Comparison of Novel Proteomic Expression Profiles for Radiation Exposure in Male and Female C57BL6 Mice.

There is a need for point-of-care diagnostics for future mass casualty events involving radiation exposure. The development of radiation exposure and dose prediction algorithms for biodosimetry is needed for screening of large populations during these scenarios, and exploration of the potential effects which sex, age, genetic heterogeneity, and physiological comorbidities may have on the utility of biodosimetry diagnostics is needed. In the current study, proteomic profiling was used to examine sex-specific differences in age-matched C57BL6 mice on the blood proteome after radiation exposure, and the usefulness of development and application of biodosimetry algorithms using both male and female samples. Male and female mice between 9-11 weeks of age received a dose of total-body irradiation (TBI) of either 2, 4 or 8 Gy and plasma was collected at days 1, 3 and 7 postirradiation. Plasma was then screened using the SomaScan v4.1 assay for ∼7,000 protein analytes. A subset panel of protein biomarkers demonstrated significant (FDR < 0.05 and |logFC| > 0.2) changes in expression after radiation exposure. All proteins were used for feature selection to build predictive models of radiation exposure using different sample and sex-specific cohorts. Both binary (prediction of any radiation exposure) and multidose (prediction of specific radiation dose) model series were developed using either female and male samples combined or only female or only male samples. The binary series (models 1, 2 and 3) and multidose series (models 4, 5 and 6) included female/male combined, female only and male only respectively. Detectable values were obtained for all ∼7,000 proteins included in the SomaScan assay for all samples. Each model algorithm built using a unique sample cohort was validated with a training set of samples and tested with a separate new sample series. Overall predictive accuracies in the binary model series was ∼100% at the model training level, and when tested with fresh samples, 97.9% for model 1 (female and male) and 100% for model 2 (female only) and model 3 (male only). When sex-specific models 2 and 3 were tested with the opposite sex, the overall predictive accuracy rate dropped to 62.5% for model 2 and remained 100% for model 3. The overall predictive accuracy rate in the multidose model series was 100% for all models at the model training level and, when tested with fresh samples, 83.3%, 75% and 83.3% for Multidose models 4-6, respectively. When sex-specific model 5 (female only) and model 6 (male only) were tested with the opposite sex, the overall predictive accuracy rate dropped to 52.1% and 68.8%, respectively. These models represent novel predictive panels of radiation-responsive proteomic biomarkers and illustrate the utility and necessity of considering sex-specific differences in development of radiation biodosimetry prediction algorithms. As sex-specific differences were observed in this study, and as use of point-of-care radiation diagnostics in future mass casualty settings will necessarily include persons of both sexes, consideration of sex-specific variation is essential to ensure these diagnostic tools have practical utility in the field.

Identifying patients suitable for targeted adjuvant therapy: advances in the field of developing biomarkers for tumor recurrence following irradiation.

Introduction: Radiation therapy (RT) is commonly used to treat cancer in conjunction with chemotherapy, immunotherapy, and targeted therapies. Despite the effectiveness of RT, tumor recurrence due to treatment resistance still lead to treatment failure. RT-specific biomarkers are currently lacking and remain challenging to investigate with existing data since, for many common malignancies, standard of care (SOC) paradigms involve the administration of RT in conjunction with other agents. Areas Covered: Established clinically relevant biomarkers are used in surveillance, as prognostic indicators, and sometimes for treatment planning; however, the inability to intercept early recurrence or predict upfront resistance to treatment remains a significant challenge that limits the selection of patients for adjuvant therapy. We discuss attempts at intercepting early failure. We examine biomarkers that have made it into the clinic where they are used for treatment monitoring and management alteration, and novel biomarkers that lead the field with targeted adjuvant therapy seeking to harness these. Expert Opinion: Given the growth of data correlating interventions with omic analysis toward identifying biomarkers of radiation resistance, more robust markers of recurrence that link to biology will increasingly be leveraged toward targeted adjuvant therapy to make a successful transition to the clinic in the coming years.

The Future of Radioactive Medicine.

The discovery of X rays in the late 19th century heralded the beginning of a new age in medicine, and the advent of channeling the power of radiation to diagnose and treat human disease. Radiation has been leveraged in medicine in a multitude of ways and is a critical element of cancer care including screening, diagnosis, surveillance, and interventional treatments. Modern radiotherapy techniques include a multitude of methodologies utilizing both externally and internally delivered radiation from a variety of approaches. This review provides a comprehensive overview of contemporary radiotherapy methodologies, the field of radiopharmaceuticals and theranostics, effects of low dose radiation and highlights the phenomena of fear of exposure to radiation and its impact in modern medicine.

Prediction of Total-Body and Partial-Body Exposures to Radiation Using Plasma Proteomic Expression Profiles.

There is a need to identify new biomarkers of radiation exposure for not only systemic total-body irradiation (TBI) but also to characterize partial-body irradiation and organ specific radiation injury. In the current study, we sought to develop novel biodosimetry models of radiation exposure using TBI and organ specific partial-body irradiation to only the brain, lung or gut using a multivariate proteomics approach. Subset panels of significantly altered proteins were selected to build predictive models of radiation exposure in a variety of sample cohort configurations relevant to practical field application of biodosimetry diagnostics during future radiological or nuclear event scenarios. Female C57BL/6 mice, 8-15 weeks old, received a single total-body or partial-body dose of 2 or 8 Gy TBI or 2 or 8 Gy to only the lung or gut, or 2, 8 or 16 Gy to only the brain using a Pantak X-ray source. Plasma was collected by cardiac puncture at days 1, 3 and 7 postirradiation for total-body exposures and only the lung and brain exposures, and at days 3, 7 and 14 postirradiation for gut exposures. Plasma was then screened using the aptamer-based SOMAscan proteomic assay technology, for changes in expression of 1,310 protein analytes. A subset panel of protein biomarkers which demonstrated significant changes (P < 0.01) in expression after irradiation were used to build predictive models of radiation exposure using different sample cohorts. Model 1 compared controls vs. all pooled irradiated samples, which included TBI and all organ specific partial irradiation. Model 2 compared controls vs. TBI vs. partial irradiation (with all organ specific partial exposure pooled within the partial-irradiated group), and model 3 compared controls vs. each individual organ specific partial-body exposure separately (brain, gut and lung). Detectable values were obtained for all 1,310 proteins included in the SOMAscan assay for all samples. Each model algorithm built using a unique sample cohort was validated with a training set of samples and tested with a separate new sample series. Overall predictive accuracies of 89%, 78% and 55% resulted for models 1-3, respectively, representing novel predictive panels of radiation responsive proteomic biomarkers. Though relatively high overall predictive accuracies were achieved for models 1 and 2, all three models showed limited accuracy at differentiating between the controls and partial-irradiated body samples. In our study we were able to identify novel panels of radiation responsive proteins useful for predicting radiation exposure and to create predictive models of partial-body exposure including organ specific radiation exposures. This proof-of-concept study also illustrates the inherent physiological limitations of distinguishing between small-body exposures and the unirradiated using proteomic biomarkers of radiation exposure. As use of biodosimetry diagnostics in future mass casualty settings will be complicated by the heterogeneity of partial-body exposure received in the field, further work remains in adapting these diagnostic tools for practical use.

AI-Driven Image Analysis in Central Nervous System Tumors-Traditional Machine Learning, Deep Learning and Hybrid Models.

The interpretation of imaging in medicine in general and in oncology specifically remains problematic due to several limitations which include the need to incorporate detailed clinical history, patient and disease-specific history, clinical exam features, previous and ongoing treatment, and account for the dependency on reproducible human interpretation of multiple factors with incomplete data linkage. To standardize reporting, minimize bias, expedite management, and improve outcomes, the use of Artificial Intelligence (AI) has gained significant prominence in imaging analysis. In oncology, AI methods have as a result been explored in most cancer types with ongoing progress in employing AI towards imaging for oncology treatment, assessing treatment response, and understanding and communicating prognosis. Challenges remain with limited available data sets, variability in imaging changes over time augmented by a growing heterogeneity in analysis approaches. We review the imaging analysis workflow and examine how hand-crafted features also referred to as traditional Machine Learning (ML), Deep Learning (DL) approaches, and hybrid analyses, are being employed in AI-driven imaging analysis in central nervous system tumors. ML, DL, and hybrid approaches coexist, and their combination may produce superior results although data in this space is as yet novel, and conclusions and pitfalls have yet to be fully explored. We note the growing technical complexities that may become increasingly separated from the clinic and enforce the acute need for clinician engagement to guide progress and ensure that conclusions derived from AI-driven imaging analysis reflect that same level of scrutiny lent to other avenues of clinical research.

Linear accelerator stereotactic radiosurgery can modulate the clinical course of Hemangioblastoma: Case series and review of the literature.

Hemangioblastomas (HB) are benign low grade vascular tumors most frequently occurring in the cerebellum, brain stem, and spinal cord. Often associated with Von Hippel Lindau disease (VHL), the lesions are often multifocal requiring complex resection and are difficult to control. Linear Accelerator (LINAC) Stereotactic Radiosurgery (SRS) has been demonstrated to provide additional tumor control. In this case series, we present our multi-center experience utilizing LINAC SRS in fourteen patients with 23 lesions. We observed a tumor control rate of 87% and found interval changes in the peritumoral enhancement to correlate with treatment outcome. In our study, SRS treatment was also well-tolerated in both cystic and noncystic patients with multifocal disease. Disease control was achieved in all but three patients post-resection and no longitudinal radiation-induced secondary malignancy was observed. SRS response correlated highly with lesion size and radiation dose. We conclude that LINAC SRS is safe and effective for patients with HB and should be considered in addition to surgery in asymptomatic, VHL patients, deep seated lesions and isolated lesions.

The addition of Valproic acid to concurrent radiation therapy and temozolomide improves patient outcome: a Correlative analysis of RTOG 0525, SEER and a Phase II NCI trial.

PURPOSE/OBJECTIVES: Valproic Acid (VPA) is an antiepileptic agent with HDACi (histone deacetylase inhibitor) activity shown to radiosensitize glioblastoma (GBM) cells. We evaluated the addition of VPA to standard radiation therapy (RT) and temozolomide (TMZ) in an open-label, phase II study (NCI-06-C-0112). The intent of the current study was to compare our patient outcomes with modern era standard of care data (RTOG 0525) and general population data (SEER 2006-2013). MATERIALS/METHODS: 37 patients with newly diagnosed GBM were treated in a phase II NCI trial with daily VPA (25 mg/kg) in addition to concurrent RT and TMZ (2006 - 2013) and 411 patients with newly diagnosed GBM were treated in the standard TMZ dose arm of RTOG 0525 (2006 - 2008). Using the SEER database, adult patients (age > 15) with diagnostic codes 9440-9443 (third edition (IDC-O-3) diagnosed between 2006 - 2013 were identified and 6083 were included in the analysis. Kaplan-Meier method was used to estimate OS and PFS. The effect of patient characteristics and clinical factors on OS and PFS was analyzed using univariate analysis and a Cox regression model. A landmark analysis was performed to correlate recurrence to OS and conditional probabilities of surviving an additional 12 months at diagnosis, 6, 12, 18, 24 and 30 months were calculated for both the trial data and the SEER data. RESULTS: Updated median OS in the NCI cohort was 30.9m (22.2- 65.6m), compared to RTOG 0525 18.9m (16.8-20.3m) (p= 0.007) and the SEER cohort of 11m. Median PFS in the NCI cohort was 11.1m (6.6 - 49.6m) compared to RTOG 0525 with a median PFS of 7.5m (6.9-8.2m) (p = 0.004). Younger age, class V RPA and MGMT status were significant for PFS in both the NCI cohort and the RTOG 0525 cohort, in addition KPS was also significant for OS. In comparison to RTOG 0525, the population in the NCI cohort had a more favorable KPS and RPA, and a higher proportion of patients receiving bevacizumab after protocol therapy however with the exception of RPA (V) (8% vs 18%) (0.026), the effects of these factors on PFS and OS were not significantly different between the two cohorts. CONCLUSION: Previously reported improvements in PFS and OS with the addition of VPA to concurrent RT and TMZ in the NCI phase II study were confirmed by comparison to both a trial population receiving standard of care (RTOG 0525) and a contemporary SEER cohort. These results provide further justification of a phase III trial of VPA/RT/TMZ.

CD44 is prognostic for overall survival in the NCI randomized trial on breast conservation with 25 year follow-up.

CD44 is a transmembrane glycoprotein involved in numerous cellular functions, including cell adhesion and extracellular matrix interactions. It is known to be functionally diverse, with alternative splice variants increasingly implicated as a marker for tumor-initiating stem cells associated with poor prognosis. Here, we evaluate CD44 as a potential marker of long-term breast cancer outcomes. Tissue specimens from patients treated on the National Cancer Institute 79-C-0111 randomized trial of breast conservation versus mastectomy between 1979 and 1987 were collected, and immunohistochemistry was performed using the standard isoform of CD44. Specimens were correlated with patient characteristics and outcomes. Survival analysis was performed using the log rank test. Fifty-one patients had evaluable tumor sections and available long-term clinical follow up data at a median follow up of 25.7 years. Significant predictors of OS were tumor size (median OFS 25.4 years for ≤2 cm vs. 7.5 years for >2 cm, p = 0.001), nodal status (median OS 17.2 years for node-negative patients vs. 6.7 years for node positive patients, p = 0.017), and CD44 expression (median OS 18.9 years for CD44 positive patients vs. 8.6 years for CD44 negative patients, p = 0.049). There was a trend toward increased PFS for patients with CD44 positive tumors (median PFS 17.9 vs. 4.3 years, p = 0.17), but this did not reach statistical significance. These findings illustrate the potential utility of CD44 as a prognostic marker for early stage breast cancer. Subgroup analysis in patients with lymph node involvement revealed CD44 positivity to be most strongly associated with increased survival, suggesting a potential role of CD44 in decision making for axillary management. As there is increasing interest in CD44 as a therapeutic target in ongoing clinical trials, the results of this study suggest additional investigation regarding the role CD44 in breast cancer is warranted.

Non-patient related variables affecting levels of vascular endothelial growth factor in urine biospecimens.

Vascular endothelial growth factor (VEGF) is an angiogenic protein proposed to be an important biomarker for the prediction of tumour growth and disease progression. Recent studies suggest that VEGF measurements in biospecimens, including urine, may have predictive value across a range of cancers. However, the reproducibility and reliability of urinary VEGF measurements have not been determined. We collected urine samples from patients receiving radiation treatment for glioblastoma multiforme (GBM) and examined the effects of five variables on measured VEGF levels using an ELISA assay. To quantify the factors affecting the precision of the assay, two variables were examined: the variation between ELISA kits with different lot numbers and the variation between different technicians. Three variables were tested for their effects on measured VEGF concentration: the time the specimen spent at room temperature prior to assay, the addition of protease inhibitors prior to specimen storage and the alteration of urinary pH. This study found that VEGF levels were consistent across three different ELISA kit lot numbers. However, significant variation was observed between results obtained by different technicians. VEGF concentrations were dependent on time at room temperature before measurement, with higher values observed 3-7 hrs after removal from the freezer. No significant difference was observed in VEGF levels with the addition of protease inhibitors, and alteration of urinary pH did not significantly affect VEGF measurements. In conclusion, this determination of the conditions necessary to reliably measure urinary VEGF levels will be useful for future studies related to protein biomarkers and disease progression.

Advances in 4D medical imaging and 4D radiation therapy.

This paper reviews recent advances in 4D medical imaging (4DMI) and 4D radiation therapy (4DRT), which study, characterize, and minimize patient motion during the processes of imaging and radiotherapy. Patient motion is inevitably present in these processes, producing artifacts and uncertainties in target (lesion) identification, delineation, and localization. 4DMI includes time-resolved volumetric CT, MRI, PET, PET/CT, SPECT, and US imaging. To enhance the performance of these volumetric imaging techniques, parallel multi-detector array has been employed for acquiring image projections and the volumetric image reconstruction has been advanced from the 2D to the 3D tomography paradigm. The time information required for motion characterization in 4D imaging can be obtained either prospectively or retrospectively using respiratory gating or motion tracking techniques. The former acquires snapshot projections for reconstructing a motion-free image. The latter acquires image projections continuously with an associated timestamp indicating respiratory phases using external surrogates and sorts these projections into bins that represent different respiratory phases prior to reconstructing the cyclical series of 3D images. These methodologies generally work for all imaging modalities with variations in detailed implementation. In 4D CT imaging, both multi-slice CT (MSCT) and cone-beam CT (CBCT) are applicable in 4D imaging. In 4D MR imaging, parallel imaging with multi-coil-detectors has made 4D volumetric MRI possible. In 4D PET and SPECT, rigid and non-rigid motions can be corrected with aid of rigid and deformable registration, respectively, without suffering from low statistics due to signal binning. In 4D PET/CT and SPECT/CT, a single set of 4D images can be utilized for motion-free image creation, intrinsic registration, and attenuation correction. In 4D US, volumetric ultrasonography can be employed to monitor fetal heart beating with relatively high temporal resolution. 4DRT aims to track and compensate for target motion during radiation treatment, minimizing normal tissue injury, especially critical structures adjacent to the target, and/or maximizing radiation dose to the target. 4DRT requires 4DMI, 4D radiation treatment planning (4D RTP), and 4D radiation treatment delivery (4D RTD). Many concepts in 4DRT are borrowed, adapted and extended from existing image-guided radiation therapy (IGRT) and adaptive radiation therapy (ART). The advantage of 4DRT is its promise of sparing additional normal tissue by synchronizing the radiation beam with the moving target in real-time. 4DRT can be implemented differently depending upon how the time information is incorporated and utilized. In an ideal situation, the motion adaptive approach guided by 4D imaging should be applied to both RTP and RTD. However, until new automatic planning and motion feedback tools are developed for 4DRT, clinical implementation of ideal 4DRT will meet with limited success. However, simplified forms of 4DRT have been implemented with minor modifications of existing planning and delivery systems. The most common approach is the use of gating techniques in both imaging and treatment, so that the planned and treated target localizations are identical. In 4D planning, the use of a single planning CT image, which is representative of the statistical respiratory mean, seems preferable. In 4D delivery, on-site CBCT imaging or 3D US localization imaging for patient setup and internal fiducial markers for target motion tracking can significantly reduce the uncertainty in treatment delivery, providing improved normal tissue sparing. Most of the work on 4DRT can be regarded as a proof-of-principle and 4DRT is still in its early stage of development.

Quantitative method to study the network formation of endothelial cells in response to tumor angiogenic factors.

To study the network formation of endothelial cells (ECs) in an extracellular matrix (ECM) environment, we have devised an EC aggregation-type model based on a diffusion limited cluster aggregation model (DLCA), where clusters of particles diffuse and stick together upon contact. We use this model to quantify EC differentiation into cord-like structures by comparing experimental and simulation data. Approximations made with the DLCA model, when combined with experimental kinetics and cell concentration results, not only allow us to quantify cell differentiation by a pseudo diffusion coefficient, but also measure the effects of tumor angiogenic factors (TAFs) on the formation of cord-like structures by ECs. We have tested our model by using an in vitro assay, where we record EC aggregation by analysing time-lapse images that provide us with the evolution of the fractal dimension measure through time. We performed these experiments for various cell concentrations and TAFs (e.g. EVG, FGF-b, and VEGF). During the first six hours of an experiment, ECs aggregate quickly. The value of the measured fractal dimension decreases with time until reaching an asymptotic value that depends solely on the EC concentration. In contrast, the kinetics depend on the nature of TAFs. The experimental and simulation results correlate with each other in regards to the fractal dimension and kinetics, allowing us to quantify the influence of each TAF by a pseudo diffusion coefficient. We have shown that the shape, kinetic aggregation, and fractal dimension of the EC aggregates fit into an in vitro model capable of reproducing the first stage of angiogenesis. We conclude that the DLCA model, combined with experimental results, is a highly effective assay for the quantification of the kinetics and network characteristics of ECs embedded in ECM proteins. Finally, we present a new method that can be used for studying the effect of angiogenic drugs in in vitro assays.

In vivo retinal gene expression in early diabetes.

PURPOSE: Studies have demonstrated a causal role for specific molecules in the pathogenesis of diabetic retinopathy. Among the implicated mediators are growth factors such as vascular endothelial growth factor (VEGF) as well as adhesion molecules and proliferation- and apoptosis-related genes. However, a coordinated large-scale investigation of gene expression in the diabetic retina has not yet been reported. Here the retinal gene expression profile of diabetic and nondiabetic animals using cDNA microarrays were analyzed and compared. METHODS: Long-Evans rats were made diabetic with streptozotocin. Retinal gene expression was analyzed over 3 weeks using high-density nylon filter-based cDNA arrays. Genes were sorted into clusters according to their temporal expression profiles. They were also grouped according to their potential pathophysiological significance. The in vivo gene expression profiles of selected genes were verified via RNase protection assay. RESULTS: The rat GeneFilter contains a total of 5147 genes, of which 1691 are known genes and 3456 are expressed sequence tags (ESTs). On day 3, the expression of 27 known genes was increased by more than twofold. On days 7 and 21, the corresponding numbers were 60 and 12, respectively. A transient upregulation (>2-fold) in expression was seen in 627 of 5147 total genes. A subset of 926 genes exhibited a modest (<2-fold) decrease in expression. No genes showed a greater than twofold decrease in expression. Overall, the identity of the genes that were upregulated suggests that the response of the retina to the diabetic challenge contains an inflammatory component. Moreover, most regulatory activity occurs during the first week of diabetes. CONCLUSIONS: The development of a rational therapy for diabetic retinopathy will be assisted by detailed knowledge regarding the molecular pathophysiology of the disease. Here, an expression profile of an underlying retinal inflammatory process in early diabetes was extracted. Beyond providing insight into the general nature of the response to a pathogenic challenge, gene expression profiling may also allow the efficient identification of potential drug targets and markers for monitoring the course of disease.

Radiation therapy to a primary tumor accelerates metastatic growth in mice.

The surgical removal of a primary tumor can result in the rapid growth of metastases. The production of angiogenesis inhibitors by the primary tumor is one mechanism for the inhibition of metastatic tumor growth. The effect of curative radiotherapy to a primary tumor known to make an inhibitor of angiogenesis and the effects on distant metastases has not been studied. We here show that the eradication of a primary Lewis lung carcinoma (LLC-LM), which is known to generate angiostatin, is followed by the rapid growth of metastases that kill the animal within 18 days after the completion of radiation therapy. The right thighs of C57BL/6 mice (n = 25) were injected s.c. with 1 x 10(6) LLC-LM cells. Animals were randomized to one of five groups: no irradiation, 40 Gy in one fraction, 30 Gy in one fraction, 40 Gy in two 20 Gy fractions, or 50 Gy in five 10 Gy fractions. Tumors were clinically eradicated in each treatment group. All of the surviving animals became dyspneic and were killed within 14-18 days after the completion of radiation therapy. Examination of their lungs revealed >46 (range, 46-62) surface metastases in the treated animals compared with 5 (range, 2-8) in the untreated animals. The lung weights had increased from 0.2 g (range, 0.19-0.22 g) in the controls to 0.58 g (range 0.44-0.84) in the experimental animals. The most effective dose regimen was 10 Gy per fraction for five fractions, and serial experiments were conducted with this fractionation scheme. Complete response of the primary tumor was seen in 25 of 35 (71%) mice. The average weight of the lungs in the nonirradiated animals was 0.22 g (range, 0.19-0.24 g) and in the irradiated animals was 0.66 g (range, 0.61-0.70 g). The average number of surface metastases increased from five per lung (range, 2-13) in the control animals to 53 per lung (range, 46-62) in the irradiated animals. Both differences were statistically significant with P < 0.001. If the nontumor-bearing leg was irradiated or the animals were sham-irradiated, no difference in the number of surface metastases or lung weights was observed between the control group and the treated group. Urinary levels of matrix metalloproteinase 2, the enzyme responsible for angiostatin processing in this tumor model, were measured and correlated with the viability and size of the primary tumor. Administration of recombinant angiostatin prevented the growth of the metastases after the treatment of the primary tumor. In this model, the use of radiation to eradicate a primary LLC-LM tumor results in the growth of previously dormant lung metastases and suggests that combining angiogenesis inhibitors with radiation therapy may control distant metastases.

Low-dose-rate ionizing irradiation for inhibition of secondary cataract formation.

INTRODUCTION: Secondary cataract formation limits visual function after cataract surgery. Various experimental methods utilizing the pharmacologic inhibition of lens epithelial cell proliferation have been proposed. However, diffusion into the anterior chamber may lead to damage of corneal endothelial cells. This study evaluated the inhibition of lens epithelial cell proliferation with a capsular bag ring, labeled with a beta-emitting radioisotope. METHODS AND MATERIALS: In vitro studies using rabbit lens epithelial cells were performed to investigate the dose-dependent effect of irradiation. Based on these results, P-32-labeled PMMA rings were implanted into the capsular bag of NZW rabbits in vivo after phacoemulsification. Animals were examined for development of posterior capsule opacification over a period of 12 weeks following surgery. Radiation damage to the surrounding ocular tissue was subsequently analyzed in histologic sections using TUNEL assay and proliferation marker. RESULTS: Irradiation of lens epithelial cells in vitro with >5 Gy resulted in a dose-dependent decrease in the number of cells. BrdU testing demonstrated a near complete inhibition of cell proliferation. In vivo, implantation of P-32-labeled PMMA rings led to inhibition of epithelial cell proliferation and secondary cataract formation but was not able to fully inhibit aberrant differentiation of some remaining cells. Histologic examination showed no evidence of radiation damage of the ciliary body or the corneal endothelium. CONCLUSIONS: Low-dose beta irradiation exhibits the potential for inhibition of lens epithelial cell proliferation both in vitro and in vivo. Further investigation of various nuclides and their radiation profiles is needed to optimize the prevention of posterior capsule opacification due to epithelial cell proliferation.

Diversity of T-cell receptor V alpha, V beta, and CDR3 expression by myelin basic protein-specific human T-cell clones.

We sequenced the cDNAs of alpha and beta T-cell receptors (TCRs), including V, J, and CDR3 regions, expressed by 54 myelin basic protein (MBP)-specific T-cell clones generated from the peripheral blood of 15 multiple sclerosis (MS) patients and three normal controls. Thirteen V alpha gene segments, 18 V beta gene segments, 23 CDR3 alpha sequences, and 30 CDR3 beta sequences were represented among these clones. Some CDR3 motifs were common to several clones that shared epitope specificity, while other motifs were common to clones with diverse epitope specificities. The extensive heterogeneity of TCR gene expression in the human immune response to MBP indicates that therapeutic strategies aimed at blocking a limited number of TCRs are unlikely to fully suppress the T-cell response to MBP in vivo.

HLA restriction and TCR usage of T lymphocytes specific for a novel candidate autoantigen, X2 MBP, in multiple sclerosis.

Previous investigations of the major 18.5-kDa isoform of myelin basic protein (MBP) as a target autoantigen in multiple sclerosis (MS) have failed to identify an epitope uniformly recognized with higher frequency in MS patients compared with controls. Because remyelination has been observed in MS plaques, we were prompted to investigate T cells specific for myelin protein isoforms with up-regulated expression during remyelination. We have recently described such T cells that recognize the exon 2-encoded region of MBP (X2 MBP), a sequence included in the 21.5- and 20.2-kDa isoforms of MBP. These cells were shown to be CD4+, HLA class II restricted, and cytolytic. In members of one multiplex MS family, X2 MBP-specific T lymphocytes were as prevalent as T cells specific for immunodominant regions within the major 18.5-kDa isoform of MBP. The present study characterizes X2 MBP-specific T cell responses in additional multiplex MS family members as well as in heterogeneous (non-familial) MS patients and in healthy controls. The frequencies of X2 MBP-specific T cells in each of the affected family members from two of three MS families were significantly increased as compared with both the heterogeneous MS group and the healthy control group. Also, X2 MBP-specific T cell lines from affected family members were primarily restricted to molecules encoded by the DR2/DQw1 allele. Although TCR usage was generally heterogeneous, there was evidence of intraindividual sequence identity. These data suggest that: 1) Myelin proteins with up-regulated expression during the course of disease should be considered as candidate autoantigens in MS. 2) The functional basis for the association of DR2/DQw1 inheritance with MS susceptibility may be related to presentation of autoantigens by this allele. 3) TCR therapy will need to be individually tailored to target the most prevalent autoantigen-specific response.