125I seeds irradiation inhibits tumor growth and induces apoptosis by Ki-67, P21, survivin, livin and caspase-9 expression in lung carcinoma xenografts.

BACKGROUND: Lung cancer is a fatal disease and a serious health problem worldwide. Patients are usually diagnosed at an advanced stage, and the effectiveness of chemotherapy for such patients is very limited. Iodine 125 seed (125I) irradiation can be used as an important adjuvant treatment for lung carcinoma. The purpose of this study was to examine the role of irradiation by 125I seeds in human lung cancer xenograft model and to determine the underlying mechanisms involved, with a focus on apoptosis. METHODS: 40 mice with A549 lung adenocarcinoma xenografts were randomly divided into 4 groups: control group (n = 10), sham seed (0 mCi) implant group (n = 10), 125I seed (0.6 mCi) implant group (n = 10) and 125I seed (0.8 mCi) implant group (n = 10), respectively. The body weight and tumor volume, were recorded every 4 days until the end of the study. Apoptotic cells were checked by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and activities of caspase-3 and caspase-8 enzyme were tested. Expression of P21, survivin, livin, caspase-9 and proliferating cell nuclear antigen (Ki-67) was detected with immunohistochemical staining. RESULTS: The results of TUNEL staining assays showed that 125I seed irradiation suppresses the growth of lung cancer xenografts in nude mice and induced apoptosis. The activity of caspase-3 and caspase-8 was significantly higher. The expression levels Ki67, survivin and livin were substantially downregulated, while P21 and caspase-9 protein expression were significantly increased following 125I seed irradiation. This study revealed that 125I seed irradiation could significantly change apoptosis-related protein in human lung cancer xenografts. CONCLUSIONS: Overall, our study demonstrates that radiation exposure by 125I seeds could be a new treatment option for lung cancer.

MicroRNA-378 enhances radiation response in ectopic and orthotopic implantation models of glioblastoma.

Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor, which is virtually incurable due to its therapeutic resistance to radiation and chemotherapy. To develop novel therapeutic approaches for treatment of GBM, we examined the role of miR-378 on tumor growth, angiogenesis, and radiation response in ectopic and orthotopic U87 glioblastoma models. Cell and tumor growth rates, in vitro and in vivo radiation sensitivities, and tumor vascular density were evaluated in U87-GFP and U87-miR-378 tumor lines. Ectopic tumor response to radiation was evaluated under normal blood flow and clamp hypoxic conditions. Results show that in vitro, miR-378 expression moderately increased cell growth rate and plating efficiency, but did not alter radiation sensitivity. U87-miR-378 tumors exhibited a higher transplantation take rate than U87-GFP tumors. In vivo, under oxygenated condition, subcutaneous U87-miR-378 tumors receiving 25 Gy showed a tendency for longer tumor growth delay (TGD) than control U87-GFP tumors. In contrast, under hypoxic condition, U87-miR-378 xenografts exhibited substantially shorter TGD than U87-GFP tumors, indicating that under normal blood flow conditions, U87-miR-378 tumors were substantially more oxygenated than U87-GFP tumors. Intracranial multi-photon laser-scanning microscopy demonstrated increased vascular density of U87-miR-378 versus control U87-GFP tumors. Finally, miR-378 increased TGD following 12 Gy irradiation in U87 intracranial xenografts, and significantly prolonged survival of U87-miR-378 tumor-bearing mice (P = 0.04). In conclusion, higher miR-378 expression in U87-miR-378 cells promotes tumor growth, angiogenesis, radiation-induced TGD, and prolongs survival of orthotopic tumor-bearing hosts. Regulation of VEGFR2 by miR-378 significantly increased vascular density and oxygenation in U87 xenografts.

Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts.

Study question: Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys? Summary answer: In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells. What is known already: Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation. Their depletion was demonstrated by morphometric and histological analyses. The effect of irradiation on expression of Sertoli and peritubular myoid cell markers, however, has not yet been described. Study design, size, duration: The testes of two prepubertal macaques (Macaca fascicularis) were dissected into testicular fragments. Fragments were randomly exposed in vitro to one of the following three doses of irradiation: 0 Gy, n = 60; 1 Gy, n = 54; 4 Gy, n = 72. Non-irradiated control fragments (0 Gy) were placed into the Faxitron for 6.6 min without irradiation. For 1 Gy and 4 Gy irradiation was applied for 1.7 and 6.6 min, respectively. Grafts were then either immediately analyzed or subcutaneously implanted under the back skin of 39 nude mice and analyzed after 6.5 months. Participants/materials setting methods: Post grafting, 133 testicular xenografts were retrieved. The body weight, serum testosterone level and seminal vesical weight of the host mice as well as the number and weight of retrieved grafts were determined. Larger grafts were used to evaluate both mRNA expression profiles and protein expression patterns. In total, 71 testicular fragments were used for morphometric and histological analysis while 68 fragments were analyzed for gene expression. For PCR arrays, M. fascicularis-specific primer sequences were employed. Irradiation-induced changes in the transcript levels of 34 marker genes were determined for each testicular graft. The effects of irradiation on peritubular myoid cells and Sertoli cells were confirmed by immunohistochemical analysis of chemokine (C-X-C motif) ligand type 11 (CXCL11), alpha smooth muscle actin (SMA) and chemokine (C-X-C motif) ligand type 12 (CXCL12). Main results and the role of chance: The four testes gave rise to 106 xenografts, which were individually analyzed, limiting the role of chance despite using only two monkeys in the study. Prior to grafting, the two donors displayed spermatogonia as the most advanced germ cell type in 95% and 70% of seminiferous tubules, respectively, while remaining tubules contained SCO. No spermatocytes were encountered prior to grafting in either monkey. After 6.5 months, non-irradiated grafts displayed spermatocytes in 15.4% and 1.8% of seminiferous tubules indicating an induction of meiosis. Irradiation resulted in a complete absence of spermatocytes. The percentage of seminiferous tubules containing spermatogonia declined in a dose-dependent manner. In non-irradiated xenografts, ~40% of tubules contained spermatogonia. This proportion was reduced to 3.4% and 4.3% in the 1 Gy treated group and to 1.3% and 0.2% in 4 Gy irradiated grafts. A dose-dependent decline in mRNA levels of selected germ cell marker genes supported the morphologically detected loss of germ cells. Irradiation had no effect on CXCL12 transcript levels. At the protein level, CXCL12-positive Sertoli cells were most abundant in the 1 Gy group compared to the 4 Gy group (P < 0.05), indicating a potential role of CXCL12 during recovery of primate spermatogenesis. The most prominent radiation-evoked changes were for CXCL11, which was localized to smooth muscle cells of blood vessels and seminiferous tubules. Transcript levels declined in a dose-dependent manner in grafts from both monkeys (MM687: P < 0.01 (0 Gy versus 4 Gy), MM627: P < 0.05 (0 Gy versus 4 Gy), P < 0.001 (1 Gy versus 4 Gy)). CXCL11 patterns of protein expression revealed irradiation-dependent changes as well. That peritubular cells are affected by X-irradiation was substantiated by changes at the transcript level between 1 and 4 Gy exposed groups (P < 0.01) and at the protein level of SMA (P < 0.05, 0 Gy versus 4 Gy). Large scale data: n/a. Limitations, reasons for caution: The spermatogonial stem cell system in primates is remarkably different from rodents. Therefore, data from a non-human primate may be more relevant to man. However, species-specific differences amongst primates cannot be fully excluded and the use of only two donors may raise concerns toward the generalization of the findings. There may also be important differences across the prepubertal period (e.g. infancy, early childhood) that are not represented by the ages included in the present study. Wider implications of the findings: This study is the first to indicate relevant testicular somatic cell responses following irradiation of prepubertal primate tissue. In addition to the well-known depletion of germ cells, the changes in Sertoli, and in particular peritubular myoid, cells may have important consequences for spermatogenic recovery. These novel findings should be taken into consideration when irradiation effects are assessed in tumor survivors. Study funding and competing interest(s): Interdisciplinary Center for Clinical Research (IZKF) Münster (Schl2/001/13) and the Excellence Cluster 'Cells in Motion' at the University Münster. There are no conflicts of interest to declare.

Effectiveness of hydrogen peroxide and electron-beam irradiation treatment for removal and inactivation of viruses in equine-derived xenografts.

Bone grafting is a common procedure for bone reconstruction in dentistry, orthopedics, and neurosurgery. A wide range of grafts are currently used, and xenografts are regarded as an interesting alternative to autogenous bone because all mammals share the same bone mineral component composition and morphology. Antigens must be eliminated from bone grafts derived from animal tissues in order to make them biocompatible. Moreover, the processing method must also safely inactivate and/or remove viruses or other potential infectious agents. This study assessed the efficacy of two steps applied in manufacturing some equine-derived xenografts: hydrogen-peroxide and e-beam sterilization treatments for inactivation and removal of viruses in equine bone granules (cortical and cancellous) and collagen and pericardium membranes. Viruses belonging to three different human viral species (Herpes simplex virus type 1, Coxsackievirus B1, and Influenzavirus type A H1N1) were selected and used to spike semi-processed biomaterials. For each viral species, the tissue culture infective dose (TCID50) on cell lines and the number of genome copies through qPCR were assessed. Both treatments were found to be effective at virus inactivation. Considering the model viruses studied, the application of hydrogen peroxide and e-beam irradiation could also be considered effective for processing bone tissue of human origin.

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization.

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.

Evaluation of porcine xenograft in collateral ligament reconstruction in beagle dogs.

This study evaluated the effectiveness of irradiated porcine tendon xenografts for lateral collateral ligament (LCL) reconstruction. Twenty healthy adult beagle dogs underwent LCL reconstruction using irradiated porcine tendons treated with poly-gamma-glutamic acid. Serological and histological assessments were performed to evaluate host immunological response at 3 and 12 months after surgery. The healing and functional integrity of the LCL reconstructions were assessed by mechanical testing and gait analysis. Histological assessment of the porcine xenografts showed gradual host cellular infiltration and graft collagen remodeling during the healing process. Porcine xenografts showed angiogenesis and no signs of inflammatory reaction. Additionally, biomechanical and gait evaluations supported graft functional integration with no differences between normal and porcine xenograft reconstruction at 12 months after surgery. Irradiated porcine xenografts showed greater cellular responses and healing properties in short- and long-term evaluations. Irradiated porcine tendons appear to be useful as xenografts for the reconstruction of damaged ligaments.

Evaluation of hyperpolarized [1-¹³C]-pyruvate by magnetic resonance to detect ionizing radiation effects in real time.

Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.

Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation.

An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.

Comparison of Iodine-125 seeds implantation and afterloading radiotherapy in murine models of human cervical cancer.

OBJECTIVE: To compare the efficacies and side effects of Iodine-125 (125I) seeds implantation with afterloading radiotherapy on tumor xenografts derived from Hela cells. METHODS: Mice bearing Hela cells were randomly divided into three groups: two therapeutic groups receiving four 125I seeds implantation and afterloading therapy, respectively, and the one control group without any intervention. Comparisons were evaluated in aspects of curative efficacies (tumor volume, tumor inhibition rate and metastasis) and side effects (body weight, ovarian endocrine functions, skin lesion surrounding the tumor). RESULTS: The xenografts tumor volume of therapeutic groups were significantly smaller than that of controls(p < 0.05),both of the 125I seeds implantation and afterloading therapy showed excellent tumor inhibition rate. Furthermore, 125I seeds implantation had milder side effects on skin, weight loss, ovarian endocrine functions. CONCLUSIONS: (125)I seed implantation may be an alternative minimally invasive therapy for cervical cancer.

11 kGy gamma irradiated demineralized bone matrix enhances osteoclast activity.

BACKGROUND: Demineralized bone matrix (DBM) allografts are widely used in orthopaedic clinics. However, the biological impact on its osteoinductivity after its sterilization process by gamma irradiation is not well studied. Furthermore, little is known about the relationship between residual calcium levels on osteoinductivity. HYPOTHESIS: We hypothesize that low-dose gamma irradiation retains the osteoinducitivity properties of DBM and causes ectopic bone formation. MATERIALS AND METHODS: A randomised animal trial was performed to compare tissue and molecular responses of low-dose (11 kGy) gamma irradiated and non-irradiated human DBM at 6 weeks post-intramuscular implantation using an athymic rat model. In addition, we correlated residual calcium levels and bone formation in gamma irradiated DBM. RESULTS: A modified haematoxylin and eosin stain identified ectopic bony capsules at all implanted sites with no significant difference on the amount of new bone formed between the groups. Statistically significantly lower ratio of alkaline phosphatase expression over tartrate-resistant acid phosphatase and/or cathepsin K expressions was found between the groups. DISCUSSION: This study found that low-dose gamma irradiated DBM, which provides a sterility assurance level of 10(-6) for bone allografts, retained osteoinductivity but exhibited significantly enhanced osteoclastic activity. Furthermore, this is the first study to find a positive correlation between residual calcium levels and bone formation in gamma irradiated DBM.

Ultraviolet B irradiation induces the expression of hornerin in xenotransplanted human skin.

Ultraviolet (UV) irradiation exerts numerous effects on the skin. Exposure of human skin to UVB at doses that induce mild sunburn reactions causes epidermal hyperproliferation and alterations in the expression of several epidermal differentiation markers. This study investigated the effects of UVB irradiation on the expression of hornerin, a member of the S100 fused-type protein family, using the xenotransplantation of normal human skin onto nude mice. Hornerin mRNA was detected in the UVB-irradiated skin on day 2 using RT-PCR. In accordance with the results of the RT-PCR, the expression of hornerin was induced in the granular layers of the UVB-exposed skin beginning two days after UVB irradiation and occurred in parallel with the expressions of cytokeratin 6 and Ki67. This finding suggests that hornerin induction in UVB-irradiated skin might be associated with epidermal hyperproliferation. This study demonstrated that hornerin is a protein whose expression is changed by UVB irradiation and suggests that the expression of hornerin might be a useful marker of acute UV damage in skin.

New bone formation around implants inserted on autologous and xenografts irradiated or not with IR laser light: a histomorphometric study in rabbits.

Use of biomaterials and light on bone grafts has been widely reported. This work assessed the influence of low-level laser therapy (LLLT) on bone volume (BV) and bone implant contact (BIC) interface around implants inserted in blocks of bovine or autologous bone grafts (autografts), irradiated or not, in rabbit femurs. Twenty-four adult rabbits were divided in 8 groups: AG: autograft; XG: xenograft; AG/L: autograft + laser; XG/L: xenograft + laser; AG/I: autograft + titanium (Ti) implant; XG/I: xenograft + Ti implant; AG/I/L: autograft + Ti implant + laser; and XG/I/L: xenograft + Ti implant + laser. The animals received the Ti implant after incorporation of the grafts. The laser parameters in the groups AG/L and XG/L were λ=780 nm, 70 mW, CW, 21.5 J/cm 2 , while in the groups AG/I/L and XG/I/L the following parameters were used: λ=780 nm, 70 mW, 0.5 cm 2 (spot), 4 J/cm 2 per point (4), 16 J/cm 2 per session, 48 h interval × 12 sessions, CW, contact mode. LLLT was repeated every other day during 2 weeks. To avoid systemic effect, only one limb of each rabbit was double grafted. All animals were sacrificed 9 weeks after implantation. Specimens were routinely stained and histomorphometry carried out. Comparison of non-irradiated and irradiated grafts (AG/L versus AG and XG/L versus XG) showed that irradiation increased significantly BV on both grafts (p=0.05, p=0.001). Comparison between irradiated and non-irradiated grafts (AG/I/L versus AG/I and XG/I/L versus XG/I) showed a significant (p=0.02) increase of the BIC in autografts. The same was seen when xenografts were used, without significant difference. The results of this investigation suggest that the use of LLLT is effective for enhancing new bone formation with consequent increase of bone-implant interface in both autologous grafts and xenografts.

New Bone Formation around Implants Inserted on Autologous and Xenografts Irradiated or not with IR Laser Light: A Histomorphometric Study in Rabbits

Use of biomaterials and light on bone grafts has been widely reported. This work assessed the influence of low-level laser therapy (LLLT) on bone volume (BV) and bone implant contact (BIC) interface around implants inserted in blocks of bovine or autologous bone grafts (autografts), irradiated or not, in rabbit femurs. Twenty-four adult rabbits were divided in 8 groups: AG: autograft; XG: xenograft; AG/L: autograft + laser; XG/L: xenograft + laser; AG/I: autograft + titanium (Ti) implant; XG/I: xenograft + Ti implant; AG/I/L: autograft + Ti implant + laser; and XG/I/L: xenograft + Ti implant + laser. The animals received the Ti implant after incorporation of the grafts. The laser parameters in the groups AG/L and XG/L were λ=780 nm, 70 mW, CW, 21.5 J/cm 2 , while in the groups AG/I/L and XG/I/L the following parameters were used: λ=780 nm, 70 mW, 0.5 cm 2 (spot), 4 J/cm 2 per point (4), 16 J/cm 2 per session, 48 h interval × 12 sessions, CW, contact mode. LLLT was repeated every other day during 2 weeks. To avoid systemic effect, only one limb of each rabbit was double grafted. All animals were sacrificed 9 weeks after implantation. Specimens were routinely stained and histomorphometry carried out. Comparison of non-irradiated and irradiated grafts (AG/L versus AG and XG/L versus XG) showed that irradiation increased significantly BV on both grafts (p=0.05, p=0.001). Comparison between irradiated and non-irradiated grafts (AG/I/L versus AG/I and XG/I/L versus XG/I) showed a significant (p=0.02) increase of the BIC in autografts. The same was seen when xenografts were used, without significant difference. The results of this investigation suggest that the use of LLLT is effective for enhancing new bone formation with consequent increase of bone-implant interface in both autologous grafts and xenografts.

O uso de biomateriais e luz em enxertos ósseos têm sido relatados. Esse trabalho avaliou a influência do laser baixa potência - LBP no volume ósseo (VO) e superfície de contato osso-implante (COI) ao redor de implantes dentários inseridos em blocos de enxerto bovino ou autólogos incorporados, irradiados ou não, em fêmures de coelho. Vinte e quatro coelhos adultos foram divididos em 8 grupos: EA: enxerto autólogo; EX: enxerto xenógeno; EA/L: enxerto autólogo + laser; EX/L: enxerto xenógeno + laser; EA/I: enxerto autólogo + implante; EX/I: enxerto xenógeno + implante; EA/I/L: enxerto autólogo + implante de titânio + laser; EX/I/L: enxerto xenógeno + implante de titânio + laser. Os animais receberam um implante de titânio após a incorporação dos enxertos. Os parâmetros de laser nos grupos EA/L e EX/L foram λ =780 nm, 70 mW, CW, 21,5 J/cm 2 ), enquanto que nos grupos EA/I/L e EX/I/L os seguintes parâmetros de laser foram utilizados: λ =780 nm, 70 mW, 0,5 cm 2 (spot), 4 J/cm 2 por ponto (4), 16 J/cm 2 por sessão, intervalo de 48 h × 12 sessões, CW, modo contato. O LBP foi repetido a cada 48 h (2 semanas). Para evitar efeito sistêmico apenas um membro de cada coelho foi duplamente enxertado. Todos os animais foram sacrificados 9 semanas após o implante. Os espécimes foram corados rotineiramente e histomorfometria foi realizada. A comparação dos enxertos não-irradiados e irradiados (EA/L versus EA e EX/L versus EX) mostrou que a irradiação aumentou significantemente (p=0,02) o VO para ambos os tipos de enxertos (p=0,05, p=0,001). A comparação dos enxertos não-irradiados e irradiados (EA/I/L versus EA/I e EX/I/L versus EX/I) mostrou um aumento significante (p=0,02) do COI nos enxertos autólogos e xenógenos sem diferença estatística. Os resultados desta investigação sugerem que o uso de LBP é efetivo para aumentar a neoformação óssea com consequente aumento do COI em enxertos autólogos e xenógenos.