Olena Palyvoda

Influence of Polymorphisms in DNA Repair Genes XPD, XRCC1 and MGMT on DNA Damage Induced by Gamma Radiation and its Repair in Lymphocytes In Vitro

Abstract Rzeszowska-Wolny, J., Polanska, J., Pietrowska, M., Palyvoda, O., Jaworska, J., Butkiewicz, D. and Hancock, R. Influence of Polymorphisms in DNA Repair Genes XPD, XRCC1 and MGMT on DNA Damage Induced by Gamma Radiation and its Repair in Lymphocytes In Vitro. Radiat. Res. 164, 132– 140 (2005). DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co γ radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp → Asn and 751Lys → Gln, XRCC1 399Arg → Gln, and MGMT 84Leu → Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (>median induced SSBs/<median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4–19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.

Raman spectroscopic differentiation of activated versus non-activated T lymphocytes: an in vitro study of an acute allograft rejection model.

Acute rejection (AR) remains a significant complication in renal transplant patients. Using serum creatinine for AR screening has proven problematic, and thus a noninvasive, highly sensitive and specific test is needed. T cells from human peripheral blood were analyzed using Raman spectroscopy. Fifty-one Mixed Lymphocyte Culture (MLC) activated T cells (ATC), 28 Mitomycin C inactivated T cells (ITC), and 35 resting T cells (RTC), were studied utilizing 785 and 514.5 nm wavelengths. Statistical analysis following subtraction of fluorescence used Students t test to quantify peak ratio differences and discriminant function analysis (DFA), with three distinct sectors assigned for grouping purposes: Sector I, ITC; Sector II, ATC; Sector III, RTC. Differences between ATC and non-activated T cells (ITC and RTC) were found at 1182 and 1195 cm-1 peak positions for both wavelengths. Significant differences in peak ratios for 785 and 514.5 nm wavelengths existed between ATC and RTC (p=0.001 and p=0.006, respectively) and ATC and ITC (p=0.001 and p=0.001, respectively), with a trend in differences observed between ITC and RTC (p=0.07 and p=0.08, respectively). Analysis of the DFA-derived sector distribution for the 785 and 514.5 nm wavelengths revealed a sensitivity of 95.7% and 89.3%, respectively, and a specificity of 100% and 93.8%, respectively. This data suggests that Raman spectroscopy can detect significant differences between activated and nonactivated T cells based upon cell-surface receptor expression, thereby establishing unique signatures that can aid in the development of a noninvasive AR screening tool with high sensitivity and specificity.

Molecular organization in SAMs used for neuronal cell growth

The attachment of cells onto solid supports is fundamental in the development of advanced biosensors or biochips. In this work, we characterize cortical neuron adhesion, growth, and distribution of an adhesive layer, depending on the molecular structure and composition . Neuronal networks are successfully grown on amino-terminated alkanethiol self-assembled monolayer (SAM) on a gold substrate without adhesion protein interfaces. Neuron adhesion efficiency was studied for amino-terminated, carboxy-terminated, and 1:1 mixed alkanethiol SAMs deposited on gold substrates. Atomic force microscopy and X-ray photoelectron spectroscopy were used to measure the roughness of gold substrate and thickness of SAM monolayers. Conformational ordering and ionic content of SAMs were characterized by vibrational sum frequency generation (VSFG) spectroscopy. Only pure amino-terminated SAMs provide efficient neuronal cell attachment. Ordering of the terminal amino groups does not affect efficiency of neuron adhesion. VSFG analysis shows that ordering of the terminal groups improves with decreasing surface roughness; however the number of gauche defects in alkane chains is independent of surface roughness. We monitor partial dissociation of carboxy groups in mixed SAMs that implies formation of NH3+ neighbors and appearance of catanionic structure. Such catanionic environment proved inefficient for neuron adhesion. Surface roughness of metal within the 0.7-2 nm range has little effect on the efficiency of neuron adhesion. This approach can be used to create new methods that help map structure-property relationships of biohybrid systems.

Differentiation of alloreactive versus CD3/CD28 stimulated T‐lymphocytes using Raman spectroscopy: A greater specificity for noninvasive acute renal allograft rejection detection

Acute rejection (AR) remains problematic in renal transplantation. As a marker, serum creatinine is limited, warranting a more effective screening tool. Raman spectroscopy (RS) can detect T‐cell activation with high sensitivity. In this study we explore its specificity. Seventy‐five inactivated, 40 alloantigen‐activated, and 75 CD3/CD28‐activated T cells were analyzed using RS. CD3/CD28‐activated peak magnitudes (PM) were 4.3% to 23.9% lower than inactivated PM at positions: 903, 1031, 1069, 1093, 1155, 1326, and 1449 cm−1, with a difference in peak ratio (PR) observed at the 1182:1195 cm−1 position (0.91 ± 0.06 vs. 1.2 ± 0.01, respectively: P = 0.006). Differences in CD3/CD28‐ and alloantigen‐activated PM were observed at: 903, 1031, 1093, 1155, 1326, and 1449 cm−1, with no PR differences at the 1182:1195 cm−1 position (0.91 ± 0.06 vs. 0.86 ± 0.09: P = 0.8). Spectral signature separation of CD3/CD28—and alloantigen‐activated groups was 100% specific and sensitive. We conclude that RS can differentiate T cells activated by different stimuli with high sensitivity and specificity.

Fibronectin adsorption to nanopatterned silicon surfaces

The possibility of using surface topography for guidance of different biological molecules and cells is a relevant topic that can be applied to a wide research activity. This study investigated the adsorption of fibronectin to a diffraction grated silicon surface. The rectangular grating profile featured a controlled surface with 350 nm period and a corrugation depth of 90 nm. Results demonstrated that the controlled surface had a significantly positive effect on the fibronectin binding. Thus, nanoscale surface topography can enhance fibronectin binding.

Relationships between acute reactions to radiotherapy in head and neck cancer patients and parameters of radiation-induced DNA damage and repair in their lymphocytes

Purpose: To study the relationship between lymphocyte radiosensitivity measured in vitro and acute reactions to radiotherapy in patients with head and neck cancer. Materials and methods: Acute reactions were measured in 34 patients using the Dische scale. Lymphocyte radiosensitivity was measured using the alkaline comet assay, the micronucleus assay, the nuclear division index and morphological assessment of apoptosis. Results: There was a weak, statistically significant correlation between in vitro radiosensitivity measured as the rate of DNA damage repair and the cumulative radiation dose exerting the maximum acute reaction scored (r = −0.366, p = 0.039, n = 34). Subgroup analyses showed that for patients with a low level of radiation-induced DNA damage there was a statistically significant relationship between lymphocyte radiosensitivity measured as inhibition of proliferation and acute toxicity (r = −0.621, p = 0.007, n = 18). For patients with a high level of residual DNA damage, there was a relationship between lymphocyte radiosensitivity measured using the micronucleus assay and acute toxicity (r = −0.597, p = 0.023, n = 14). Conclusions: Combining two measures of radiosensitivity improves the ability to correlate in vitro lymphocyte radiosensitivity and acute radiotherapy toxicity data.

Raman Spectroscopy in the Diagnosis of Ulcerative Colitis

INTRODUCTION At present, the diagnosis of ulcerative colitis (UC) requires the histologic demonstration of characteristic mucosal inflammatory changes. A rapid and noninvasive diagnosis would be of value, especially if it could be adapted to a simple rectal probe. Raman spectroscopy creates a molecular fingerprint of substances by detecting laser light scattered from asymmetric, vibrating, and chemical bonds. We hypothesize that Raman spectroscopy can distinguish UC from non-UC colon tissue rapidly and accurately. MATERIALS AND METHODS Colon tissue specimens were obtained from patients operated at the Childrens Hospital of Michigan, United States, including UC colon and non-UC colon. The samples were examined with a Renishaw inVia Raman microscope (Gloucestershire, United Kingdom) with a 785 nm laser. Principal component analysis and discriminant function analysis were used to classify groups. Final classification was evaluated against histologic diagnoses using leave-one-out cross-validation at a spectral level. RESULTS We compared Raman spectroscopy examination of colon specimens from four patients with UC and four patients without UC. A total of 801 spectra were recorded from colon specimens. We evaluated 100 spectra each from the mucosal and serosal surfaces of patients with UC and 260 spectra from the mucosal surface and 341 spectra from the serosal surface of the patients who did not have UC. For samples from the mucosal surface, the Raman analysis had a sensitivity of 82% and a specificity of 89%. For samples from the serosal surface, Raman spectroscopy had a sensitivity of 87% and a specificity of 93%. When considering each tissue sample and deciding the diagnosis based on the majority of spectra from that sample, there were no errors in the diagnosis. CONCLUSIONS Raman spectroscopy can distinguish UC from normal colon tissue rapidly and accurately. This technology offers the possibility of real-time diagnosis as well as the ability to study changes in UC-afflicted colon tissue that do not appear histologically.

Surface modification and neural tissue culture of thin film electrode materials

Good interfaces between electrodes and neural tissue are very important in chronic in vivo stimulation/recording. In order to study the effect of electrode materials and surface structure on neural interfaces, we cultured neurons on thin films of electrode materials that are expected to be biocompatible, such as platinum, and iridium oxide. We used both flat film surfaces and laser micro-structured ones. The laser micro-structuring consisted of creating regular arrays of micro-bumps with height of about 1μm and diameter of 2-3 microns. We have found conditions for fabrication of such micro-bumps on platinum and iridium thin films on borosilicate glass substrate (Pyrex 7740) by mask-projection irradiation with single nano-second pulses from a KrF excimer laser (λ=248nm). Laser micro-structured iridium films were coated with IrO 2 by pulsed DC reactive sputtering to obtain micro-structured IrO 2 films. Two types of iridium oxide films were studied: amorphous (reactively sputtered at room substrate temperature) and polycrystalline (reactively sputtered at 300°C). Cortical neurons isolated from rat embryo brain were cultured onto these thin film surfaces. Cells were more than 98% viable as determined by trypan blue exclusion tests. Poly-D-Lysine coated surfaces were used as positive controls for cell. Regular optical and fluorescent microscopy techniques were used to image the cells after they were cultured. To differentiate between live and dead cells a viability test with fluorescein diacetate (FDA) and propidium iodide was carried out. Also, immunocytochemistry analysis of neuron cells was performed using antibody for neuron-specific enolase (NSE) staining. A qualitative and quantitative comparison was carried out between the different types of modified electrode surfaces to study the neuronal growth in order to explore the feasibility of micro-bumps as stimulating/recording neural interfaces. These results are intended for use in optimization of future electrical stimulation/recording experiments that we plan to carry out.

Raman spectroscopic modeling of early versus late T-lymphocyte activation via differential spectral detection of receptor expression

The proven efficacy of renal transplantation has made it the definitive treatment for end-stage renal disease. Despite its wide acceptance, transplantation has been limited by organ shortages. In the face of this, preservation of allograft longevity is essential. The predominately T cell-driven process of acute rejection (AR) can lead to graft dysfunction and even graft loss. As a marker for AR screening, serum creatinine has a low sensitivity and specificity. This has warranted the development of more accurate screening/diagnostic tools such as Raman Spectroscopy (RS) which has been demonstrated in previous studies to accurately quantify T cell activation. In this study we further explore its application by modeling the dynamic process of cell surface receptor expression during T cell activation. 50 mitogen (Concanavalin A and pokeweed) activated T cells were stained with CD69, CD25, and CD71 monoclonal antibodies (mAbs) at 48 and 72 hour time points. In parallel, 50 activated T cells were analyzed using RS at these same time periods. At 4 8h there was high expression of the CD69 cell surface receptor detected via mAb staining with no appreciable binding of CD25/CD71 fluorescent tag. In conjunction, 48 hour RS-analyzed T cells demonstrated a significant peak difference at the 1585 cm(-1) position which represented a 63% (p=0.01) increase in peak magnitude when compared with the 72 hour samples. By contrast, the 72 hour data demonstrated an attenuation of the CD69 expression and increased CD25/CD71 expression. The corresponding RS analysis showed two significant peak differences at the 903 cm(-1) and 1449 cm(-1) positions that were not present at 48 h. These differences in Raman shifts resulted in a 40% (p=0.04) and a 59% (p=0.001) increase in peak magnitudes at these positions, respectively. This study serves to further validate RS as a screening modality capable of not only detecting T cells early in the activation process through the spectral signatures associated with CD69, but also quantifying the persistent expression of CD25 and CD71. This provides a foundation for the development of a system capable of the accurate assessment of acute and maintenance immunosuppression efficacy at the molecular level.