Suspension-Induced Stem Cell Transition: A Non-Transgenic Method to Generate Adult Stem Cells from Mouse and Human Somatic Cells.

Adult stem cells (ASCs) can be cultured with difficulty from most tissues, often requiring chemical or transgenic modification to achieve adequate quantities. We show here that mouse primary fibroblasts, grown in suspension, change from the elongated and flattened morphology observed under standard adherent culture conditions of generating rounded cells with large nuclei and scant cytoplasm and expressing the mesenchymal stem cell (MSC) marker (Sca1; Ly6A) within 24 h. Based on this initial observation, we describe here a suspension culture method that, irrespective of the lineage used, mouse fibroblast or primary human somatic cells (fibroblasts, hepatocytes and keratinocytes), is capable of generating a high yield of cells in spheroid form which display the expression of ASC surface markers, circumventing the anoikis which often occurs at this stage. Moreover, mouse fibroblast-derived spheroids can be differentiated into adipogenic and osteogenic lineages. An analysis of single-cell RNA sequence data in mouse fibroblasts identified eight distinct cell clusters with one in particular comprising approximately 10% of the cells showing high levels of proliferative capacity expressing high levels of genes related to MSCs and self-renewal as well as the extracellular matrix (ECM). We believe the rapid, high-yield generation of proliferative, multi-potent ASC-like cells via the process we term suspension-induced stem cell transition (SIST) could have significant implications for regenerative medicine.

The impact of fingolimod on Treg function in brain ischaemia.

Fingolimod has generally shown neuroprotective effects in stroke models. Here, we tested the hypothesis that fingolimod modulates T-cell cytokine production towards a regulatory phenotype. Second, we investigated how fingolimod altered the Treg suppressive function and the sensitivity of effector T cells to regulation. Mice that had underwent the permanent electrocoagulation of the left middle cerebral artery received saline or fingolimod (0.5 mg/kg) daily for 10-days post-ischaemia. Fingolimod improved neurobehavioural recovery compared to saline control and increased Treg frequency in the periphery and brain. Tregs from fingolimod-treated animals had a higher expression of CCR8. Fingolimod increased the frequencies of CD4+ IL-10+ , CD4+ IFN-γ+ and CD4+ IL-10+ IFN-γ+ cells in spleen and blood, and CD4+ IL-17+ cells in the spleen, with only minor effects on CD8+ T-cell cytokine production. Treg from post-ischaemic mice had reduced suppressive function compared to Treg from non-ischaemic mice. Fingolimod treatment rescued this function against saline-treated but not fingolimod-treated CD4+ effector T cells. In conclusion, fingolimod seems to improve the suppressive function of Treg post-stroke while also increasing the resistance of CD4+ effector cells to this suppression. Fingolimod's capacity to increase both effector and regulatory functions may explain the lack of consistent improvement in functional recovery in experimental brain ischaemia.

Adaptation of transgene mRNA translation boosts the anticancer efficacy of oncolytic HSV1.

BACKGROUND: Transgenes deliver therapeutic payloads to improve oncolytic virus immunotherapy. Transgenes encoded within oncolytic viruses are designed to be highly transcribed, but protein synthesis is often negatively affected by viral infection, compromising the amount of therapeutic protein expressed. Studying the oncolytic herpes simplex virus-1 (HSV1), we found standard transgene mRNAs to be suboptimally translated in infected cells. METHODS: Using RNA-Seq reads, we determined the transcription start sites and 5'leaders of HSV1 genes and uncovered the US11 5'leader to confer superior activity in translation reporter assays. We then incorporated this 5'leader into GM-CSF expression cassette in oncolytic HSV1 and compared the translationally adapted oncolytic virus with the conventional, leaderless, virus in vitro and in mice. RESULTS: Inclusion of the US11 5'leader in the GM-CSF transgene incorporated into HSV1 boosted translation in vitro and in vivo. Importantly, treatment with US11 5'leader-GM-CSF oncolytic HSV1 showed superior antitumor immune activity and improved survival in a syngeneic mouse model of colorectal cancer as compared with leaderless-GM-CSF HSV1. CONCLUSIONS: Our study demonstrates the therapeutic value of identifying and integrating platform-specific cis-acting sequences that confer increased protein synthesis on transgene expression.

Recombinant pregnancy-specific glycoprotein-1-Fc reduces functional deficit in a mouse model of permanent brain ischaemia.

Background: The well-characterised role of the immune system in acute ischaemic stroke has prompted the search for immunomodulatory therapies. Pregnancy-specific glycoproteins (PSGs) are a group of proteins synthesised by placental trophoblasts which show immunomodulatory properties. The aim of this study was to determine whether a proposed PSG1-based therapeutic enhanced recovery in a mouse model of brain ischaemia and to explore possible immunomodulatory effects. Methods: Mice underwent permanent electrocoagulation of the left middle cerebral artery (pMCAO). They received saline (n = 20) or recombinant pregnancy-specific glycoprotein-1-alpha "fused" to the Fc domain of IgG1 (rPSG1-Fc) (100 µg) (n = 22) at 1 h post-ischaemia. At 3 and 5 days post-ischaemia, neurobehavioural recovery was assessed by the grid-walking test. At 5 days post-ischaemia, lesion size was determined by NeuN staining. Peripheral T cell populations were quantified via flow cytometry. Immunohistochemistry was used to quantify ICAM-1 expression and FoxP3+ cell infiltration in the ischaemic brain. Immunofluorescence was employed to determine microglial activation status via Iba-1 staining.Results: rPSG1-Fc significantly enhanced performance in the grid-walking test at 3 and 5 days post-ischaemia. No effect on infarct size was observed. A significant increase in circulating CD4+ FoxP3+ cells and brain-infiltrating FoxP3+ cells was noted in rPSG1-Fc-treated mice. Among CD4+ cells, rPSG1-Fc enhanced the expression of IL-10 in spleen, blood, draining lymph nodes, and non-draining lymph nodes, while downregulating IFN-γ and IL-17 in spleen and blood. A similar cytokine expression pattern was observed in CD8+ cells. rPSG1-Fc reduced activated microglia in the infarct core. Conclusion: The administration of rPSG1-Fc improved functional recovery in post-ischaemic mice without impacting infarct size. Improved outcome was associated with a modulation of the cytokine-secreting phenotype of CD4+ and CD8+ T cells towards a more regulatory phenotype, as well as reduced activation of microglia. This establishes proof-of-concept of rPSG1-Fc as a potential stroke immunotherapy.

Preclinical Evaluation of Fingolimod in Rodent Models of Stroke With Age or Atherosclerosis as Comorbidities.

Preclinical data indicate that fingolimod improves outcome post-ischaemia. This study used a rigorous study design in normal male C57BL/6JOlaHsd mice and in mice with common stroke comorbidities to further evaluate the translational potential of fingolimod. Stroke was induced via middle cerebral artery electrocoagulation in 8-9-week old mice (young mice), 18 month old mice (aged mice), and in high-fat diet-fed 22-week old ApoE-/- mice (hyperlipidaemic mice). Recovery was evaluated using motor behavioural tests 3 and 7 days after stroke. Tissue damage was evaluated at 7 days. A lower dose of fingolimod, 0.5 mg/kg, but not 1 mg/kg, increased lesion size but decreased ipsilateral brain atrophy in younger mice, without an effect on behavioural outcomes. Fingolimod-treated aged mice showed a significant improvement over saline-treated mice in the foot fault test at 7 days. Fingolimod-treated hyperlipidaemic mice showed a decreased infarct size but no difference in behavioural performance. Increasing fingolimod treatment time to 10 days showed no benefit in young mice. Pooled data showed that fingolimod improved performance in the foot fault test. Flow cytometry studies showed that fingolimod had marked effects on T cell frequencies in various tissues. The results show that the effects of fingolimod in stroke are less robust than the existing literature might indicate and may depend on the inflammatory status of the animals.

Autophagy in Tumor Immunity and Viral-Based Immunotherapeutic Approaches in Cancer.

Autophagy is a fundamental catabolic process essential for the maintenance of cellular and tissue homeostasis, as well as directly contributing to the control of invading pathogens. Unsurprisingly, this process becomes critical in supporting cellular dysregulation that occurs in cancer, particularly the tumor microenvironments and their immune cell infiltration, ultimately playing a role in responses to cancer therapies. Therefore, understanding "cancer autophagy" could help turn this cellular waste-management service into a powerful ally for specific therapeutics. For instance, numerous regulatory mechanisms of the autophagic machinery can contribute to the anti-tumor properties of oncolytic viruses (OVs), which comprise a diverse class of replication-competent viruses with potential as cancer immunotherapeutics. In that context, autophagy can either: promote OV anti-tumor effects by enhancing infectivity and replication, mediating oncolysis, and inducing autophagic and immunogenic cell death; or reduce OV cytotoxicity by providing survival cues to tumor cells. These properties make the catabolic process of autophagy an attractive target for therapeutic combinations looking to enhance the efficacy of OVs. In this article, we review the complicated role of autophagy in cancer initiation and development, its effect on modulating OVs and immunity, and we discuss recent progress and opportunities/challenges in targeting autophagy to enhance oncolytic viral immunotherapy.

Histological, behavioural and flow cytometric datasets relating to acute ischaemic stroke in young, aged and ApoE-/- mice in the presence and absence of immunomodulation with fingolimod.

In this work, the sphingosine-1-phosphate receptor modulator fingolimod was assessed as a preclinical candidate for the treatment of acute ischaemic stroke according to the Stroke Therapy Academic Industry Roundtable (STAIR) preclinical recommendations. Young (15-17 weeks), aged (72-73 weeks), and ApoE-/- mice (20-21 weeks) fed a high fat diet (all C57BL/6 mice) underwent permanent electrocoagulation of the left middle cerebral artery. Mice received either saline or fingolimod (0.5 mg/kg or 1 mg/kg) at 2-, 24-, and 48-hours post-ischaemia via intraperitoneal (i.p.) injection. Another cohort of young mice (8-9 and 17-19 weeks) received short-term (5 days) or long-term (10 days) fingolimod (0.5 mg/kg) treatment in a treatment duration study. For young, aged, and ApoE-/- mice, motor behavioural tests (cylinder and grid-walking) were performed at days 0, 3, and 7 post-ischaemia to evaluate neurobehavioural recovery. In the treatment duration study, the grid-walking test was performed at days 0, 2, 5 and 10 post-ischaemia. Brain tissue sections were stained with haematoxylin and eosin (H&E), and NeuN to quantify tissue damage. Flow cytometry was used to quantify T cell populations in blood, spleen, and lymph nodes. The data presented in this article improves our understanding of the potential neuroprotective and immunomodulatory effects of fingolimod in a mouse model of brain ischaemia. Such data may be significant in the design of future preclinical and clinical stroke studies for fingolimod.

The effect of fingolimod on regulatory T cells in a mouse model of brain ischaemia.

BACKGROUND: The role of the immune system in stroke is well-recognised. Fingolimod, an immunomodulatory agent licensed for the management of relapsing-remitting multiple sclerosis, has been shown to provide benefit in rodent models of stroke. Its mechanism of action, however, remains unclear. We hypothesised fingolimod increases the number and/or function of regulatory T cells (Treg), a lymphocyte population which promotes stroke recovery. The primary aim of this study was to rigorously investigate the effect of fingolimod on Tregs in a mouse model of brain ischaemia. The effect of fingolimod in mice with common stroke-related comorbidities (ageing and hypercholesteremia) was also investigated. METHODS: Young (15-17 weeks), aged C57BL/6 mice (72-73 weeks), and ApoE-/- mice fed a high-fat diet (20-21 weeks) underwent permanent electrocoagulation of the left middle cerebral artery. Mice received either saline or fingolimod (0.5 mg/kg or 1 mg/kg) at 2, 24, and 48 h post-ischaemia via intraperitoneal injection. Another cohort of young mice (8-9, 17-19 weeks) received short-term (5 days) or long-term (10 days) fingolimod (0.5 mg/kg) treatment. Flow cytometry was used to quantify Tregs in blood, spleen, and lymph nodes. Immunohistochemistry was used to quantify FoxP3+ cell infiltration into the ischaemic brain. RESULTS: Fingolimod significantly increased the frequency of Tregs within the CD4+ T cell population in blood and spleen post-ischaemia in all three mouse cohorts compared to untreated ischemic mice. The highest splenic Treg frequency in fingolimod-treated mice was observed in ApoE-/- mice (9.32 ± 1.73% vs. 7.8 ± 3.01% in young, 6.09 ± 1.64% in aged mice). The highest circulating Treg frequency was also noted in ApoE-/- mice (8.39 ± 3.26% vs. 5.43 ± 2.74% in young, 4.56 ± 1.60% in aged mice). Fingolimod significantly increased the number of FoxP3+ cells in the infarct core of all mice. The most pronounced effects were seen when mice were treated for 10 days post-ischaemia. CONCLUSIONS: Fingolimod increases Treg frequency in spleen and blood post-ischaemia and enhances the number of FoxP3+ cells in the ischaemic brain. The effect of fingolimod on this regulatory cell population may underlie its neuroprotective activity and could be exploited as part of future stroke therapy.

Minimally invasive lateral approaches for the treatment of spinal tumors: single-position surgery without the "flip".

Although primary tumors of the spine and neural elements are rare, metastatic disease to the spine is quite common. Traditionally, surgical treatment for spinal tumor patients involves open decompression with or without stabilization. The single-position minimally invasive (MIS) lateral approach, which has been recently described over the recent decade, allows simultaneous access to the anterior and posterior columns with the patient positioned in the lateral decubitus position. Herein, we review the application of single-position MIS lateral surgery for the treatment of spinal neoplasm. The aim was to review the evolution, operative technique, outcomes, and complications associated with MIS lateral approaches for spinal tumors. The history of spinal tumor diagnosis and management are reviewed and discussed as well as the author's experience and literature regarding spinal tumor treatment outcome and surgical complications, with particular attention to single-position, MIS lateral approaches. In addition, the author's surgical technique is outlined in detail for thoracic, thoracolumbar and lumbar tumors. Furthermore, there are specific indications and complications associated with the surgical treatment of spinal tumors, and the MIS, single-position lateral approach, when applied appropriately, allows for concurrent access to the anterior and posterior column while mitigating the complications associated with traditional, open posterior-based approaches. In the treatment of spinal neoplasms, the goals of surgery are dictated by a number of tumor-specific and patient-specific factors. Therefore, operative treatment of tumors in the future may be a consolidation of historical surgical techniques and MIS, single-position lateral approaches. Regardless, multidisciplinary management is imperative for the individualized treatment of the patient and optimization of outcome.

Acute Treatment With Fingolimod Does Not Confer Long-Term Benefit in a Mouse Model of Intracerebral Haemorrhage.

Intracerebral haemorrhage (ICH) has no specific treatment, but accounts for up to 15% of all strokes and has the highest mortality. Fingolimod (FTY720) is an immunomodulator approved for the management of multiple sclerosis, with abundant evidence of efficacy in experimental ischemic stroke, and more limited evidence in experimental ICH. The goal of this study was to confirm the efficacy of fingolimod in experimental ICH using rigorous and statistically well-powered studies. ICH was induced in C57BL/6JOlaHsd male and female mice by intrastriatal bacterial collagenase injection. Fingolimod (0.5 mg/kg) or saline was administered intraperitoneally after 0.5, 24 and 72 h, in a randomized and blinded manner. Functional improvement with cylinder, wire hanging, and foot fault tests was evaluated one and two weeks later. Lesion volume and hemispheric atrophy were quantified at the 14-day endpoint. There was a higher mortality in saline-treated females compared to fingolimod-treated females and saline-treated males. There was no treatment- or gender-related difference in the behavioural tests. Histological outcome measures did not differ between any of the groups. These results, contrasting with those of previous studies of fingolimod in experimental ICH, emphasize the importance of rigorous testing of this agent in models more representative of the clinical situation.

Sequencing of Androgen-Deprivation Therapy With External-Beam Radiotherapy in Localized Prostate Cancer: A Phase III Randomized Controlled Trial.

PURPOSE: Dose-escalated radiotherapy (RT) with androgen-deprivation therapy (ADT) is a standard definitive treatment of localized prostate cancer (LPCa). The optimal sequencing of these therapies is unclear. Our phase III trial compared neoadjuvant versus concurrent initiation of ADT in combination with dose-escalated prostate RT (PRT). PATIENTS AND METHODS: Patients with newly diagnosed LPCa with Gleason score ≤ 7, clinical stage T1b to T3a, and prostate-specific antigen < 30 ng/mL were randomly allocated to neoadjuvant and concurrent ADT for 6 months starting 4 months before RT (neoadjuvant group) or concurrent and adjuvant ADT for 6 months starting simultaneously with RT (concurrent group). The primary end point was biochemical relapse-free survival (bRFS). Stratified log-rank test was used to compare bRFS and overall survival (OS). Incidence of grade ≥ 3 late RT-related toxicities was compared by log-rank test. RESULTS: Overall, 432 patients were randomly assigned to the neoadjuvant (n = 215) or concurrent group (n = 217). At 10 years, bRFS rates for the two groups were 80.5% and 87.4%, respectively. Ten-year OS rates were 76.4% and 73.7%, respectively. There was no significant difference in bRFS (P = .10) or OS (P = .70) between the two groups. Relative to the neoadjuvant group, the hazard ratio for the concurrent group was 0.66 (95% CI, 0.41 to 1.07) for bRFS and 0.94 (95% CI, 0.68 to 1.30) for OS. No significant difference was observed in the 3-year incidence of late RT-related grade ≥ 3 GI (2.5% v 3.9%) or genitourinary toxicity (2.9% v 2.9%). CONCLUSION: In our study, there was no statistically significant difference in bRFS between the two treatment groups. Similarly, no difference was seen in OS or late RT-related toxicities. On the basis of these results, both neoadjuvant and concurrent initiations of short-term ADT with dose-escalated PRT are reasonable standards of care for LPCa.

Immunomodulatory Therapeutic Strategies in Stroke.

The role of immunity in all stages of stroke is increasingly being recognized, from the pathogenesis of risk factors to tissue repair, leading to the investigation of a range of immunomodulatory therapies. In the acute phase of stroke, proposed therapies include drugs targeting pro-inflammatory cytokines, matrix metalloproteinases, and leukocyte infiltration, with a key objective to reduce initial brain cell toxicity. Systemically, the early stages of stroke are also characterized by stroke-induced immunosuppression, where downregulation of host defences predisposes patients to infection. Therefore, strategies to modulate innate immunity post-stroke have garnered greater attention. A complementary objective is to reduce longer-term sequelae by focusing on adaptive immunity. Following stroke onset, the integrity of the blood-brain barrier is compromised, exposing central nervous system (CNS) antigens to systemic adaptive immune recognition, potentially inducing autoimmunity. Some pre-clinical efforts have been made to tolerize the immune system to CNS antigens pre-stroke. Separately, immune cell populations that exhibit a regulatory phenotype (T- and B- regulatory cells) have been shown to ameliorate post-stroke inflammation and contribute to tissue repair. Cell-based therapies, established in oncology and transplantation, could become a strategy to treat the acute and chronic stages of stroke. Furthermore, a role for the gut microbiota in ischaemic injury has received attention. Finally, the immune system may play a role in remote ischaemic preconditioning-mediated neuroprotection against stroke. The development of stroke therapies involving organs distant to the infarct site, therefore, should not be overlooked. This review will discuss the immune mechanisms of various therapeutic strategies, surveying published data and discussing more theoretical mechanisms of action that have yet to be exploited.

The immune system and stroke: from current targets to future therapy.

Stroke is a major cause of morbidity and mortality worldwide. Despite the intensive search for new therapies, hundreds of agents targeting various pathophysiological mechanisms have failed clinical trials, and the thrombolytic agent tissue plasminogen activator is currently the only FDA-approved medication for the treatment of acute ischemic stroke. The immune system is involved in all stages of stroke, from the pathogenesis of risk factors to neurotoxicity, to tissue remodeling and repair. There is a bidirectional interaction between the brain and the immune system, with stroke-induced immunosuppression and subsequent infection a principal source of patient mortality. Newer work also points to a role for the gut microbiota in the immune response to stroke, while clinical sequelae such as dementia might now also be explained in immune terms. However, the exact roles of innate and adaptive components have not been fully elucidated, with studies reporting both detrimental and beneficial functions. Time is a key determinant in defining whether immunity and inflammation are neuroprotective or neurotoxic. The local inflammatory milieu also has a clear influence on many proposed treatments. This review examines the individual components of the immune response to stroke, highlighting the most promising future stroke immunotherapies.

Proposal of a post-prostatectomy clinical target volume based on pre-operative MRI: volumetric and dosimetric comparison to the RTOG guidelines.

BACKGROUND: Recurrence rates following radiotherapy for prostate cancer in the post-operative adjuvant or salvage setting remain substantial. Previous work from our institution demonstrated that published prostate bed CTV guidelines frequently do not cover the pre-operative MRI defined prostate. Inadequate target delineation may contribute to the high recurrence rates, but increasing target volumes may increase dose to organs at risk. METHODS: We propose guidelines for delineating post-prostatectomy target volumes based upon an individual's co-registered pre-operative MRI. MRI-based CTVs and PTVs were compared to those created using the RTOG guidelines in 30 patients. Contours were analysed in terms of absolute volume, intersection volume (Jaccard Index) and the ability to meet the RADICALS and QUANTEC rectal and bladder constraints (tomotherapy IMRT plans with PTV coverage of V98% ≥98%). RESULTS: CTV MRI was a mean of 18.6% larger than CTV RTOG: CTV MRI mean 138 cc (range 72.3 - 222.2 cc), CTV RTOG mean 116.3 cc (range 62.1 - 176.6 cc), (p < 0.0001). The difference in mean PTV was only 4.6%: PTV MRI mean 386.9 cc (range 254.4 - 551.2), PTV RTOG mean 370 cc (range 232.3 - 501.6) (p = 0.05). The mean Jaccard Index representing intersection volume between CTVs was 0.72 and 0.84 for PTVs. Both criteria had a similar ability to meet rectal and bladder constraints. Rectal DVH: 77% of CTV RTOG cases passed all RADICALS criteria and 37% all QUANTEC criteria; versus 73% and 40% for CTV MRI (p = 1.0 for both). Bladder DVH; 47% of CTV RTOG cases passed all RADICALS criteria and 67% all QUANTEC criteria, versus 57% and 60% for CTV MRI (p = 0.61for RADICALS, p = 0.79 for QUANTEC). CTV MRI spares more of the lower anterior bladder wall than CTV RTOG but increases coverage of the superior lateral bladder walls. CONCLUSION: CTV contours based upon the patient's co-registered pre-operative MRI in the post-prostatectomy setting may improve coverage of the individual's prostate bed without substantially increasing the PTV size or dose to bladder/ rectum compared to RTOG CTV guidelines. Further evaluation of whether the use of pre-operative MRI improves local control rates is warranted.

Creation of 3-dimensional prostate cancer maps: methodology and clinical and research implications.

CONTEXT: The creation of 3-dimensional prostate cancer maps could assist with surgical intervention, radiotherapy treatment planning and for correlative pathology-imaging research. OBJECTIVES: To develop methodology for creating detailed, 3-dimensional, prostate cancer maps (3DPCM) of tumor location, extra prostatic extension sites, and positive margins and to assess the adequacy of current clinical target volumes for postoperative radiotherapy to the prostate using 3DPCM coregistered with preoperative magnetic resonance imaging. DESIGN: Parallel slices of prostatectomy specimens were created with ProCUT, and 2-dimensional cancer maps were generated as line diagrams after microscopic examination of each slice. The 2-dimensional cancer maps were aligned and stacked to create a 3DPCM, which was coregistered with the preoperative magnetic resonance imaging scan. The map was exported to the radiotherapy planning system and was used to determine the areas at greater risk, which were then compared against the current Radiation Therapy Oncology Group guidelines for contouring postoperative clinical target volumes to assess the adequacy of coverage. RESULTS: Twenty-eight patients with a mean age of 66 years (range, 52-73) underwent radical prostatectomy and postoperative radiotherapy. Seventeen patients (61%) received adjuvant radiotherapy for pT3 disease and/or positive margins, and the rest underwent salvage radiotherapy. Thirty-nine percent (11 of 28) of the patients had Gleason scores of 8 or 9. The contours based on the Radiation Therapy Oncology Group guidelines for postoperative radiotherapy resulted in inadequate coverage of extraprostatic extensions in 79% (22 of 28) and positive margins in 64% (18 of 28) of the cases. CONCLUSIONS: We have developed a methodology for creation of 3DPCM. Modification of the radiotherapy contours, based on the 3DPCM coregistered with pretreatment magnetic resonance imaging, covers the areas at high risk of recurrence. The 3DPCM could become an important clinical and research tool for urologists, pathologists, radiologists, and oncologists.

Biomechanical Evaluation of a Novel Autogenous Bone Interbody Fusion Cage for Posterior Lumbar Interbody Fusion in a Cadaveric Model.

STUDY DESIGN: A human cadaveric biomechanical study of a novel, prefabricated autogenous bone interbody fusion (ABIF) cage. OBJECTIVE: To evaluate the biomechanical properties of the ABIF cage in a single-level construct with and without transpedicular screw and rod fixation. SUMMARY OF BACKGROUND DATA: In current practice, posterior lumbar interbody fusion is generally carried out using synthetic interbody spacers or corticocancellous iliac crest bone graft (ICBG) in combination with posterior instrumentation. However, questions remain concerning the use of synthetic intervertebral implants as well as the morbidity ICBG harvesting. Therefore, ABIF cage has been developed to obviate some of the challenges in conventional posterior lumbar interbody fusion instrumentation and to facilitate the fusion process. METHODS: Eighteen adult cadaveric lumbosacral (L3-S1) specimens were tested. Test conditions included single lumbosacral segments across (1) intact, (2) decompressed, (3) intervertebral cage alone, and (4) intervertebral cage with bilateral transpedicular fixation. Range of motion (ROM), neutral zone (NZ), and axial failure load were tested for each condition. RESULTS: The ICBG, polyetheretherketone cage, or ABIF cage alone exhibited a significantly lower (P < 0.05) ROM and NZ than the decompressed spine. In comparison with the intact spine, all 3 test conditions without supplemental fixation were able to decrease ROM and NZ to near intact levels. When stabilized with pedicle screws, the ROM was significantly less and the NZ was significantly lower (P < 0.05) for each group both compared with the intact spine. In axial compression testing, the failure load of polyetheretherketone cage was the highest, with no significant difference between the ICBG and the ABIF cage. CONCLUSION: These data suggest that the novel ABIF cage can bear the physiological intervertebral peak load, similar to ICBG. When combined with pedicle screw and rod fixation, it exhibits similar biomechanical properties as the polyetheretherketone cage plus posterior instrumentation. Based on the biomechanical properties of ABIF cage, the prospect of these cages in clinical practice is expected.

Investigating chromosome damage using fluorescent in situ hybridization to identify biomarkers of radiosensitivity in prostate cancer patients.

PURPOSE: In order to evaluate fluorescent in situ hybridization (FISH) as a method for predicting radiosensitivity, this study examined the incidence of translocations, after exposure to in vitro radiation, in both normally responding patients and those exhibiting severe late effects after radiotherapy treatment. MATERIALS AND METHODS: Patients were selected from a randomized trial for intermediate-risk prostate cancer. Of the patients entered on trial with mature follow-up, 3% developed grade 3 late proctitis. Blood samples were taken from this radiosensitive cohort along with matched control patients with no late proctitis. Whole blood samples were exposed to 0 or 4 Gy and cultured according to the International Atomic Energy Agency (IAEA) recommended methods. Colour junctions were evaluated in the resulting metaphases and scored according to the Protocol for Aberration Identification and Nomenclature Terminology (PAINT) system. RESULTS: Both groups were statistically similar at 0 Gy. After 4 Gy in vitro radiation, the radiosensitive group had significantly higher rates of chromosome damage in the number of colour junctions per cell (p = 0.002), the number of deletions per cell (p = 0.01) and the number of dicentrics per cell (p = 0.005). CONCLUSIONS: These results indicate that the analysis of translocations using FISH after in vitro irradiation correlates with clinical response to radiation. This cytogenetic assay should be considered as a potential predictor of radiosensitivity.

Chromosome damage and cell proliferation rates in in vitro irradiated whole blood as markers of late radiation toxicity after radiation therapy to the prostate.

PURPOSE: In vitro irradiated blood samples from prostate cancer patients showing late normal tissue damage were examined for lymphocyte response by measuring chromosomal aberrations and proliferation rate. METHODS AND MATERIALS: Patients were selected from a randomized trial evaluating the optimal timing of dose-escalated radiation and short-course androgen deprivation therapy. Of 438 patients, 3% experienced grade 3 late radiation proctitis and were considered to be radiosensitive. Blood samples were taken from 10 of these patients along with 20 matched samples from patients with grade 0 proctitis. The samples were irradiated at 6 Gy and, along with control samples, were analyzed for dicentric chromosomes and excess fragments per cell. Cells in first and second metaphase were also enumerated to determine the lymphocyte proliferation rate. RESULTS: At 6 Gy, there were statistically significant differences between the radiosensitive and control cohorts for 3 endpoints: the mean number of dicentric chromosomes per cell (3.26 ± 0.31, 2.91 ± 0.32; P=.0258), the mean number of excess fragments per cell (2.27 ± 0.23, 1.43 ± 0.37; P<.0001), and the proportion of cells in second metaphase (0.27 ± 0.10, 0.46 ± 0.09; P=.0007). CONCLUSIONS: These results may be a valuable indicator for identifying radiosensitive patients and for tailoring radiation therapy.

Investigating γ H2AX as a Biomarker of Radiosensitivity Using Flow Cytometry Methods.

Background and Purpose. This project examined the in vitro γ H2AX response in lymphocytes of prostate cancer patients who had a radiosensitive response after receiving radiotherapy. The goal of this project was to determine whether the γ H2AX response, as measured by flow cytometry, could be used as a marker of individual patient radiosensitivity. Materials and Methods. Patients were selected from a randomized clinical trial evaluating the optimal timing of Dose Escalated Radiation and short-course Androgen Deprivation Therapy. Of 438 patients, 3% developed Grade 3 late radiation proctitis and were considered to be radiosensitive. Blood was drawn from 10 of these patients along with 20 matched samples from patients with Grade 0 proctitis. Dose response curves up to 10 Gy along with time response curves after 2 Gy (0-24 h) were generated for each sample. The γ H2AX response in lymphocytes and lymphocyte subsets was analyzed by flow cytometry. Results. There were no significant differences between the radiosensitive and control samples for either the dose course or the time course. Conclusions. Although γ H2AX response has previously been demonstrated to be an indicator of individual patient radiosensitivity, flow cytometry lacks the sensitivity necessary to distinguish any differences between samples from control and radiosensitive patients.