Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells.

Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site-specific endonucleases. Two genomic sites within the MBS85 and chemokine (C-C motif) receptor 5 (CCR5) genes (AAVS1 and CCR5 zinc-finger nuclease (CCR5-ZFN) sites, respectively) have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells (HSCs). Matrix chromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptional inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and HSCs. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a ZFN was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.

The evolution of inquilinism, host-plant use and mitochondrial substitution rates in Tamalia gall aphids.

We used mitochondrial DNA data to infer phylogenies for 28 samples of gall-inducing Tamalia aphids from 12 host-plant species, and for 17 samples of Tamalia inquilinus, aphid 'inquilines' that obligately inhabit galls of the gall inducers and do not form their own galls. Our phylogenetic analyses indicate that the inquilines are monophyletic and closely related to their host aphids. Tamalia coweni aphids from different host plants were, with one exception, very closely related to one another. By contrast, the T. inquilinus aphids were strongly genetically differentiated among most of their host plants. Comparison of branch lengths between the T. coweni clade and the T. inquilinus clade indicates that the T. inquilinus lineage evolves 2.5-3 times faster for the cytochrome oxidase I gene. These results demonstrate that: (1) Tamalia inquilines originated from their gall-inducing hosts, (2) communal (multi-female) gall induction apparently facilitated the origin of inquilinism, (3) diversification of the inquilines has involved rapid speciation along host-plant lines, or the rapid evolution of host-plant races, and (4) the inquilines have undergone accelerated molecular evolution relative to their hosts, probably due to reduced effective population sizes. Our findings provide insight into the behavioural causes and evolutionary consequences of transitions from resource generation to resource exploitation.

Chemical oxygen demand analysis of wastewater using trivalent manganese oxidant with chloride removal by sodium bismuthate pretreatment.

Current chemical oxygen demand (COD) analyses generate wastes containing hexavalent and trivalent chromium, mercury, and silver. Waste disposal is difficult, expensive, and poses environmental hazards. A new COD test is proposed that eliminates these metals and shortens analysis time, where trivalent manganese oxidant replaces hexavalent chromium (dichromate). A silver catalyst is not required. Optional pretreatment removes chloride via oxidation to chlorine using sodium bismuthate, eliminating the need for mercury. Sample aqueous and solid components are separated for chloride removal, then recombined for total COD measurement. Soluble and nonsoluble COD can be determined separately. Digestion at 150 degrees C is complete in 1 hour. Results are determined by titration or by spectrophotometric reading. Test wastes contain none of the metals regulated for disposal under the Resource Conservation and Recovery Act. Results are shown for selected organic compounds and various wastewaters. Statistical comparisons are made with dichromate COD and biochemical oxygen demand (BOD5) test values.

Psychosocial aspects of cancer genetics: women at high risk for breast and ovarian cancer.

In the past five years the advent of cancer genetic testing has created concern about the negative psychosocial sequelae of genetic counseling and testing. Research indicates that the women most likely to seek genetic testing are anxious about carrying a gene mutation and developing breast cancer. Women who are at high risk have poor knowledge and the expectation of being a gene-mutation carrier. High levels of distress have been shown to interfere with decision-making about genetic testing. Further, individuals who decline genetic testing may be at increased risk for depressive symptoms even more than those who are found to be gene-mutation carriers. There is great concern that inappropriate candidates will seek genetic testing. Improved education and access to genetic counseling are essential to help women make appropriate decisions about genetic testing. Strategies for the prevention of breast and ovarian cancer are explored, and methods to reduce the adverse psychosocial effects of decision-making about genetic testing and preventive treatment strategies are suggested.

Corticosteroids as an antiangiogenic agent for histoplasmosis-related subfoveal choroidal neovascularization.

The purpose of this study was to evaluate the role of corticosteroids in managing subfoveal choroidal neovascularization (CNV) secondary to the presumed ocular histoplasmosis syndrome. The cases of eighteen patients with histoplasmosis-related subfoveal CNV treated with corticosteroids were reviewed. Ten patients received oral prednisone for 4 to 6 weeks, and eight received a single sub-Tenon's injection of triamcinalone. Visual acuity outcomes were analyzed along with side effect profiles. At two-week follow-up, the prednisone group showed a median improvement in Snellen visual acuity of +2.0 lines, while the triamcinalone group remained essentially stable with a 0.5 line median loss. At treatment end (4 to 6 weeks), both groups showed no significant change in median acuity at 0.0 and -1.0 lines, respectively. Median final vision at 3 months also remained essentially stable at -0.5 lines for each group. Three patients reported anxiety, all of whom were taking prednisone 80 mg daily. Two patients reported increased appetite and weight gain on regimens of prednisone 80 and 100 mg daily. There were no adverse effects reported in the other patients receiving oral prednisone or in any patient receiving sub-Tenon's triamcinalone. The results suggest a beneficial effect of corticosteroids in stabilizing subfoveal CNV secondary to ocular histoplasmosis. In this small series, oral prednisone resulted in a short-term improvement in visual acuity, which stabilized over longer follow-up. The sub-Tenon's triamcinalone group achieved similar final stabilization without the initial improvement. Corticosteroids may be particularly valuable in managing neovascularization in patients who are awaiting interventions currently under development, in preventing recurrence after subfoveal surgery, or in treating non-surgical candidates. Further study is warranted to define the precise role of corticosteroids in this condition.

DNA repair and mutagen sensitivity in patients with triple primary cancers.

The purpose of this study was to measure DNA repair capacity and mutagen sensitivity in patients who have had three or more primary forms of cancer. It was hypothesized that, if abnormalities in DNA repair and mutagen sensitivity were cancer susceptibility factors, such findings would be seen with regularity in individuals with multiple primary cancers. DNA repair capacity was measured by determining repair of UV-irradiated plasmid DNA (pCMVCAT) transfected into peripheral blood lymphocytes. Results from 18 patients and a like number of age- and sex-matched controls demonstrated a significant difference in DNA repair capacity (P < 0.0001; odds ratio = 14). Mutagen sensitivity was measured by determining the mean number of chromatid breaks per cell after in vitro exposure to either bleomycin or 4-nitroquinoline-1-oxide. The difference in mean bleomycin- or 4-nitroquinoline-1-oxide-induced mutagen sensitivity between cases and controls was not statistically significant. Fourteen of the 18 patients had positive family histories of cancer; in 10, the history was compatible with cancer susceptibility syndromes. Although the numbers were small, there was no suggestion in this study that treatment or the presence of cancer was the cause of the DNA repair abnormalities encountered. These findings support the concept of diminished DNA repair capacity as an underlying feature in the development of a mutator phenotype.

Psychological counseling strategies for women at risk of breast cancer.

Women with family histories of breast cancer have a much higher risk of developing the disease than women in the general population. In the absence of primary prevention for breast cancer, secondary prevention in the form of early detection is our best bet against premature morbidity and mortality. This article describes the most salient psychological issues for high-risk women as well as ways for improving screening behaviors. Based on our work and other studies in the literature, we found that there were several key variables related to psychological distress and surveillance behaviors. Barriers to screening were a major reason why women did not engage in any breast cancer prevention behaviors. Cognitive deficits, in terms of lack of knowledge, and breast cancer misbeliefs contributed to poor adherence to screening. Most important, anxiety or emotional distress not only interfered with adherence to screening but also affected quality of life negatively in that many women needed psychological counseling. In developing psychological counseling strategies for high-risk women, we focused on the treatment outcomes of reducing emotional distress, decreasing perceived vulnerability, and improving adherence to screening behaviors. We conducted a preliminary study by piloting a group psychoeducational intervention for 6 consecutive weeks. This intervention was found to significantly reduce perception of risk (P < .02) and to increase adherence to screening behaviors (P < .01). If proven effective in a randomized controlled trial, this intervention can be proposed to other cancer centers and prevention programs for implementation and enhancement of the behaviors among high-risk women that will assure early detection and decrease breast cancer mortality.

A family of retroviruses that utilize related phosphate transporters for cell entry.

The amphotropic murine retrovirus receptor Ram-1 shows significant sequence similarity to the gibbon ape leukemia virus (GALV) receptor Glvr-1, and both of these cell surface virus receptors normally function as sodium-dependent phosphate symporters. However, Ram-1 from humans or rats does not serve as a receptor for GALV, and Glvr-1 from humans does not serve as a receptor for amphotropic virus. Here we show that the murine retrovirus 10A1 can enter cells by using either Glvr-1 or Ram-1. Furthermore, we have constructed Ram-1/Glvr-1 hybrid receptors that allow entry of both GALV and amphotropic virus. While GALV and amphotropic virus are in separate interference groups when assayed on human cells, they do interfere with each other in cells expressing the hybrid receptor. These results indicate a close functional relationship between retroviruses that utilize members of this newly defined receptor family and provide a molecular explanation for nonreciprocal and cell type-specific interference observed for some retrovirus classes.

Long-term responses of women to indole-3-carbinol or a high fiber diet.

We test the hypothesis that the estrogen metabolite ratio 2-OH-estrone:estriol can be raised via dietary indole-3-carbinol (I3C) and that this higher ratio can be sustained over a 3-month test period. We also explore the possible role of pure fiber on estradiol metabolism. Using a randomized clinical trial with three arms, each containing 20 subjects, arm 1 received 400 mg/day of I3C daily for 3 months, arm 2 received 20 g of alpha-cellulose daily for the same time period as a source of added fiber, and arm 3 received a placebo dose. Blood levels of a variety of biochemical parameters were measured. The urinary 2-OH-estrone:estriol estrogen metabolite ratio was measured monthly at the same time of the menstrual cycle. While no changes were observed in the control and alpha-cellulose-treated arms, a substantial mean increase in the ratio was observed in the I3C-treated arm at month 1; that increase was maintained over the 3-month time period. Three of the 20 subjects in this I3C-treated group differed from the others in that no significant change in the metabolite ratio was observed at any time point. The results suggest that I3C can serve to increase the 2-OH-estrone:estriol metabolite ratio in a sustained manner without detectable side effects and that some individuals may be resistant to such change.

Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.

Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and murine amphotropic retrovirus (Ram-1) are distinct but related proteins having multiple membrane-spanning regions. Distant homology with a putative phosphate permease of Neurospora crassa suggested that these receptors might serve transport functions. By expression in Xenopus laevis oocytes and in mammalian cells, we have identified Glvr-1 and Ram-1 as sodium-dependent phosphate symporters. Two-electrode voltage-clamp analysis indicates net cation influx, suggesting that phosphate is transported with excess sodium ions. Phosphate uptake was reduced by > 50% in mouse fibroblasts expressing amphotropic envelope glycoprotein, which binds to Ram-1, indicating that Ram-1 is a major phosphate transporter in these cells. RNA analysis shows wide but distinct tissue distributions, with Glvr-1 expression being highest in bone marrow and Ram-1 in heart. Overexpression of Ram-1 severely repressed Glvr-1 synthesis in fibroblasts, suggesting that transporter expression may be controlled by net phosphate accumulation. Accordingly, depletion of extracellular phosphate increased Ram-1 and Glvr-1 expression 3- to 5-fold. These results suggest simple methods to modulate retroviral receptor expression, with possible applications to human gene therapy.

Breast-gut connection: origin of chenodeoxycholic acid in breast cyst fluid.

The notion that a breast-gut connection might modulate the microenvironment of breast tissue was supported by the finding that breast cyst fluid contains bile acids that are characteristically found in the intestines. To establish that the gut, rather than circulating steroid precursors, is the source of bile acids in breast cyst fluid, we gave two patients deuterium-labelled chenodeoxycholic acid (three 200 mg doses by mouth), starting 9 days before aspiration of breast cysts. The chenodeoxycholic acid concentration of seven samples of aspirated cyst fluid ranged from 42 to 94 mumol/L. The corresponding serum concentrations of chenodeoxycholic acid on the same day were 0.8 and 2.9 mumol/L, of which the labelled compound comprised 13.0% (0.38 mumol/L) and 28.2% (0.23 mumol/L). The deuterated chenodeoxycholic acid concentrations in cyst fluid were 0.79 and 1.26 mumol/L in two samples from patient 1 and 3.22 mumol/L in patient 2; these values are equivalent to 11-17% of the serum concentrations [corrected]. This study shows that intestinal bile acids rapidly gain access to cyst fluid. Further studies should investigate the mechanisms that govern the exchange processes and the maintenance of the high cyst fluid to plasma concentration gradients, and the biological half-lives of individual constituents.

Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes.

Cultured vascular smooth muscle cells (SMCs) containing retrovirally introduced genes are a potential vehicle for gene replacement therapy. Because the cultured SMCs are selected for their ability to proliferate in vitro, it is possible that the SMCs might be permanently altered and lose their capacity to respond to growth-suppressing conditions after being seeded back into blood vessels. To investigate this possibility we measured SMC proliferation and intimal thickening in balloon-injured Fischer 344 rat carotid arteries seeded with SMCs stained with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) and infected with replication-defective retrovirus expressing human adenosine deaminase or human placental alkaline phosphatase. The majority of the seeded SMCs remained in the intima while a few of the cells appeared to migrate into the first layer of the media. Intimal SMC proliferation returned to background levels (< 0.1% thymidine labeling index) by 28 d. At late times (1 and 12 mo) the morphological appearance of the intima was the same for balloon-injured arteries with or without seeded SMC, except that the seeded arteries continued to express human adenosine deaminase or alkaline phosphatase. These results support the conclusion that cultured SMC infected with a replication-defective virus containing human adenosine deaminase or alkaline phosphatase are not phenotypically altered and do not become transformed. After seeding onto the surface of an injured artery, they stop replicating but continue to express the introduced human genes even over the long term.

erbB-2 and myc oncoproteins in sera and tumors of breast cancer patients.

This study compares the prevalence of elevated serological levels of erbB-2 and myc proteins in 36 breast cancer patients and 25 healthy, ambulatory female controls. The controls were frequency matched to the cases by age and ethnicity. Oncoprotein levels were determined blind to the "case-control status" of the individual from whom the specimen was derived. Corresponding tissue levels were examined in tumors of the 13 cases from whom sufficient tissue was available. Serum oncoproteins were elevated as follows: erbB-2 in one control (4%) compared with nine cases (25%; PFisher's exact = 0.03); myc in no control (0%) compared with seven cases (19%; PFisher's exact = 0.02). Elevated serum levels of erbB-2 or myc oncoproteins were detected in four of the seven cases (57.1%) of in situ cancer without evidence of infiltration. In all cases with elevated serum oncoproteins where tumor tissue was available, the corresponding protein was elevated in the tumor. The three cases who had elevated preoperative serum oncoprotein levels and from whom it was possible to procure postoperative specimens had normal postoperative serum oncoprotein levels. We conclude that (a) erbB-2 and myc oncoproteins are elevated in a proportion of breast cancer patients, (b) the tumor seems to be the source of the serum elevation, and (c) these proteins may be useful as part of a panel of biomarkers of early malignant disease.

Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus.

The host and tissue specificity of retrovirus infection is largely determined by specific cellular receptors that mediate virus entry. Genes encoding these receptors are widely distributed in the genome, and the receptors identified to date show no sequence similarity. We have identified the cellular receptor for amphotropic murine retroviruses, Ram-1, by screening a rat cDNA expression library introduced into amphotropic virus-resistant hamster cells. The 656-amino acid receptor is homologous to the gibbon ape leukemia virus receptor at both hydrophobic termini but is highly divergent in the central hydrophilic region. Both receptors appear to be integral membrane proteins having multiple membrane-spanning regions. Identification of this family of receptors will help define the evolutionary relationship between retroviruses and their cellular receptors.

Inhibitors of retrovirus infection are secreted by several hamster cell lines and are also present in hamster sera.

We have previously shown that Chinese hamster ovary (CHO) cells are resistant to infection by gibbon ape leukemia virus and amphotropic pseudotype retroviral vectors because of the secretion of factors that inhibit retrovirus infection. Such factors were not secreted by any mouse or human cell lines tested. Secretion of the inhibitors and resistance to infection are abrogated by treatment of CHO cells with the glycosylation inhibitor tunicamycin. Here we show that the inhibitory activities against gibbon ape leukemia virus and amphotropic viruses are partially separable and that glycosylation mutations in CHO cells mimic the effects of tunicamycin treatment. We find that several hamster cell lines derived from both Chinese and Syrian hamsters secrete inhibitors of retrovirus infection, showing that these inhibitors are not unique to the CHO cell line. Inhibitory factors are also present in the sera of Chinese and Syrian hamsters but were not detected in bovine serum. These results suggest the presence of specific factors that function to inhibit retrovirus infection in hamsters.

Screening for colorectal cancer with fecal occult blood testing and sigmoidoscopy.

BACKGROUND: The high incidence of and mortality from colorectal cancer (160,000 new cases and 60,000 deaths in the United States each year) are compelling public health concerns. Following the evolution of effective surgery for this disease since the 1960s, the focus has been on improving methods of detection and integrating them into effective screening programs. PURPOSE: This was the first study to evaluate the effectiveness, in a setting of comprehensive medical examinations, of using the fecal occult blood test in conjunction with sigmoidoscopy, rather than sigmoidoscopy alone, to screen for colorectal cancer. Our end points were extent of compliance with fecal occult blood test and sigmoidoscopy, numbers of cancers detected, and mortality rate. METHODS: From 1975 through 1979, a total of 21,756 patients (aged 40 and older) who presented at the Preventive Medicine Institute-Strang Clinic for routine medical examinations were enrolled by calendar period into study and control groups. Study patients were offered annually both rigid sigmoidoscopy examinations and fecal occult blood tests requiring two stool specimens per day for 3 days, while control patients were offered only annual sigmoidoscopy. The majority of fecal occult blood test cards were not rehydrated before assay. Patients with positive tests were referred for double-contrast barium enema and colonoscopy. Two distinct trials were carried out. Trial I was primarily a demonstration of feasibility of using the fecal occult blood test as a supplemental screening method. Of the 9277 participants, 7168 (77%) were assigned to the study group and offered the fecal occult blood test. In trial II, approximately half of the 12,479 patients were assigned to each group. Patients in both trials had follow-up through 1984. RESULTS: Compliance with the fecal occult blood test was initially high in both trials, but diminished such that only 56% of study patients in trial I and 20% of those in trial II returned for second tests. On the initial (prevalence) screen, a substantial number of early-stage cancers were detected by the fecal occult blood test, primarily in trial II. In trial II, survival probability was significantly greater (P < .001) in the study group than in the controls (70% versus 48%), and colorectal cancer mortality was lower (0.36 versus 0.63) with borderline significance (P = .053, one-sided). CONCLUSIONS AND IMPLICATIONS: The screening of average-risk individuals (aged 50 and older) for colorectal cancer through use of the fecal occult blood test in conjunction with sigmoidoscopy can increase the likelihood of early detection of this disease. This practice, coupled with prompt diagnostic work-up following positive tests, will result in treatment of earlier stage cancers and increased survival after treatment.

Reporting bilaterality status in first-degree relatives with breast cancer: a validity study.

The objective of this study was to validate reports on the bilaterality status of breast cancer in first-degree relatives of women with a strong family history of the disease; i.e., women with 1) two first-degree relatives who have, or have had, breast cancer; 2) one first- and one second-degree affected relative; or 3) one first-degree relative with diagnosis of breast cancer before the age of 50 years and/or bilateral breast cancer. We were able to obtain hospital records for 94 affected relatives of 83 patients who agreed to participate in the study. The accounts of these women were compared to the bilaterality status indicated in the hospital records of the affected relatives. Inconsistencies that might have been attributed to incomplete medical records were resolved through personal interviews with the participants, and when indicated, with other family members or the physician of the affected relative. Overall, 89.4% (84/94) of the reports validated in this manner were correct. Participants who reported unilateral breast cancer in a first-degree relative were correct 94.4% (68/72) of the time. Similarly, 94.0% (47/50) of the accounts concerning affected living relatives were accurate, regardless of whether the participant had indicated unilateral or bilateral disease. However, participants who reported bilateral breast cancer in a deceased relative were accurate only 61.5% (8/13) of the time. Incorrect reports were associated with misunderstanding of medical terminology, especially if the participant was young at the time of the diagnosis of her relative.

Urinary prostate specific antigen levels: role in monitoring the response of prostate cancer to therapy.

We have shown that prostate specific antigen (PSA) levels can be as readily obtained from voided urine as from serum samples. This procedure was found to give stable and reproducible results. PSA analyses were performed on voided urine collected from 42 patients with benign prostatic hypertrophy (BPH), 27 with stage D2 prostate cancer and 57 after radical prostatectomy. The 42 BPH samples had a mean urinary PSA level of 216 ng./ml., which did not correlate with estimated prostate size. For 4 of 5 patients with stage D2 disease who presented before hormonal therapy urinary PSA levels were greater than 50 ng./ml. For 22 stage D2 patients seen after initiation of hormonal therapy the majority had low urinary PSA levels. After initiation of hormonal therapy in most cases low urinary PSA levels were found in conjunction with high serum PSA values. However, in other cases we found high urinary PSA with low serum PSA levels. Of 43 patients who underwent radical prostatectomy for stages A to C disease it was noteworthy that 77% had elevated urinary PSA levels, while only 33% had elevated serum levels. Therefore, close to 80% of these patients have prostate tissue remaining locally after this operation.

Steroidal nonspecific esterase metabolism of N-hydroxy-2-acetylaminofluorene: evidence for selective activation by the cellular reductant NADPH.

The significance of the nonspecific esterases of human mononuclear leukocytes (HMLs) in arylamine carcinogenesis is suggested by data showing that the metabolically formed hydroxamic acid derivative of 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, is a substrate for this class of enzymes. A viable cell assay for the nonspecific esterases using alpha-naphthyl acetate as substrate is described, and data showing this activity to be sensitive to already known substrates for HML esterases as measured by three previously described assays are presented. All four assays of the same esterase activity are shown to be highly sensitive to up- and down-regulation by addition of NADPH or NADP to viable HML cultures. Selective activation of a purified rabbit nonspecific esterase by NADPH, but not by the other cellular reductants, NADH and glutathione, was demonstrated. Cytosols prepared from normal human tissue samples of liver, breast, colon, and brain were also activated by the presence of NADPH. These data do not indicate that steroidal nonspecific esterases are redox-modulated by the presence of mixed disulfides in their structure. Instead, they support the direct and specific influence of NADPH as a widespread activator of esterase activity by a mechanism not yet understood.