Detection of 1, N2-propanodeoxyguanosine adducts as potential endogenous DNA lesions in rodent and human tissues

Superantigen-activated T cells can be targeted by monoclonal antibodies (mAb) to lyse MHC class II negative tumour cells. In this study we determined the susceptibility of the T-lymphoblastoid leukaemic cell line CCRF-CEM and its multidrug resistant sublines CCRF VCR100, CCRF VCR1000 and CCRF ADR5000 to lysis by monoclonal antibody-targeted and superantigen-activated T cells (superantigen-dependent cellular cytotoxicity, SDCC). A recombinant fusion protein of protein A and the superantigen Staphylococcus enterotoxin A (SEA) was used together with the mAbs anti-CD7, anti-CD38, anti-CD45RA and 4E3 (anti-P-glycoprotein) to correlate susceptibility to SDCC with expression of the MDR1-gene product. Our results demonstrated SDCC to be independent of MDR1-gene expression. This was further confirmed by blocking the function of Pgp in the leukaemic cell lines with a cyclosporine A derivative, which had no influence on SDCC. As expected, expression of the respective cell surface antigens on target cells had a strong impact on SDCC, although other factors seem to influence efficiency of SDCC as well.     

Three-dimensional visualization and measurement of conformal dose distributions using magnetic resonance imaging of BANG polymer gel dosimeters

PURPOSE/OBJECTIVE: The measurement of complex dose distributions (those created by irradiation through multiple beams, multiple sources, or multiple source dwell positions) requires a dosimeter that can integrate the dose during a complete treatment. Integrating dosimeter devices generally are capable of measuring only dose at a point (ion chamber, diode, TLD) or in a plane (film). With increasing use of conformal dose distributions requiring shaped, noncoplanar beams, there will be an increased requirement for a dosimeter that can record and display a 3D dose distribution. The use of a 3D dosimeter will be required to confirm the accuracy of treatment plans produced by the current generation of 3D treatment-planning computers. METHODS AND MATERIALS: The use of a Fricke-infused gel and magnetic resonance imaging (MRI) to demonstrate the localization of stereotactic beams has been demonstrated (11). The recently developed BANG polymer gel dosimetry system (MGS Research, Inc., Guilford, CT), based on radiation-induced chain polymerization of acrylic monomers dispersed in a tissue-equivalent gel, surpasses the Fricke-gel method by providing accurate, quantitative dose distribution data that do not deteriorate with time (6, 9). The improved BANG2 formulation contains 3% N,N'-methylene-bisacrylamide, 3% acrylic acid, 1% sodium hydroxide, 5% gelatin, and 88% water, where all percentages are by weight. The gel was poured into volumetric flasks, of dimensions comparable to a human head. The gels were irradiated with complex beam arrangements, similar to those used for conformal radiation therapy. Images of the gels were acquired using a Siemens 1.5T imager and a Hahn spin-echo pulse sequence (90 degrees-tau-180 degrees-tau-acquire, for different values of tau). The images were transferred via network to a Macintosh computer for which a data analysis and display program was written. The program calculates R2 maps on the basis of multiple TE images, using a monoexponential nonlinear least-squares fit based on the Levenberg-Marquardt algorithm. The program also creates a dose-to-R2 calibration function by fitting a polynomial to a set of dose and R2 data points, obtained from gels irradiated in test tubes to known doses. This function can then be applied to any other R2 map, so that a dose map can be computed and displayed. RESULTS: Through exposure to known doses of radiation, the gel has been shown to respond linearly with dose in the range of 0 to 10 Gy, and its response is independent of the beam energy or modality. Dose distributions have been imaged in orthogonal planes, and can be displayed in a convenient form for comparison with isodose plans. The response of the gel is stable; the gel can be irradiated at any time after its manufacture, and imaging can be conducted any time following a brief interval after irradiation. CONCLUSION: The polymer gel dosimeter has been shown to be a valuable device for displaying three-dimensional dose distributions. The imaged dose distribution can be compared easily with calculated dose distributions, to validate a treatment planning system. In the future, gels may be prepared in anthropomorphic phantoms, to confirm unique patient dose distributions.     

Nonenzymatic glycation of type IV collagen and matrix metalloproteinase susceptibility

The glomerular basement membrane (GBM) is damaged in diabetes through complex mechanisms that are not fully understood. Prominent among them is nonenzymatic protein glycation leading to the formation of so-called advanced glycation end products (AGEs). We examined the effects of in vitro glycation of intact collagen type IV in bovine lens capsule (LBM) and kidney glomerular (GBM) basement membranes on their susceptibility to matrix metalloproteinases, using stromelysin 1 (MMP-3) and gelatinase B (MMP-9). Sites of cleavage of unmodified LBM collagen were located in the triple helical region. In vitro glycation by glucose severely inhibited the release of soluble collagen cleavage peptides by MMP-3 and MMP-9. The distribution of AGEs within the three domains of collagen IV (7S, triple helical, and noncollagenous NC1) were compared for LBM glycation using AGE fluorescence, pentosidine quantitation, and immunoreactivity towards anti-AGE antibodies that recognize the AGE carboxymethyllysine (CML). Marked asymmetry was observed, with the flexible triple helical domain having the most pentosidine and fluorescent AGEs but the least CML. The in vivo relevance of these findings is supported by preliminary studies of AGE distribution in renal basement membrane (RBM) collagen IV domains from human kidneys of two insulin-dependent diabetics and one normal subject. Pentosidine and fluorescent AGE distributions of diabetic RBM were similar to LBM, but the CML AGE in diabetic kidney was less in the triple helical domain than in NC1. Our results support the hypothesis that nonenzymatic glycation of collagen IV contributes to the thickening of basement membranes, a hallmark of diabetic nephropathy.     

Social phobia. Longitudinal course and long-term outcome of cognitive-behavioral treatment

Interest in social phobia has increased dramatically in the past decade, and our knowledge of this previously understudied disorder has increased as well. We now know that social phobia is a chronic condition and that patients with this disorder are unlikely to experience significant improvement without intervention. It is also a highly prevalent condition affecting as many as 13% of the adult population of the Unites States. Although our understanding of the causes of social phobia remains limited, we do know that it is associated with serious impairment and disability in multiple spheres. Thus, the development of treatments with proven long-term efficacy is an important research goal. In this article, we have reviewed studies that examined either exposure, cognitive restructuring, social skills training, or some combination of these treatments. Here, we summarize the major findings of this review. Exposure has fared well as a treatment for social phobia and, in every case, within-group analyses show that patients have improved after treatment. Methodologic problems in some studies, however, limit the conclusions that can be drawn about the comparative efficacy of exposure, social skills training, and relaxation therapy. Conceptual models of social phobia have stressed the importance of cognitive processes in the development and maintenance of social phobia and much attention has been directed at the long-term efficacy of cognitive-behavioral techniques. It has been hypothesized that exposure plus cognitive restructuring would be a particularly effective combination and several methodologically sound studies have examined this combination. These studies have demonstrated consistently clinically significant within-group changes and superiority to control conditions. Heimberg's CBGT is probably the most widely studied of these treatments. CBGT has been shown to be more effective than an equally credible attention-placebo group. Patients receiving CBGT have maintained their advantage over patients in the attention-placebo group, even 5 years after treatment although flaws in that follow-up study limit generalizability of its results. Generalized and nongeneralized social phobic patients respond equivalently to this highly integrated treatment, and it has been applied effectively by researchers outside the center where it was developed. Despite the successes of combined exposure and cognitive restructuring treatments, it remains unclear as to what the effective component(s) of these and similar treatments are and, therefore, whether or not the integration of therapy components is really necessary. A number of the studies reviewed addressed this question with mixed results. Three studies showed that the combination therapy was superior to either treatment alone. There is also evidence that patients treated with exposure only may show some deterioration during follow-up whereas patients treated with cognitive restructuring and exposure may continue to improve. Still, other studies found no differences in long-term outcome among exposure alone, RET, or the combined treatment. Hope et al found that exposure alone was as effective as an integrated treatment combining exposure and cognitive restructuring (CBGT), and Taylor and colleagues (submitted for publication, 1995) reported that exposure was not enhanced by initial treatment with cognitive restructuring. These results are disappointing in light of all that has been written about the likely benefits of combining cognitive and behavioral therapy in the treatment of social phobia. For example, it has been hypothesized that fear of negative evaluation is a key factor in social phobia and that change in this construct should be the goal of treatment. There is some research that supports this claim and other evidence that suggests that exposure alone is not particularly effective in producing those changes. Butler concluded that the treatment of social phobia is made more difficult whe     

Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.     

A gap detection tactility test for sensory deficits associated with carpal tunnel syndrome

An automated gap detection tactility test was investigated for quantifying sensory deficits associated with carpal tunnel syndrome (CTS). The test, which involved sensing a tiny gap in an otherwise smooth surface by probing with the finger, had functional resemblance to many work-related tactile activities such as detecting scratches or surface defects. Gap detection thresholds were measured using the converging staircase method of limits paradigm. Sixteen normal subjects between 21 and 66 years of age were tested for studying important factors affecting gap detection thresholds. Actively probing with the index finger had a threshold almost an order of magnitude more sensitive (mean = 0.19 mm, SD = 0.11 mm) than passive touch (mean = 1.63 mm, SD = 0.62 mm), which was similar to two-point discrimination. Average thresholds decreased by 24% as contact force increased from 25 to 75 g. Performance in this tactility test quickly stabilized and showed little learning effects over the period of the test, as evidenced by the lack of significant differences between six replicates. The results were highly repeatable. No significant threshold differences were observed between test and retest trials on different days, or between dominant and non-dominant hands. A contact force of 50 g was recommended as optimal for this test since it required moderate force but resulted in a smaller threshold compared with 25 or 75 g. A companion study was conducted using eight normal subjects and ten subjects diagnosed as having CTS. Average gap detection threshold, when finger probing was allowed, was 0.20 mm (SD = 0.11 min) for the normal subjects and increased two-fold to 0.40 mm (SD = 0.19 mm) for the CTS subjects. Average gap detection threshold, when the finger probing was not allowed, was 1.71 mm (SD = 0.53 mm) for the normal subjects and increased by 48% to 2.53 mm (SD = 0.87 mm) for the CTS subjects. The results suggest that people suffering from CTS may experience similar functional deficits in daily living and work activities. The small inter-subject variability makes this test a candidate for having utility as a monitoring test for loss of cutaneous tactile sensitivity.     

Multiple recurrences of cervical intraepithelial neoplasia in women with the human immunodeficiency virus

OBJECTIVE: To evaluate the long-term outcomes after treatment of cervical intraepithelial neoplasia (CIN) in women infected with the human immunodeficiency virus (HIV). METHODS: Human immunodeficiency virus-infected and HIV-negative women treated for CIN by ablation or excision were followed-up prospectively by cytology and colposcopy for periods of up to 73 months. RESULTS: Among 127 HIV-infected CIN patients, 62% developed recurrent CIN by 36 months after treatment, compared with 18% of the 193 HIV-negative CIN patients. Recurrence rates reached 87% in 41 HIV-infected women with CD4 counts less than 200 cells/mm3. Progression to higher-grade neoplasia, including one invasive cancer, occurred by 36 months in 25% of HIV-infected and 2% of HIV-negative women. After adjusting for age, CIN severity, and treatment type, predictors of recurrence included HIV infection (rate ratio 4.4), and, in HIV-positive women, low CD4 count (rate ratio 2.2). In patients treated by excision, predictors of recurrence included HIV infection (rate ratio 2.0) and residual CIN after treatment (rate ratio 2.7). After a second treatment,a second CIN recurrence developed in 14 of 33 HIV-infected and in one of 17 HIV-negative women. After a third treatment, three of six HIV-infected women developed a third recurrence. With long-term follow-up, 45% of treated HIV-infected CIN patients had chronic condylomatous changes in the cervix compared with 5% of HIV-negative women. CONCLUSION: In HIV-infected women, CIN may recur despite multiple treatments, and chronic condylomatous changes are common. Innovative therapies for controlling CIN in HIV-infected women are needed.