Comparison of IL-2-antibody to IL-2-Fc with or without stereotactic radiation therapy in CEA immunocompetent mice with CEA positive tumors.

BACKGROUND: The potent immune effects of interleukin-2 (IL-2) for cancer therapy can be increased by genetic fusion of IL-2 to the Fc domain of an antibody (IL-2-Fc) or tumor targeted by genetic fusion to a whole antibody known as an immunocytokine (ICK). METHODS: An anti-CEA ICK (M5A-IL-2) was compared to an IL-2-Fc fusion protein using tumor therapy and PET imaging in CEA transgenic immunocompetent mice bearing CEA positive colon or breast tumors. Combination with stereotactic radiation therapy (SRT) was performed with either ICK or IL-2-Fc. RESULTS: ICK and IL-2-Fc had comparable antitumor effects in both tumor models, although ICK had higher tumor uptake and slower blood clearance than an IL-2-Fc. Analysis of IFNγ+ /CD8+ and FoxP3+ /CD4+ T cells revealed higher levels of IFNγ-producing CD8+ T cells in ICK treated mice versus more efficient Treg elimination in IL-2-Fc treated mice. No significant or lasting toxicity was detected for either agent. Combination therapies with SRT revealed comparable efficacy and induction of immune memory for both ICK and IL-2-Fc when mice were rechallenged post-therapy. CONCLUSIONS: IL-2-Fc had comparable antitumor efficacy to CEA-targeted M5A-IL-2 ICK, while both fusion proteins induced immune memory when combined with SRT. Differences in the therapeutic mechanisms of both agents were observed.

Total marrow irradiation reduces organ damage and enhances tissue repair with the potential to increase the targeted dose of bone marrow in both young and old mice.

Total body irradiation (TBI) is a commonly used conditioning regimen for hematopoietic stem cell transplant (HCT), but dose heterogeneity and long-term organ toxicity pose significant challenges. Total marrow irradiation (TMI), an evolving radiation conditioning regimen for HCT can overcome the limitations of TBI by delivering the prescribed dose targeted to the bone marrow (BM) while sparing organs at risk. Recently, our group demonstrated that TMI up to 20 Gy in relapsed/refractory AML patients was feasible and efficacious, significantly improving 2-year overall survival compared to the standard treatment. Whether such dose escalation is feasible in elderly patients, and how the organ toxicity profile changes when switching to TMI in patients of all ages are critical questions that need to be addressed. We used our recently developed 3D image-guided preclinical TMI model and evaluated the radiation damage and its repair in key dose-limiting organs in young (~8 weeks) and old (~90 weeks) mice undergoing congenic bone marrow transplant (BMT). Engraftment was similar in both TMI and TBI-treated young and old mice. Dose escalation using TMI (12 to 16 Gy in two fractions) was well tolerated in mice of both age groups (90% survival ~12 Weeks post-BMT). In contrast, TBI at the higher dose of 16 Gy was particularly lethal in younger mice (0% survival ~2 weeks post-BMT) while old mice showed much more tolerance (75% survival ~13 weeks post-BMT) suggesting higher radio-resistance in aged organs. Histopathology confirmed worse acute and chronic organ damage in mice treated with TBI than TMI. As the damage was alleviated, the repair processes were augmented in the TMI-treated mice over TBI as measured by average villus height and a reduced ratio of relative mRNA levels of amphiregulin/epidermal growth factor (areg/egf). These findings suggest that organ sparing using TMI does not limit donor engraftment but significantly reduces normal tissue damage and preserves repair capacity with the potential for dose escalation in elderly patients.

Development and characterization of a preclinical total marrow irradiation conditioning-based bone marrow transplant model for sickle cell disease.

Sickle cell disease (SCD) is a serious global health problem, and currently, the only curative option is hematopoietic stem cell transplant (HCT). However, myeloablative total body irradiation (TBI)-based HCT is associated with high mortality/morbidity in SCD patients. Therefore, reduced-intensity (2-4 Gy) total body radiation (TBI) is currently used as a conditioning regimen resulting in mixed chimerism with the rescue of the SCD disease characteristic features. However, donor chimerism gradually reduces in a few years, resulting in a relapse of the SCD features, and organ toxicities remained the primary concern for long-term survivors. Targeted marrow irradiation (TMI) is a novel technique developed to deliver radiation to the desired target while sparing vital organs and is successfully used for HCT in refractory/relapsed patients with leukemia. However, it is unknown if TMI will be an effective treatment for a hematological disorder like SCD without adverse effects seen on TBI. Therefore, we examined preclinical feasibility to determine the tolerated dose escalation, its impact on donor engraftment, and reduction in organ damage using our recently developed TMI in the humanized homozygous Berkley SCD mouse model (SS). We show that dose-escalated TMI (8:2) (8 Gy to the bone marrow and 2 Gy to the rest of the body) is tolerated with reduced organ pathology compared with TBI (4:4)-treated mice. Furthermore, with increased SCD control (AA) mice (25 million) donor BM cells, TMI (8:2)-treated mice show successful long-term engraftment while engraftment failed in TBI (2:2)-treated mice. We further evaluated the benefit of dose-escalated TMI and donor cell engraftment in alleviating SCD features. The donor engraftment in SCD mice completely rescues SCD disease features including recovery in RBCs, hematocrit, platelets, and reduced reticulocytes. Moreover, two-photon microscopy imaging of skull BM of transplanted SCD mice shows reduced vessel density and leakiness compared to untreated control SCD mice, indicating vascular recovery post-BMT.

Feasibility of a Novel Sparse Orthogonal Collimator-Based Preclinical Total Marrow Irradiation for Enhanced Dosimetric Conformality.

Total marrow irradiation (TMI) has significantly improved radiation conditioning for hematopoietic cell transplantation in hematologic diseases by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. Recently, preclinical three-dimensional image-guided TMI has been developed to enhance mechanistic understanding of the role of TMI and to support the development of experimental therapeutics. However, a dosimetric comparison between preclinical and clinical TMI reveals that the preclinical TMI treatment lacks the ability to reduce the dose to some of the vital organs that are very close to the skeletal system and thus limits the ability to evaluate radiobiological relevance. To overcome this limit, we introduce a novel Sparse Orthogonal Collimator (SOC)-based TMI and evaluate its ability to enhance dosimetric conformality. The SOC-TMI-based dose modulation technique significantly improves TMI treatment planning by reducing radiation exposures to critical organs that are close to the skeletal system that leads to reducing the gap between clinical and preclinical TMI.

Infrared spectrometric biomarkers for ulcerative colitis screening using human serum samples.

This study uses infrared spectrometry coupled with data analysis techniques to understand colitis-induced alterations in the molecular components of serum samples. Using samples from 18 ulcerative colitis patients and 28 healthy volunteers, we assessed features such as absorbance values at wavenumbers of 1033 and 1076 cm-1 , and the ratios at 1121 versus 1020 cm-1 and 1629 versus 1737 cm-1 . Through the deconvolution of the amide I band, protein secondary structure analysis was performed. Colitis-induced alterations are reflected as fluctuations in the vibrational modes, and are used to identify associated spectral signatures. The results of the study show statistically significant differences in five identifying spectral signatures. Among them, the sensitivity and specificity of the spectral signature, I1121 /I1020 , were 100% and 86%, respectively. These findings resemble our earlier proof-of-concept investigations in mouse models and provide preliminary evidence that this could be a reliable diagnostic test for human patients.

TEM study of chronic alcoholism effects on early carcinogenesis by probing the nanoscale structural alterations of cell nuclei.

Nanoscale structural alteration in the nuclei of cells with the progression of carcinogenesis is due to the rearrangements of the basic building blocks in the cell such as DNA, RNA, lipids, etc. Although epigenetic modifications underlie the development of cancer, exposure to carcinogenic chemicals such as alcohol also enhances the development of cancer. We report the effects of chronic alcoholism on early-carcinogenesis based on changes in the degree of nanoscale structural alterations (L d) in nuclei. For this, transmission electron microscopy (TEM) imaging of the nuclei of colonic cells is performed for the following four mouse models: control mice; chronic alcoholic mice treated with ethanol (i.e., EtOH mice); mice treated with colonic carcinogen azoxymethane (AOM) and dextran sulfate sodium (DSS) that induced colitis (i.e., AOM + DSS mice); and chronic alcoholic or EtOH treated mice, together with AOM and DSS treatment (i.e., AOM + DSS + EtOH mice). The disordered optical lattices are constructed from their respective TEM images of thin colonic cell nuclei and the L d values are calculated using the inverse participation ratio (IPR) technique from the spatially localized eigenfunctions of these lattices. Results show no significant difference in the average L d value of the colon cell nuclei of alcohol treated mice relative to its control [i.e., L d(C) ∼ L d(EtOH)]; however, an increase in the L d value of alcohol treated precancerous cells [i.e., L d(AOM + DSS + EtOH) > L d(AOM + DSS)], indicating that alcohol accelerates the early carcinogenic process.

Optimizing infrared spectral discrimination to enhance disease diagnostics: monitoring the signatures of inflammatory bowel diseases with anti-TNFα therapy.

This study presents an application of infrared spectroscopy of sera for monitoring the efficacy of anti-TNFα therapy for inflammatory bowel diseases. Understanding the therapeutic response includes the analysis of absorption bands representing constituent molecules. Interleukin-10 knockout mouse model of the diseases with anti-TNFα treatment was used. The discrimination potential is optimized by analyzing data with curve fitting. It shows; antibody therapy markedly ameliorated the disease, concurring with earlier mucosal immunology and pathophysiologic studies. This technique may thus also be useful for the evaluation of mucosal healing or other therapeutic modalities of gastrointestinal tract diseases keeping the endoscopic tests as confirmatory.

Protein Conformational Changes in Breast Cancer Sera Using Infrared Spectroscopic Analysis.

Protein structural alterations, including misfolding and aggregation, are a hallmark of several diseases, including cancer. However, the possible clinical application of protein conformational analysis using infrared spectroscopy to detect cancer-associated structural changes in proteins has not been established yet. The present study investigates the applicability of Fourier transform infrared spectroscopy in distinguishing the sera of healthy individuals and breast cancer patients. The cancer-associated alterations in the protein structure were analyzed by fitting the amide I (1600-1700 cm-1) band of experimental curves, as well as by comparing the ratio of the absorbance values at the amide II and amide III bands, assigning those as the infrared spectral signatures. The snapshot of the breast cancer-associated alteration in circulating DNA and RNA was also evaluated by extending the spectral fitting protocol to the complex region of carbohydrates and nucleic acids, 1140-1000 cm-1. The sensitivity and specificity of these signatures, representing the ratio of the α-helix and ß-pleated sheet in proteins, were both 90%. Likewise, the ratio of amides II and amide III (I1556/I1295) had a sensitivity and specificity of 100% and 80%, respectively. Thus, infrared spectroscopy can serve as a powerful tool to understand the protein structural alterations besides distinguishing breast cancer and healthy serum samples.

Recent Progress on Extended Wavelength and Split-Off Band Heterostructure Infrared Detectors.

The use of multilayer semiconductor heterojunction structures has shown promise in infrared detector applications. Several heterostructures with innovative compositional and architectural designs have been displayed on emerging infrared technologies. In this review, we aim to illustrate the principles of heterostructure detectors for infrared detection and explore the recent progress on the development of detectors with the split-off band and threshold wavelength extension mechanism. This review article includes an understanding of the compositional and the architectural design of split-off band detectors and to prepare a database of their performances for the wavelength extension mechanism. Preparing a unique database of the compositional or architectural design of structures, their performance, and penetrating the basics of infrared detection mechanisms can lead to significant improvements in the quality of research. The brief outlook of the fundamentals of the infrared detection technique with its appropriateness and limitations for better performance is also provided. The results of the long-term study presented in this review article would be of considerable assistance to those who are focused on the heterostructure infrared detector development.

Clinical significance of MUC13 in pancreatic ductal adenocarcinoma.

BACKGROUND: Poor prognosis of pancreatic cancer (PanCa) is associated with lack of an effective early diagnostic biomarker. This study elucidates significance of MUC13, as a diagnostic/prognostic marker of PanCa. METHODS: MUC13 was assessed in tissues using our in-house generated anti-MUC13 mouse monoclonal antibody and analyzed for clinical correlation by immunohistochemistry, immunoblotting, RT-PCR, computational and submicron scale mass-density fluctuation analyses, ROC and Kaplan Meir curve analyses. RESULTS: MUC13 expression was detected in 100% pancreatic intraepithelial neoplasia (PanIN) lesions (Mean composite score: MCS = 5.8; AUC >0.8, P < 0.0001), 94.6% of pancreatic ductal adenocarcinoma (PDAC) samples (MCS = 9.7, P < 0.0001) as compared to low expression in tumor adjacent tissues (MCS = 4, P < 0.001) along with faint or no expression in normal pancreatic tissues (MCS = 0.8; AUC >0.8; P < 0.0001). Nuclear MUC13 expression positively correlated with nodal metastasis (P < 0.05), invasion of cancer to peripheral tissues (P < 0.5) and poor patient survival (P < 0.05; prognostic AUC = 0.9). Submicron scale mass density and artificial intelligence based algorithm analyses also elucidated association of MUC13 with greater morphological disorder (P < 0.001) and nuclear MUC13 as strong predictor for cancer aggressiveness and poor patient survival. CONCLUSION: This study provides significant information regarding MUC13 expression/subcellular localization in PanCa samples and supporting the use anti-MUC13 MAb for the development of PanCa diagnostic/prognostic test.

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer-stage detection.

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct "optical lattices," which represents 2-dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called "disorder strength." The method showed a promising potential in differentiating cancerous and non-cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.

Protein secondary structure analysis of dried blood serum using infrared spectroscopy to identify markers for colitis screening.

There remains a great need for diagnosis of inflammatory bowel disease, for which the current technique, colonoscopy, is costly and also has risks for complications. Attenuated total reflectance Fourier transform infrared spectroscopy is a new screening technique to evaluate colitis. Using second derivative spectral deconvolution of the absorbance spectra, a full set of spectral markers were identified based on statistical analysis. Using this method, Amide I group frequencies, (specifically, α-helix to ß-sheet ratio of the protein secondary structure) were identified in addition to the previously reported glucose and mannose signatures in sera of chronic and acute mice models of colitis. We also used the same technique to demonstrate that these spectral markers (α-helix/ß-sheet ratio, glucose and mannose) are recovering to basal levels upon anti-TNFα therapy. Hence, this technique will be able to identify changes in the sera due to diseases.

ATR-FTIR spectral discrimination between normal and tumorous mouse models of lymphoma and melanoma from serum samples.

This study presents, attenuated total reflection Fourier transforms infrared spectroscopy of dried serum samples in an effort to assess biochemical changes induced by non-Hodgkin's lymphoma and subcutaneous melanoma. An EL4 mouse model of non-Hodgkin lymphoma and a B16 mouse model of subcutaneous melanoma are used to extract a snapshot of tumor-associated alteration in the serum. The study of both cancer-bearing mouse models in wild types and their corresponding control types, emphasizes the diagnostic potential of this approach as a screening technique for non-Hodgkin lymphoma and melanoma skin cancer. Infrared absorbance values of the different spectral bands, hierarchical clustering and integral values of the component bands by curve fitting, show statistically significant differences (student's t-test, two-tailed unequal variance p-value < 0.05) between spectra representing healthy and tumorous mouse. This technique may thus be useful for having individualized route maps for rapid evaluation of lymphoma and melanoma status and associated therapeutic modalities.

Light localization properties of weakly disordered optical media using confocal microscopy: application to cancer detection.

We have developed a novel technique to quantify submicron scale mass density fluctuations in weakly disordered heterogeneous optical media using confocal fluorescence microscopy. Our method is based on the numerical evaluation of the light localization properties of an 'optical lattice' constructed from the pixel intensity distributions of images obtained with confocal fluorescence microscopy. Here we demonstrate that the technique reveals differences in the mass density fluctuations of the fluorescently labeled molecules between normal and cancer cells, and that it has the potential to quantify the degree of malignancy of cancer cells. Potential applications of the technique to other disease situations or characterizing disordered samples are also discussed.

Quantitative analysis of nanoscale intranuclear structural alterations in hippocampal cells in chronic alcoholism via transmission electron microscopy imaging.

Chronic alcoholism is known to alter the morphology of the hippocampus, an important region of cognitive function in the brain. Therefore, to understand the effect of chronic alcoholism on hippocampal neural cells, we employed a mouse model of chronic alcoholism and quantified intranuclear nanoscale structural alterations in these cells. Transmission electron microscopy (TEM) images of hippocampal neurons were obtained, and the degree of structural alteration in terms of mass density fluctuation was determined using the light-localization properties of optical media generated from TEM imaging. The results, which were obtained at length scales ranging from ~30 to 200 nm, show that 10-12 week-old mice fed a Lieber-DeCarli liquid (alcoholic) diet had a higher degree of structural alteration than control mice fed a normal diet without alcohol. The degree of structural alteration became significantly distinguishable at a sample length of ~100 nm, which is the typical length scale of the building blocks of cells, such as DNA, RNA, proteins and lipids. Interestingly, different degrees of structural alteration at such length scales suggest possible structural rearrangement of chromatin inside the nuclei in chronic alcoholism.