CHARIOT: a phase I study of berzosertib with chemoradiotherapy in oesophageal and other solid cancers using time to event continual reassessment method.

BACKGROUND: Berzosertib (M6620) is a highly potent (IC50 = 19 nM) and selective, first-in-class ataxia telangiectasia-mutated and Rad3-related protein kinase (ATR) inhibitor. This trial assessed the safety, preliminary efficacy, and tolerance of berzosertib in oesophageal cancer (A1 cohort) with RT and advanced solid tumours (A2 cohort) with cisplatin and capecitabine. METHODS: Single-arm, open-label dose-escalation (Time-to-Event Continual Reassessment Method) trial with 16 patients in A1 and 18 in A2. A1 tested six dose levels of berzosertib with RT (35 Gy over 15 fractions in 3 weeks). RESULTS: No dose-limiting toxicities (DLTs) in A1. Eight grade 3 treatment-related AEs occurred in five patients, with rash being the most common. The highest dose (240 mg/m2) was determined as the recommended phase II dose (RP2D) for A1. Seven DLTs in two patients in A2. The RP2D of berzosertib was 140 mg/m2 once weekly. The most common grade ≥3 treatment-related AEs were neutropenia and thrombocytopenia. No treatment-related deaths were reported. CONCLUSIONS: Berzosertib combined with RT is feasible and well tolerated in oesophageal cancer patients at high palliative doses. Berzosertib with cisplatin and capecitabine was well tolerated in advanced cancer. Further investigation is warranted in a phase 2 setting. CLINICAL TRIALS IDENTIFIER: EU Clinical Trials Register (EudraCT) - 2015-003965-27 ClinicalTrials.gov - NCT03641547.

Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND AND OBJECTIVES: Leishmania is transmitted by the bite of the phlebotomine sandfly or by transfusion of infected blood products. Leishmaniasis currently poses a significant problem in several parts of the world, and is an emerging problem in others. The Mirasol PRT technology is based on the use of riboflavin and ultraviolet light to generate chemical reactions in the nucleic acids of pathogens, which prevents replication and leads to inactivation. The intent of this study was to examine the ability of the Mirasol PRT System to kill the Leishmania parasite in human plasma and platelet concentrates. MATERIALS AND METHODS: In visceral Leishmaniasis, amastigotes are present in the blood and in the reticuloendothelial system within monocytes. For each unit of plasma or platelets treated, isolated mononuclear cells obtained from 100 ml of normal donor whole blood were incubated with 1.0 x 10(8) Leishmania donovani infantum promastigotes to produce amastigote-laden macrophages. The infected macrophages were added to 250 ml of human plasma or to 250 ml of platelet concentrates. Infected units were cultured pretreatment in 10-fold serial dilutions to determine the limits of detection. Thirty millilitres of 500 microM riboflavin was added to each unit, which was then illuminated with 5.9 J/cm2 of ultraviolet light (6.24 J/ml). After treatment and after 2 months of frozen storage, plasma units were cultured in 10-fold serial dilutions. Platelets were cultured on the day of treatment and on day 5 of storage post-illumination. RESULTS: A 5 log reduction of Leishmania was demonstrated in five of six units of plasma, and a 7 log reduction of Leishmania was demonstrated in one plasma unit. A 5 log reduction of Leishmania was demonstrated in five of six units of platelets, and a 6 log reduction of Leishmania was demonstrated in one unit. CONCLUSIONS: There is no donor screen for Leishmania and other pathogens constantly emerging in our blood supply. The Mirasol PRT System for Platelets and Plasma is an effective means of killing Leishmania and other emerging pathogens in these blood products.

Ovarian follicular growth and atresia: the relationship between cell proliferation and survival.

Growth factors and steroids play an important role in the regulation of ovarian follicular development. In cattle, two of the earliest detectable differences between the healthy dominant follicle selected for development to the ovulatory stage and subordinate follicles destined to undergo atresia are the greater availability of IGF and the greater capacity to produce estradiol in the dominant follicle. We have shown that IGF-I and estradiol stimulate the proliferation of bovine granulosa cells in vitro and promote granulosa cell survival by increasing resistance to apoptosis. Furthermore, the ability of IGF-I and estradiol to increase resistance to apoptosis is tied to their ability to promote progression through the cell cycle. Blocking the cell cycle at the transition between the first gap phase and the DNA synthesis phase using a specific inhibitor prevented the protective effects of IGF-I and estradiol against apoptosis. Further experiments showed that the protective effect of IGF-I against apoptosis is mediated by the stimulation of phosphatidylinositol 3-kinase and its downstream target, protein kinase B/Akt. Constitutive activation of Akt by the infection of granulosa cells with a recombinant Akt adenovirus protected against apoptosis, and this effect also depended on cell cycle progression. These experiments show that the protective effect of estradiol and IGF-I against apoptosis depends on unperturbed progression through the cell cycle. Once follicles have developed to the preovulatory stage, the LH surge induces terminal differentiation of granulosa cells and withdrawal from the cell cycle. Bovine granulosa cells withdraw from the cell cycle by 12 h after the LH surge and become resistant to apoptosis, even in the absence of growth factors. Treatment with a progesterone receptor antagonist in vitro caused reentry of granulosa cells into the cell cycle and susceptibility to apoptosis, suggesting that induction of progesterone receptor expression by the LH surge is required for cell cycle withdrawal and resistance to apoptosis. In summary, the susceptibility of granulosa cells to apoptosis depends on the cell cycle. Proliferating granulosa cells in growing follicles depend on growth factors for survival, whereas cells that have terminally differentiated in response to the LH surge are resistant to apoptosis and relatively independent of growth factors for survival.

White particulate matter found in blood collection bags consist of platelets and leukocytes.

BACKGROUND: In late January 2003, some blood centers and hospitals throughout the US voluntarily sus-pended the use of some RBC and plasma units for trans-fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets. STUDY DESIGN AND METHODS: All AS-5 nonleuko-reduced RBCs and plasma units were visually inspected for WPM by placing the bags on a flat counter, undisturbed, for approximately 10 minutes and then perform-ing a visual examination for particles. Particles were isolated and placed on microscope slides or in plastic tubes for further analysis. Electron microscopy, bright field microscopy, differential interference contrast microscopy, infrared spectroscopy, and flow cytometry procedures were performed to establish the nature of the particulate matter. In addition, leukoreduction filters and blood transfusion sets were used on RBCs units with WPM. RESULTS: The particles were mostly composed of PLTs and WBCs, and fragments of these cells. All macroscopic WPM was removed from RBCs with leukoeduction and transfusion filters. CONCLUSIONS: WPM originated from PLTs and WBCs. Foreign matter (e.g., plastic) was not observed in any of the units. Leukoreduction and transfusion filters can be used to remove macroscopic WPM.

Determinants of outcome in melanoma patients with cerebral metastases.

PURPOSE: To analyze prognostic factors, effects of treatment, and survival for patients with cerebral metastases from melanoma. PATIENTS AND METHODS: All melanoma patients with cerebral metastases treated at the Sydney Melanoma Unit between 1952 and 2000 were identified. From 1985 to 2000, patients were diagnosed and treated using consistent modern techniques and this cohort was analyzed in detail. Multivariate analysis of prognostic factors for survival was performed. RESULTS: A total of 1137 patients with cerebral metastases were identified; 686 were treated between 1985 and 2000. For these 686 patients, the median time from primary diagnosis to cerebral metastasis was 3.1 years (range, 0 to 41 years). A total of 646 patients (94%) have died as a result of melanoma. The median survival from the time of diagnosis of cerebral metastasis was 4.1 months (range, 0 to 17.2 years). Treatment was as follows: surgery and postoperative radiotherapy, 158 patients; surgery alone, 47 patients; radiotherapy alone, 236 patients; and supportive care alone, 210 patients. Median survival according to treatment received for these four groups was 8.9, 8.7, 3.4, and 2.1 months, respectively; the differences between surgery and nonsurgery groups were statistically significant. On multivariate analysis, significant factors associated with improved survival were surgical treatment (P <.0001), no concurrent extracerebral metastases (P <.0001), younger age (P =.0007), and longer disease-free interval (P =.036). Prognostic factors analysis confirmed the important influence of patient selection on treatment received. CONCLUSION: This large series documents the characteristics of patients who developed cerebral metastases from melanoma. Median survival was dependent on treatment, which in turn was dependent on patient selection.

Susceptibility of ovarian granulosa cells to apoptosis differs in cells isolated before or after the preovulatory LH surge.

The luteinizing hormone (LH) surge initiates the final stages of ovarian follicle development, and induces ovulation and luteinization of preovulatory follicles. To investigate whether exposure to the LH surge alters follicle cell susceptibility to apoptosis, granulosa and theca cells were isolated from bovine preovulatory follicles before and 14 h after injection of GnRH to induce an LH surge. Granulosa cells isolated before the LH surge were susceptible to apoptosis induced by soluble Fas ligand or serum withdrawal, while cells isolated after the LH surge were resistant to apoptosis. Resistance to Fas-mediated apoptosis was not associated with decreased Fas mRNA or protein levels. Pretreatment of granulosa cells isolated after the LH surge with the protein synthesis inhibitor cycloheximide rendered the cells susceptible to Fas-mediated apoptosis, indicating that inhibition of apoptosis was mediated by expression of labile survival factors. Theca cells were sensitive to Fas-mediated apoptosis before and after exposure to the LH surge. Resistance to apoptosis of granulosa cells from preovulatory follicles after the LH surge may be important for normal ovulation and luteinization.

Relationship of Fas ligand expression and atresia during bovine follicle development.

The Fas antigen (Fas) is a cell surface receptor that may be involved in the initiation and progression of follicle cell apoptosis during atresia. Fas initiates apoptosis in sensitive cells after binding Fas ligand (FasL). Other experiments have shown that expression of Fas mRNA and responsiveness to Fas-mediated apoptosis vary in bovine granulosa and theca cells during follicle development. In the present study, FasL mRNA content was measured and Fas and FasL protein expression was examined in bovine granulosa and theca cells of healthy dominant follicles and the two largest atretic subordinate follicles on day 5 of the oestrous cycle (day 0 = oestrus), and of dominant follicles from the first wave of follicle development after they had become atretic and showed no growth for 4 days. FasL mRNA content was higher in granulosa cells from atretic compared with healthy follicles. FasL mRNA content was also higher in theca cells from atretic subordinate compared with healthy dominant follicles on day 5, but did not differ between theca cells from healthy and atretic dominant follicles. Immunohistochemical staining for FasL was more intense in theca compared with granulosa cells and in atretic compared with healthy follicles. Immunohistochemical staining for Fas was more intense in granulosa compared with theca cells and in atretic subordinate compared with healthy dominant follicles on day 5. Immune cells, known to express Fas and FasL, were localized in the theca, but not the granulosa, cell layer of all follicles. Higher concentrations of Fas and FasL in cells from atretic follicles, together with the previous demonstration of increased responsiveness of granulosa cells from subordinate follicles to FasL-induced apoptosis, support a potential role for FasL-mediated apoptosis during ovarian follicle atresia.

Apoptosis of bovine granulosa cells after serum withdrawal is mediated by Fas antigen (CD95) and Fas ligand.

Ovarian follicular atresia occurs by apoptosis of granulosa and theca cells. The Fas antigen (Fas), a cell surface receptor that triggers apoptosis when activated by Fas ligand (FasL), may be involved in this process. A possible role of the Fas pathway in mediating serum withdrawal-induced apoptosis of granulosa cells was examined. Granulosa cells collected from 5- to 10-mm bovine follicles were cultured in DMEM-F12 containing serum for 3 days, deprived of serum, and live cells were counted at various times after serum withdrawal. Cell death increased significantly 6 h after serum withdrawal (21% +/- 7%; P: < 0.05 vs. 0 h) and continued to increase until 24 h (43% +/- 6%). No further increases in cell death were observed through 72 h. Detection of the translocation of phosphatidylserine to the outer surface of the cell membrane by annexin V binding indicated that cells died by apoptosis. Quantitative reverse transcriptase-polymerase chain reaction assays showed no changes in Fas mRNA levels but a 4.7-fold increase in FasL mRNA 3 h after serum withdrawal (P: < 0.05 vs. 0 h). FasL mRNA remained elevated through 24 h and returned to basal levels at 48 h. Immunohistochemical staining showed that both Fas and FasL protein increased on the cell surface within 3 h and remained elevated through 12 h (the last time point tested). Binding of FasL to Fas was blocked with two reagents that bind to the extracellular domain of FasL: an anti-FasL antibody and Fas:Fc, a chimeric protein consisting of the Fc portion of human immunoglobulin G and the extracellular domain of human Fas. Cell death 24 h after serum withdrawal was reduced 55% +/- 10% and 34% +/- 12% by anti-FasL antibody and Fas:Fc, respectively (P: < 0.05 vs. no blocking protein). In conclusion, serum withdrawal-induced apoptosis of bovine granulosa cells is mediated at least partially by Fas/FasL interactions. These results are consistent with a potential role of Fas in an autocrine or paracrine pathway to trigger ovarian follicular atresia.

Activities of contemporary Mayan elders.

Social gerontology researchers have investigated various aspects of elder activities in diverse culture contexts. This project assessed the knowledge that Mayan people have about the activities of their elders. Data were collected through two interview procedures. The first procedure, free-listing, was conducted with an initial sample of Mayan subjects (n=31), who provided terms representing their cultural knowledge of elder Mayan activities. A second sample (n=56) performed a pile sort task using 18 of those activities. The pile sort findings were subjected to multidimensional scaling, and that technique produced a two-dimensional visual representation of three distinct clusters of activities: productive work, serious socio-religious undertakings and frivolous ventures. Besides the coherent multidimensional scaling representation for that total sample of 56 subjects, scalings for subsamples based on generational affiliation were run. Two-dimensional pictures or visual maps for the aged, middle-aged and young adult subjects are remarkably similar to each other and to the picture for the total sample. From a methodological perspective the research findings demonstrate that elder Mayan activities, and presumably those of elders in other societies, are amenable to interview procedures that have high validity and high reliability.

Regulation of Fas antigen (Fas, CD95)-mediated apoptosis of bovine granulosa cells by serum and growth factors.

Our previous studies have shown that bovine granulosa cells cultured in basal media supplemented with 5% fetal bovine serum (BM-FBS) are resistant to apoptosis induced by recombinant Fas ligand (FasL) unless pretreated with interferon-gamma (IFN). Experiments were conducted to test the hypothesis that serum and growth factors alter the susceptibility of granulosa cells to FasL-induced apoptosis. Granulosa cells were cultured in BM-FBS, BM containing insulin, transferrin, selenium, and BSA (BM-ITS), and in BM-ITS supplemented with insulin-like growth factor-I (IGF). Cells were susceptible to FasL-induced killing in BM-ITS (27% killing) but were resistant in BM-FBS and in BM-ITS containing IGF (P < 0.05 vs. killing in BM-ITS). Exposure of phosphatidylserine residues on the outer cell membrane, an early marker of apoptosis, was stimulated by FasL and prevented in the presence of IGF. Neutralization of IGF activity in serum with IGF binding protein 3 reduced the protective effect of FBS on FasL-induced killing (P < 0.05), suggesting that IGF is an inhibitory component in FBS. Cotreatment with IFN overcame the inhibitory effects of serum and IGF on FasL-induced killing (31% and 29% killing, respectively, P > 0.05), but IFN did not potentiate killing of cells cultured in BM-ITS. IFN increased expression of Fas antigen (Fas, the receptor for FasL) mRNA five- to sevenfold (P: < 0. 05) and increased immunostaining for Fas protein similarly in all types of media. Addition of the growth factors epidermal growth factor or basic fibroblast growth factor to BM-ITS also inhibited FasL-induced killing (P < 0.05), whereas keratinocyte growth factor, transforming growth factor, platelet-derived growth factor, FSH, and LH had no effect. In summary, FasL-induced killing is inhibited by FBS and certain growth factors. IFN increased expression of Fas similarly in all types of media but was required for FasL-induced killing only in BM containing FBS or IGF. Therefore, modulation of responsiveness to FasL-induced apoptosis by growth factors and IFN is not directly related to the level of Fas expression.

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis.

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.

Expression and activity of the Fas antigen in bovine ovarian follicle cells.

The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.

Reoperative parathyroid surgery in the era of sestamibi scanning and intraoperative parathyroid hormone monitoring.

HYPOTHESIS: Results of reoperative parathyroid surgery (RPS) have improved with the advent of sestamibi parathyroid subtraction scanning and intraoperative parathyroid hormone (IOPTH) monitoring. DESIGN: Retrospective review of patient histories, preoperative localization studies, operative data, including IOPTH monitoring, and outcomes for patients undergoing recent RPS at a single institution. Follow-up was complete (mean, 20 months). SETTING: Tertiary care referral center. PATIENTS: All patients undergoing RPS for benign persistent or recurrent primary hyperparathyroidism during the period 1989 to 1997. MAIN OUTCOME MEASURES: Overall cure rate and operative morbidity from RPS; sensitivity and accuracy of preoperative localization studies; and prediction of cure from IOPTH monitoring. RESULTS: The study group included 124 patients (87 women and 37 men). Hypercalcemia was corrected in 109 patients (88%). Permanent recurrent laryngeal nerve injury occurred in 0.8% and permanent hypoparathyroidism in 13% of patients. Test sensitivities and accuracies, respectively, were as follows: ultrasound with biopsy, 90% and 82%; sestamibi parathyroid subtraction scanning, 82% and 67%; and ultrasound alone, 75% and 65%. Level of IOPTH was predictive of cure in all patients with a 70% or greater fall from baseline at 20 minutes after excision. Persistent multigland disease was the major cause for reoperative failure (73%). CONCLUSIONS: Neither cure rates nor operative morbidity have changed appreciably over the past 2 decades, despite the introduction of sestamibi parathyroid subtraction scanning and IOPTH monitoring. Multigland disease continues to represent the principal cause of failure in RPS despite the routine use of preoperative localization studies. Thus far, increasing the stringency of IOPTH monitoring from a 50% to 70% decline from baseline levels has been predictive of cure, even in multigland disease. Most missed abnormal glands reside in normal anatomic locations, and the need for multiple operations, not just the reoperation, results in the increased morbidity seen with RPS.

Primary mucosal and glans penis melanomas: the Sydney Melanoma Unit experience.

BACKGROUND: Melanomas that arise on mucosal surfaces and the glans penis are rare. METHODS: A retrospective study of the Sydney Melanoma Unit experience with 69 patients treated since 1956 for these types of melanomas was undertaken to determine primary lesion site, sex, age at diagnosis, symptoms, clinical stage at first presentation. histopathology, treatment and outcome. RESULTS: Primary lesion sites were: nasal cavity (n = 9), oral cavity (n = 16), vulva/vagina (n = 25), anus/rectum (n = 13) and glans penis (n = 6). At diagnosis, 55 patients had local disease only, eight had regional lymph node metastases and six had widespread disease. Local recurrence as the first sign of relapse developed in 15 of the 55 stage I patients (three-stage system). Prognosis for the entire group was poor, only 10% being disease free 3 years after diagnosis and overall 3- and 5-year actuarial survival being 40% and 23%. respectively. The only statistically significant factor influencing survival was stage of disease at diagnosis (P = 0.002). CONCLUSIONS: Possible reasons for poor survival include: (i) non-specific symptoms resulting in late presentation; (ii) locally advanced disease not being recognized by a clinician as a rare form of melanoma, resulting in a delay in treatment; (iii) anatomical constraints precluding surgery with generous margins and consequently resulting in a high incidence of local recurrence. Also, rich vascularity and multiple lymphatic drainage pathways may mean a predisposition to early dissemination. Prompt diagnosis and referral to a specialist unit for treatment and follow up are essential. Adequate surgery remains the cornerstone of treatment for these types of melanoma until more effective systemic therapies become available.

Toxicological properties of several novel oligonucleotide analogs in mice.

The toxicological properties of ISIS 3082, a phosphorothioate oligonucleotide, and five structurally related analogs of ISIS 3082, were examined in Balb/c mice. Comparisons were made between the uniform phosphorothioate oligonucleotide (ISIS 3082), and a 2' propoxy modified phosphodiester (ISIS 9044), a 2' propoxy phosphorothioate (ISIS 9045), a chimeric oligonucleotide comprised of 2' propoxy diester wings and phosphorothioate deoxy center (ISIS 9046), a 5' C18 amine phosphorothioate (ISIS 9047), or a 5' cholesterol modified phosphorothioate (ISIS 8005) oligonucleotide. Oligonucleotides were administered at 50 mg/kg by i.v. bolus injection (tail vein) every other day for 14 days. In general, the spectrum of alterations observed for ISIS 3082 and all of the analogs were relatively similar. Balb/c mice treated with ISIS 3082 were observed to have increases in liver transaminases and a decrease in triglycerides consistent with results from previous studies performed in CD-1 mice. Spleen weights were also increased in ISIS 3082-treated mice, but no histopathological alterations were noted. ISIS 9046 resulted in a toxicity profile that was very similar to that described for ISIS 3082 with the exception of a slightly lower cholesterol level. Alterations induced by ISIS 9045, ISIS 9047 and ISIS 8005 were qualitatively similar to ISIS 3082, but in general more pronounced, with greater reductions in cholesterol and platelet counts, or increases in blood urea nitrogen relative to ISIS 3082. Red blood cell (RBC) counts and hematocrit were also reduced in mice treated with ISIS 9046, ISIS 9047 and ISIS 8005 relative to the ISIS 3082 treatment group. Kupffer cell hypertrophy and basophilic inclusions in Kupffer cells were observed in mice treated with ISIS 9045, ISIS 9047 and ISIS 8005, but not in ISIS 3082-treated mice. A unique renal lesions was noted in mice treated with ISIS 9044 only that was characterized as mild atrophy of proximal convoluted tubules associated with interstitial fibrosis. With the exception of the renal lesions observed in ISIS 9044 treated mice, the toxicity profiles of various oligonucleotide analogs examined in this study were similar to that observed for ISIS 3082.