined with pulsed primary ion beams, using an analytical model developed to derive the distributions from the experimental data. A series of probability distribution functions was investigated for ion impacts of C60 with sub-MeV energies (0.12-0.54 MeV), which can provide sufficient SIs per impact to determine the functions. Their complicated and undefined SI emission processes were characterized based on the determined functions.

Latent ion tracks were finally observed in diamond.

Injecting high-energy heavy ions in the electronic stopping regime into solids can create cylindrical damage zones called latent ion tracks. Although these tracks form in many materials, none have ever been observed in diamond, even when irradiated with high-energy GeV uranium ions. Here we report the first observation of ion track formation in diamond irradiated with 2-9 MeV C60 fullerene ions. Depending on the ion energy, the mean track length (diameter) changed from 17 (3.2) nm to 52 (7.1) nm. High resolution scanning transmission electron microscopy (HR-STEM) indicated the amorphization in the tracks, in which π-bonding signal from graphite was detected by the electron energy loss spectroscopy (EELS). Since the melting transition is not induced in diamond at atmospheric pressure, conventional inelastic thermal spike calculations cannot be applied. Two-temperature molecular dynamics simulations succeeded in the reproduction of both the track formation under MeV C60 irradiations and the no-track formation under GeV monoatomic ion irradiations.

In vivo delivery of interferon-α gene enhances tumor immunity and suppresses immunotolerance in reconstituted lymphopenic hosts.

T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c(+) cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c(+) cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c(+) cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.

Ion tracks in silicon formed by much lower energy deposition than the track formation threshold.

Damaged regions of cylindrical shapes called ion tracks, typically in nano-meters wide and tens micro-meters long, are formed along the ion trajectories in many insulators, when high energy ions in the electronic stopping regime are injected. In most cases, the ion tracks were assumed as consequences of dense electronic energy deposition from the high energy ions, except some cases where the synergy effect with the nuclear energy deposition plays an important role. In crystalline Si (c-Si), no tracks have been observed with any monomer ions up to GeV. Tracks are formed in c-Si under 40 MeV fullerene (C60) cluster ion irradiation, which provides much higher energy deposition than monomer ions. The track diameter decreases with decreasing the ion energy until they disappear at an extrapolated value of ~ 17 MeV. However, here we report the track formation of 10 nm in diameter under C60 ion irradiation of 6 MeV, i.e., much lower than the extrapolated threshold. The diameters of 10 nm were comparable to those under 40 MeV C60 irradiation. Furthermore, the tracks formed by 6 MeV C60 irradiation consisted of damaged crystalline, while those formed by 40 MeV C60 irradiation were amorphous. The track formation was observed down to 1 MeV and probably lower with decreasing the track diameters. The track lengths were much shorter than those expected from the drop of Se below the threshold. These track formations at such low energies cannot be explained by the conventional purely electronic energy deposition mechanism, indicating another origin, e.g., the synergy effect between the electronic and nuclear energy depositions, or dual transitions of transient melting and boiling.

Historical record of polychlorinated biphenyls in a sediment core from Lake Biwa, Japan: Significance of unintentional emission and weathering signals revealed by full congener-specific analysis.

The occurrence of 209 PCB congeners was determined in a sediment core dated between 1930 and 2019 from Lake Biwa, a typical temperate monomictic lake in Japan. Concentrations of total PCBs ranged from 5.3 to 48 ng/g dry weight (dw), showing a highest peak at the 1960s to 1970s. The temporal trend of total PCBs in this sediment core generally matched with Japanese PCB production and emission pattern (i.e., increasing from the 1950s, peaking at 1970, and gradually decreasing since 1972). The vertical PCB profiles in our core were affected by physical mixing and bioturbation. By using a detailed and comprehensive analytical method, we have found elevated concentrations and special historical profiles of several congeners such as CB-7, -11, -47/48/75, -51, -68, and -209, which are still rarely included in routine PCB analysis. Some tetra-CB congeners like CB-47/48/75, -51, and -68 showed their concentration peaks at the early 2010s, which may be unintentionally produced during polymer manufacturing processes. PCB homolog- and congener-specific profiles in our sediment core samples have experienced weathering with higher proportions of penta- and hexa-CBs as compared to the Kanechlor usage pattern (i.e., dominated by tri- and tetra-CBs). Both intentional (i.e., technical mixtures) and unintentional (e.g., PCB-containing polymers and pigments) sources of PCBs were suggested from congener-specific analysis.

Determination of brominated flame retardants including polybrominated diphenyl ethers, pentabromoethylbenzene, hexabromobiphenyl, and decabromodiphenyl ethane in sediment samples: Validation of a rapid and efficient clean-up method and application to a sediment core from Lake Biwa, Japan.

The development of rapid and efficient analytical method for the determination of legacy and current-use brominated flame retardants (BFRs) has been performed due to environmental concern related to these pollutants. In the present study, we used an automated clean-up device equipped with pre-packed micro-column sets (containing sulfuric acid impregnated silica gel and silver-modified alumina) to develop an effective purification method for polybrominated diphenyl ethers (PBDEs), pentabromoethylbenzene, hexabromobiphenyl, and decabromodiphenyl ethane (DBDPE) in sediment extracts. Matrix-spiked sediments (n = 6) and the Standard Reference Material® 1944 samples (n = 6) were tested. Our method showed acceptable accuracy, repeatability, and sensitivity for almost all the target compounds with reduced processing time, labor requirement, and solvent amounts as compared to conventional clean-up method (e.g., sulfuric acid treatment and self-packed chromatographic columns). The validated method was applied to sediment core samples (n = 16) collected in 2019 from Lake Biwa, the largest lake in Japan. PBDEs were detected in sediment samples of 0-13 cm depth (dated between 1990 and 2019) at relatively low concentrations (median 5.7; range 2.6-9.4 ng/g dry weight). PBDE profiles were dominated by BDE-209, which accounted for 91 ± 10% of total PBDEs. Among other BFRs, only DBDPE was found in sediment layers of 0-9 cm depth (deposited between 2005 and 2019). DBDPE concentrations ranged from 0.43 to 1.6 (median 0.71) ng/g and showed increasing trend toward shallower depths.

C60 ions of 1 MeV are slow but elongate nanoparticles like swift heavy ions of hundreds MeV.

This study reports that high fluence fullerene ion (C60+) irradiation of 1-6 MeV, which was made possible by a new-type of high-flux ion source, elongates metal nanoparticles (NPs) in amorphous SiO2 as efficiently as swift heavy ions (SHIs) of 200 MeV Xe14+, i.e., two orders of the magnitude higher energy ions. Comparing the irradiation effects induced by both the beams, the stopping processes of C60 ions in SiO2 are discussed in this paper. Despite of having almost the same elongation efficiency, the C60+ irradiation induced ~10 times more efficient sputtering due to the clustering enhancement and/or the synergy effect. Ion tracks of ~10.4 nm in diameter and 60-80 nm in length were observed in crystalline SiO2 under 4 MeV C60 irradiation. While the track diameter was comparable to those by SHIs of the same electronic stopping, much shorter track lengths than those predicted by a rigid C60 molecule model indicates that the fragmentation occurred due to nuclear collisions. The elongation of the metal NPs was induced only down to the depth where the tracks were observed but not beyond.

Historical changes in the ecosystem condition of a small mountain lake over the past 60 years as revealed by plankton remains and Daphnia ephippial carapaces stored in lake sediments.

To examine if changes in species composition of a plankton community in the past due to anthropogenic activities can be clarified in lakes without any monitoring data, we analyzed genetically ephippial carapaces of Daphnia with plankton remains stored in the bottom sediments of Lake Hataya Ohunma in Japan. In the lake, abundance of most plankton remains in the sediments was limited and TP flux was at low levels (2-4 mg/m2/y) before 1970. However TP flux increased two-fold during the period from 1980s to 1990 s. In parallel with this increase, abundance of most plankton remains increased although abundance of benthic testate amoebae's remains decreased, indicating that the lake trophic condition had changed from oligo- to mesotrophic for the past 60 years. According to cluster analysis, the stratigraphic sediments were divided into two periods with different features of the phytoplankton composition. Chronological comparison with events in the watershed suggested that eutrophication occurred because of an increase in visitors to the watershed and deposition of atmospheric dust. In this lake more than 50% of resting eggs produced by Daphnia over the past 60 years hatched. However, genetic analysis of the ephippial carapaces (remains) showed that the Daphnia population was originally composed of D. dentifera but that D. galeata, or its hybrid with D. dentifera, invaded and increased the population density when the lake was eutrophied. Subsequently, large D. pulex established populations in the 1980s when largemouth bass were anonymously introduced. These results indicated that the Lake Hataya Ohunma plankton community underwent significant changes despite the fact that there were no notable changes in land cover or land use in the watershed. Since increases in atmospheric deposition and release of fish have occurred in many Japanese lakes, the changes in the plankton community described here may be widespread in these lakes.

Administration of wild-type p53 adenoviral vector synergistically enhances the cytotoxicity of anti-cancer drugs in human lung cancer cells irrespective of the status of p53 gene.

Recombinant adenovirus mediated p53 gene transfer combined with anti-cancer drugs has clinical potential for gene therapy of lung cancer. We constructed a recombinant adenoviral vector expressing wild-type p53 cDNA (Ad-p53), and assessed the efficacy of a combined treatment with Ad-p53 and six anti-cancer drugs (cisplatin, 5-fluorouracil, doxorubicin, docetaxel, irinotecan, and etoposide) for human lung cancer cell lines, H1299 (with deleted p53), RERF-LC-OK (with mutant p53), and A549 (with wild-type p53). The infection of the Ad-p53 vector into H1299 cells, RERF-LC-OK cells, or A549 cells increased the sensitivity to all six drugs regardless of the cellular p53 status, and a synergism was observed by the isobolic method in combination studies (D<1). We conclude that our strategy using adenoviral mediated p53 gene transfer to cancer cells can enhance the cytotoxic effect of anti-cancer drugs, which leading to an improvement of lung cancer chemotherapy.

Cloning of 67-kDa laminin receptor cDNA and gene expression in normal and malignant cell lines of the human lung.

Cell-adhesive protein laminin and its specific receptor play an important role in the processes of cancer proliferation, invasion and metastasis. In the present study, we cloned the cDNAs of the 67-kDa laminin receptor both from a human lung cell line (IMR90) and from a human lung cancer cell line (SBC3), and determined the nucleotide sequences. In comparison with both cDNA sequences of the protein-coding region, three nucleotide differences were found. These differences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67-kDa-laminin receptor mRNA was higher in SBC3 than in IMR90.

Expression of the 37-kDa laminin binding protein in murine lung tumor cell correlates with tumor angiogenesis.

Expression of the 37-kDa laminin binding protein (37LBP), a precursor protein of the 67-kDa laminin receptor, correlates well with the biological aggressiveness of cancer cells. Previously, we have established murine lung cancer cell lines T11 and T15, in which 37LBP expression was remarkably diminished, and reported that the mean survival time of the T11 and the T15-recipients was significantly prolonged compared with that of the control cell lines (P29 and T42). In the present study, immunohistochemical findings of the tumors demonstrated that the microvessel density in the T11 (28. 1+/-7.2/mm(2)) and in the T15 tumor (29.7+/-6.5/mm(2)) were significantly lower than that observed in P29 (46.3+/-8.7/mm(2)) or in T42 (50.5+/-4.4/mm(2)). Expression of vascular endothelial growth factor (VEGF) was repressed in T11 and T15 compared with its expression in P29 and T42. It was also shown that conditioned media of T11 and T15 cells exhibited significantly reduced proliferation and migration of the capillary endothelial cells. These results suggest that decreased expression of 37LBP in antisense-RNA transfectant may relate to its low tumorigenicity, and that this effect may be partly caused by the diminished tumor angiogenesis of murine lung cancer.

Immunohistochemical detection of the laminin receptor polypeptide, a putative precursor of 67-kda-laminin receptor, in human lung-cancer.

Laminin receptor polypeptide was immunodetected in human lung cancer with a polyclonal antibody raised against a 20-mer peptide of the putative high-affinity laminin receptor of 67 kDa (Wewer et al: Cancer Res 47: 5691-5698, 1987). As a result, immunoreactivity was recognized specifically in cancer cells both in freshly-prepared cell samples and in paraffin-embedded tissue specimens. It was shown that immunodetection of the laminin receptor polypeptide with this antipeptide antibody could be applied to diagnostic use for lung cancer. The results also suggest that the laminin receptor polypeptide is not necessarily a membrane-associated protein and may function without further processing to the 67 kDa-laminin receptor.

Variety of laminin expressions in murine neoplastic cell-lines - neuroblastoma na cells produce only laminin-b2 chain.

In the present study, we investigated the expression of laminin in three murine neoplastic cell lines; 3LL-SA (Lewis lung carcinoma), NA (neuroblastoma) and F9 (teratocarcinoma). Both Western and Northern blot analyses demonstrated that parietal endoderm-like F9 expressed three laminin chains A, B1 and B2. On the other hand, 3LL-SA cells synthesized two laminin chains B1 and B2, and NA cells only B2 chain. The analyses of the restriction fragment length polymorphism indicated that the genes for coding regions of all chains were present and grossly intact both in 3LL-SA and in NA just as in F9. These findings suggest that expression of laminin seems to be transcriptionally regulated in each neoplastic cell line specifically. Since these cell lines produce different forms of laminin, they can be used for investigation of the multifunctions of laminin molecule.

Exogenous control of cardiac gene therapy: evidence of regulated myocardial transgene expression after adenovirus and adeno-associated virus transfer of expression cassettes containing corticosteroid response element promoters.

OBJECTIVE: Because of the relative inaccessibility of the heart for repeated gene therapy, it would be useful to regulate the expression of transgenes delivered in a single dose of a gene therapy vector. Incorporation into the vector of a regulatable promoter that is responsive to pharmacologic agents that are widely used and well tolerated in clinical practice represents such a control strategy. METHODS: A replication-deficient adenovirus or an adeno-associated virus containing a chimeric promoter composed of 5 glucocorticoid response elements and the murine thrombopoietin complementary DNA (AdGRE.mTPO or AAVGRE.mTPO) was administered to the hearts of Sprague-Dawley rats. Platelet levels were evaluated as a reporter of transgene activity with or without dexamethasone. For comparison, rats received a control adenovirus vector, AdCMV.mTPO or AdCMV.Null, and the control adeno-associated virus vector AAVCMV.luc, which encodes for the firefly luciferase (luc) gene. RESULTS: Platelet elevation in the AdGRE.mTPO group peaked 4 days after dexamethasone administration, with a return to baseline 1 week after the initial corticosteroid dose. Subsequent dexamethasone administration at 2 and 4 weeks resulted in similar but progressively decreased responses. The AAVGRE.mTPO group had 5 peak platelet levels to a minimum of 2.2-fold with respect to baseline without diminution with subsequent dexamethasone administrations out to 169 days. In contrast, the AdCMV.Null and AAVCMV.luc groups demonstrated no increase in platelet counts and the AdCMV.mTPO group demonstrated a slow rise to a single peak platelet count independent of dexamethasone administration. CONCLUSION: It may be possible to control on demand the expression of a gene transferred to the heart. This strategy should be useful in cardiac gene therapy.

The effect of anti-CD3 on the induction of non-MHC restricted cytolytic activity.

The effects of an anti-CD3 mAb on induction of non-MHC restricted cytolysis was investigated. Peripheral blood mononuclear cells (PBMC) from normal donors (29) and cancer patients (18) were cultured in 100 U/ml of interleukin-2 (rIL-2) with and without anti-CD3 mAb (OKT3, 10 ng/ml) for the first 48 hours of incubation. Thereafter, both PBMC cultures were maintained on rIL-2 up to 20 days. PBMC proliferation was enhanced 17-fold in number by day 20 when anti-CD3 mAb and rIL-2 was present during the first 48 hours but only 3-fold by day 20 when rIL-2 alone was present. Concomitantly anti-CD3 mAb but not Lym-1, an isotype matched control, inhibited the induction of lytic activity against both NK sensitive (K562) and NK resistant (Raji) target cell lines. Thus the inhibitory effect is dependent on anti-CD3 mAb stimulating the CD3/TCR T-cell receptor complex. While lytic activity was dependent on the concentration of rIL-2, inhibition of the induction phase of non-MHC restricted lytic activity was independent of the concentration of rIL-2. Flow cytometry analysis indicated that treatment with the anti-CD3 mAb increased the percentage of CD3 positive cells, CD4 positive cells and especially CD25 positive cells, but decreased th percentage of CD56 positive cells. Supernatants from anti-CD3 mAb stimulated cultures also inhibited the induction of non-MHC restricted lytic activity. Lymphokine analysis showed that supernatants of anti-CD3 mAb stimulated cultures had higher levels of TNF-alpha and IFN-gamma. However, TNF-alpha and IFN-gamma alone or in combination could not mediate the inhibitory effect. The inhibitory factor(s) was partially purified by sequential chromatography on matrices of controlled pore glass and Sepharose CL-6B. The molecular weight of the inhibitory factor(s) was less than 67K. These studies have identified a novel regulatory pathway controlling non-MHC restricted cytolysis. Perturbation of the T-cell CD3/TCR complex with the anti-CD3 mAb results in the secretion of a soluble mediator that down-regulates the induction of rIL-2 dependent non-MHC restricted cytolysis.

Successful case of adult ABO-incompatible liver transplantation: beneficial effects of intrahepatic artery infusion therapy: a case report.

BACKGROUND: In Japan ABO-incompatible liver transplantation has been done on >100 occasions up to 2003. However, <30% are cases involving adults. The difficultly of ABO-incompatible liver transplantation is associated with the high frequency of humoral rejection and local disseminated intravascular coagulation (DIC), leading to many postoperative complications. We report a successful case of adult ABO-incompatible liver transplantation with the use of an intrahepatic artery infusion. METHODS: A 36-year-old man with Wilson disease, underwent living donor liver transplantation from an ABO-incompatible donor. The immunosuppressive therapy included multiple perioperative plasmaphereses, splenectomy, and treatment with tacrolimus, methylprednisolone, and cyclophosphamide. The dose and blood level of tacrolimus were the same as in ABO-compatible cases. In addition to these therapies, we administered an intrahepatic arterial infusion with prostaglandin (PG) E1 alone. RESULTS: After perioperative plasmapheresis and cyclophosphamide, antidonor blood group antibody titers remained undiluted and without vascular complications throughout the postoperative course, but there was a tendency for bleeding that continued for 10 days after transplantation. On postoperative day 10, a reexploration was performed for intraabdominal bleeding. During another operation on postoperative day 59 a biloma was found and drained. The patient has now survived for 120 days after transplantation with normal liver function. CONCLUSIONS: Beneficial effect of intrahepatic artery infusion with PGE1 seems to be useful in adult ABO-incompatible liver transplantation.

[Non-small-cell lung cancer (NSCLC) in an elderly patient--a case of squamous cell carcinoma successfully treated with chemotherapy using vinorelbine].

We report an elderly patient with squamous cell carcinoma who was successfully treated with chemotherapy using vinorelbine. A 76-year-old man was referred to our hospital for evaluation of a nodular shadow in the left lung. Chest CT scam showed a 3-cm tumor shadow in left S9 and a 1-cm small nodule in right S2. Transbronchial lung biopsy yielded a diagnosis of squamous cell carcinoma. The clinical stage was IV (cT2N2M1). The patient first underwent chemotherapy consisting of cisplatin (CDDP) 80 mg/m2 on day 1 and vinorelbine (VNB) 20 mg/m2 on days 1, 8, and 15, which generated tumor shrinkage of 48% as well as transient elevation of grade 1 in serum creatinine. The 2 cycles of chemotherapy using vinorelbine only (VNB 20 mg/m2 on days 1, 8, 15) produced a tumor reduction of 70% with grade-1 decrease of granulocytes. The low grade of toxicity enabled us to treat the patient in our outpatient office for the second cycle of the regimen. This case suggests that chemotherapy using low-dose vinorelbine might be suitable to treat elderly patients with NSCLC in outpatient settings.

In vivo comet assay of a novel galacto-oligosaccharide in rats.

A novel galacto-oligosaccharide (GOS) manufactured by a two-step enzyme reaction of lactose was examined in a comet assay for its potential to induce DNA damage in vivo by estimating the DNA fragmentation level in the cellular nuclei of the glandular stomach, colon, and peripheral blood. GOS was orally administered at doses of 0 (vehicle alone), 500, 1000, and 2000 mg/kg/day to five male Crl: CD(Sprague Dawley) rats per group three times (48, 24, and 3 h before the animals were terminated). The specimens were prepared in accordance with the standard protocol (version 14.2) of the "International Validation of the In Vivo Rodent Alkaline Comet Assay for the Detection of Genotoxic Carcinogens" organized by the Japanese Center for the Validation of Alternative Methods. No significant differences in the percentage of DNA in the tail were observed between the GOS-treated groups and vehicle controls in any of the organs evaluated. Additionally, no GOS-related clinical signs or effects on body weight were seen. Based on these results, the comet assay of GOS in the glandular stomach, colon, and peripheral blood using rats was judged negative. Therefore, it is concluded that GOS did not induce DNA damage in vivo under the conditions employed in this study.