BACKGROUND

Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are man-made, persistent organic pollutants widely spread throughout the environment and human populations. They have been found to interfere with fetal growth in some animal models, but whether a similar effect is seen in humans is uncertain.

OBJECTIVE

We investigated the association between plasma levels of PFOS and PFOA in pregnant women and their infants' birth weight and length of gestation.

METHODS

We randomly selected 1,400 women and their infants from the Danish National Birth Cohort among those who completed all four computer-assisted telephone interviews, provided the first blood samples between gestational weeks 4 and 14, and who gave birth to a single live-born child without congenital malformation. PFOS and PFOA were measured by high performance liquid chromatography-tandem mass spectrometer.

RESULTS

PFOS and PFOA levels in maternal plasma were on average 35.3 and 5.6 ng/mL, respectively. Only PFOA levels were inversely associated with birth weight (adjusted beta = -10.63 g; 95% confidence interval, -20.79 to -0.47 g). Neither maternal PFOS nor PFOA levels were consistently associated with the risk for preterm birth or low birth weight. We observed no adverse effects for maternal PFOS or PFOA levels on small for gestational age.

CONCLUSIONS

Our nationwide cohort data suggest an inverse association between maternal plasma PFOA levels and birth weight. Because of widespread exposure to these chemicals, our findings may be of potential public health concern.

Periodontal disease infection with oral biofilm microorganisms initiates host immune response and signs of periodontitis, including bone resorption. This review delineates some mechanisms underlying the host immune response in periodontal infection and alveolar bone resorption. Activated T lymphocytes have been historically implicated in experimental periodontal bone resorption. An experimental rat adoptive transfer/gingival challenge periodontal disease model has been demonstrated to require antigen-specific T lymphocytes and gingival instillation of antigen and LPS for bone resorption. Interference with costimulatory interactions between T cells and antigen-presenting cells abrogated bone resorption, further emphasizing the significance of immune response in periodontal disease. Receptor activator of nuclear factor kappaB ligand (RANKL), a critical osteoclast differentiation factor, is expressed on T lymphocytes in human periodontal disease as determined by immunohistochemical and confocal microscopic analyses. Interference with RANKL by systemic administration of osteoprotegerin (OPG), the decoy receptor for (and inhibitor of) RANKL, resulted in abrogation of periodontal bone resorption in the rat model. This finding indicated that T cell-mediated bone resorption is RANKL-dependent. In additional experiments, treatment of T cell-transferred rats with kaliotoxin (a scorpion venom potassium channel inhibitor) resulted in decreases in T-cell RANKL expression, diminished induction of RANKL-dependent osteoclastogenesis, and abrogation of bone resorption, implicating an important role of immune response/RANKL expression in osteoclastogenesis/bone resorption. In other experiments, adoptive transfer of antigen-specific, RANKL-expressing B cells, and infection with the antigen-bearing Actinobaccillus actinomycetemcomitans gave rise to periodontal bone resorption, indicating that B cells also have the capacity to mediate bone resorption, probably via RANKL expression. In humans, prominent T lymphocytes have been identified in periodontal disease, and diseased tissues showed elevated RANKL mRNA expression, as well as decreased OPG mRNA expression. Mononuclear cells from periodontal lesions involving T cells and B cells of patients induced osteoclastogenesis in vitro. In summary, a biofilm interface initiates immune cell infiltration, stimulating osteoclastogenesis/bone resorption in periodontal disease. This resorption can be ameliorated by inhibition of RANKL activity or by diminishing immune cell stimulation. These two procedures, if localized, have the potential to lead to the prevention or therapeutic management of periodontal disease and therefore require further study.

BACKGROUND

The goal of the gene normalization task is to link genes or gene products mentioned in the literature to biological databases. This is a key step in an accurate search of the biological literature. It is a challenging task, even for the human expert; genes are often described rather than referred to by gene symbol and, confusingly, one gene name may refer to different genes (often from different organisms). For BioCreative II, the task was to list the Entrez Gene identifiers for human genes or gene products mentioned in PubMed/MEDLINE abstracts. We selected abstracts associated with articles previously curated for human genes. We provided 281 expert-annotated abstracts containing 684 gene identifiers for training, and a blind test set of 262 documents containing 785 identifiers, with a gold standard created by expert annotators. Inter-annotator agreement was measured at over 90%.

RESULTS

Twenty groups submitted one to three runs each, for a total of 54 runs. Three systems achieved F-measures (balanced precision and recall) between 0.80 and 0.81. Combining the system outputs using simple voting schemes and classifiers obtained improved results; the best composite system achieved an F-measure of 0.92 with 10-fold cross-validation. A 'maximum recall' system based on the pooled responses of all participants gave a recall of 0.97 (with precision 0.23), identifying 763 out of 785 identifiers.

CONCLUSIONS

Major advances for the BioCreative II gene normalization task include broader participation (20 versus 8 teams) and a pooled system performance comparable to human experts, at over 90% agreement. These results show promise as tools to link the literature with biological databases.

BACKGROUND

We have recently introduced liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 25-hydroxyvitamin D(2) (25OHD(2)) and 25OHD(3) testing. During subsequent clinical use, we identified significantly elevated results in some infants. We hypothesized this might represent assay interference caused by C-3 epimers of 25OHD(2) or 25OHD(3).

OBJECTIVE

Our aims were to 1) determine the prevalence of C-3 epimers of 25OHD(2) or 25OHD(3) in human serum, and 2) identify the patient populations that might be affected.

METHODS

We modified our LC-MS/MS method to allow detection of C-3 epimers. We retested specimens from four patient groups with the new method and an extracted RIA: 1) children less than 1 yr old, 2) children 1-18 yr old, 3) adults aged 20-87 yr with liver disease, and 4) adults aged 19-91 yr without liver disease.

RESULTS

In 172 children from group 1 with detectable 25OHD(2) or 25OHD(3), we identified C-3 epimers in 39 (22.7%). The epimers contributed 8.7-61.1% of the total 25-OHD. There was an inverse relationship between patient age and epimer percentage (r = 0.48; P < 0.002). The RIA gave accurate 25-OHD results that correlated with the modified LC-MS/MS method. No C-3 epimers were detected in any of the other groups.

CONCLUSIONS

Significant concentrations of C-3 epimers of 25OHD(2) or 25OHD(3) are commonly found in infants. This can lead to overestimation of 25-OHD levels. Measurements in children less than 1 yr should therefore be performed with an assay that allows accurate detection of 25-OHD in the presence of its C-3 epimers.

The goal of the study was to establish if there was a relationship between molecular patterns and virus evolution. Therefore the complete genome sequence of two distinct apathogenic Newcastle disease virus (NDV) strains was determined and a third genome size category, containing 15,198 nucleotides, was recognized. Phylogenetic analysis revealed that two major separations resulting in three genome size categories occurred during the history of NDV. An ancient division in the primordial reservoir (wild waterbird species) led to two basal sister clades, class I and II, with genome sizes 15,198 (due to a 12 nucleotide insert in the phosphoprotein gene) and 15,186 nucleotides, respectively. Ancestors of only class II viruses colonized chicken populations and subsequently converted to virulent forms. These took place more than once and resulted in an early lineage [including genotypes I-IV and H33(W)] with genome size of 15,186 nucleotides. A second division occurred in the 20th century in the secondary (chicken) host. This gave rise to the branching-off of a clade (including recent genotypes V-VIII consisting of only pathogenic viruses) with the concomitant insertion of six nucleotides into the 5' non-coding region of the nucleoprotein gene thereby increasing the genome size to 15,192 nucleotides.

Five algorithms proposed in the literature for library search identification of unknown compounds from their low resolution mass spectra were optimized and tested by matching test spectra against reference spectra in the NIST-EPA-NIH Mass Spectral Database. The algorithms were probability-based matching (PBM), dot-product, Hertz et al. similarity index, Euclidean distance, and absolute value distance. The test set consisted of 12,592 alternate spectra of about 8000 compounds represented in the database. Most algorithms were optimized by varying their mass weighting and intensity scaling factors. Rank in the list of candidatc compounds was used as the criterion for accuracy. The best performing algorithm (75% accuracy for rank 1) was the dot-product function that measures the cosine of the angle between spectra represented as vectors. Other methods in order of performance were the Euclidean distance (72%), absolute value distance (68%) PBM (65%), and Hertz et al. (64%). Intensity scaling and mass weighting were important in the optimized algorithms with the square root of the intensity scale nearly optimal and the square or cube the best mass weighting power. Several more complex schemes also were tested, but had little effect on the results. A modest improvement in the performance of the dot-product algorithm was made by adding a term that gave additional weight to relative peak intensities for spectra with many peaks in common.

The perception of self-motion direction, or heading, relies on integration of multiple sensory cues, especially from the visual and vestibular systems. However, the reliability of sensory information can vary rapidly and unpredictably, and it remains unclear how the brain integrates multiple sensory signals given this dynamic uncertainty. Human psychophysical studies have shown that observers combine cues by weighting them in proportion to their reliability, consistent with statistically optimal integration schemes derived from Bayesian probability theory. Remarkably, because cue reliability is varied randomly across trials, the perceptual weight assigned to each cue must change from trial to trial. Dynamic cue reweighting has not been examined for combinations of visual and vestibular cues, nor has the Bayesian cue integration approach been applied to laboratory animals, an important step toward understanding the neural basis of cue integration. To address these issues, we tested human and monkey subjects in a heading discrimination task involving visual (optic flow) and vestibular (translational motion) cues. The cues were placed in conflict on a subset of trials, and their relative reliability was varied to assess the weights that subjects gave to each cue in their heading judgments. We found that monkeys can rapidly reweight visual and vestibular cues according to their reliability, the first such demonstration in a nonhuman species. However, some monkeys and humans tended to over-weight vestibular cues, inconsistent with simple predictions of a Bayesian model. Nonetheless, our findings establish a robust model system for studying the neural mechanisms of dynamic cue reweighting in multisensory perception.

BACKGROUND

Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques.

METHODS

Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe - Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals).

RESULTS

The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores.

CONCLUSIONS

The results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real-time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS-PCR, SSOP-ELISA, and HOLA are all reasonable alternatives with the SSOP-ELISA approach having the highest throughput.

OBJECTIVE

To assess outside a clinical trial the psychological outcome of different treatment policies in women with early breast cancer who underwent either mastectomy or breast conservation surgery depending on the surgeon's opinion or the patient's choice. To determine whether the extent of psychiatric morbidity reported in women who underwent breast conservation surgery was associated with their participation in a randomised clinical trial.

METHODS

Prospective, multicentre study capitalising on individual and motivational differences among patients and the different management policies among surgeons for treating patients with early breast cancer.

METHODS

12 District general hospitals, three London teaching hospitals, and four private hospitals.

METHODS

269 Women under 75 with a probable diagnosis of stage I or II breast cancer who were referred to 22 different surgeons.

METHODS

Surgery and radiotherapy or adjuvant chemotherapy, or both, depending on the individual surgeon's stated preferences for managing early breast cancer.

METHODS

Anxiety and depression as assessed by standard methods two weeks, three months, and 12 months after surgery.

RESULTS

Of the 269 women, 31 were treated by surgeons who favoured mastectomy, 120 by surgeons who favoured breast conservation, and 118 by surgeons who offered a choice of treatment. Sixty two of the women treated by surgeons who offered a choice were eligible to choose their surgery, and 43 of these chose breast conserving surgery. The incidences of anxiety, depression, and sexual dysfunction were high in all treatment groups. There were no significant differences in the incidences of anxiety and depression between women who underwent mastectomy and those who underwent lumpectomy. A significant effect of surgeon type on the incidence of depression was observed, with patients treated by surgeons who offered a choice showing less depression than those treated by other surgeons (p = 0.06). There was no significant difference in psychiatric morbidity between women treated by surgeons who offered a choice who were eligible to choose their treatment and those in the same group who were not able to choose. Most of the women (159/244) gave fear of cancer as their primary fear rather than fear of losing a breast. The overall incidences of psychiatric morbidity in women who underwent mastectomy and those who underwent lumpectomy were similar to those found in the Cancer Research Campaign breast conservation study. At 12 months 28% of women who underwent mastectomy in the present study were anxious compared with 26% in the earlier study, and 27% of women in the present study who underwent lumpectomy were anxious compared with 31% in the earlier study. In both the present and earlier study 21% of women who underwent mastectomy were depressed, and 19% of women who underwent lumpectomy in the present study were depressed compared with 27% in the earlier study.)

CONCLUSIONS

There is still no evidence that women with early breast cancer who undergo breast conservation surgery have less psychiatric morbidity after treatment than those who undergo mastectomy. Women who surrender autonomy for decision making by agreeing to participate in randomised clinical trials do not experience any different psychological, sexual, or social problems from those women who are treated for breast cancer outside a clinical trial.

The process of cell death caused by influenza virus infection in cultured MDCK and HeLa cells was analysed. This infection gave rise to nuclear fragmentation and chromatin condensation accompanied by chromosomal DNA fragmentation into oligonucleosomes. Chromosomal DNA fragmentation progressed concomitantly with cell lysis of MDCK cells and HeLa cells, producing high and low yields of virus particles, respectively, indicating that the extent of cell lysis was not proportional to the virus production. The endonuclease inhibitor zinc blocked DNA fragmentation in MDCK cells. Cycloheximide inhibited DNA fragmentation as well as cell lysis. Inhibition occurred when the drug was added to the medium within 2 h after infection but not efficiently at 4 h or later. Infection induced the Fas Ag gene, which encodes a possible apoptosis-mediating molecule, in the early infectious stage followed by the expression of Fas Ag on the cell surface. These results suggested that influenza virus infection causes apoptotic death of cultured cells, and their fate might be determined at an early stage of the infection by induction of an apoptotic gene.

1. Two mutants of Escherichia coli K 12 were isolated which, although able to grow on glucose, are unable to grow with succinate or d-lactate as the sole source of carbon. 2. Genetic mapping of these mutants showed that they both contain a mutation in a gene (designated uncA) mapping at about minute 73.5 on the E. coli chromosome. 3. The uncA(-) alleles were transferred by bacteriophage-mediated transduction into another strain of E. coli and the transductants compared with the parent strain to determine the nature of the biochemical lesion in the mutants. 4. The mutants gave low aerobic growth yields when grown on limiting concentrations of glucose, but oxidase activities in membranes from both the mutants and the normal strain were similar. 5. Measurement of P/O ratios with d-lactate as substrate indicated that a mutation in the uncA gene causes uncoupling of phosphorylation associated with electron transport. 6. Determination of the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activities in the mutant and normal strains indicated that the uncA gene is probably the structural gene for Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 7. Mg(2+),Ca(2+)-stimulated adenosine triphosphatase therefore appears to be essential for oxidative phosphorylation in E. coli.

Growth of mature B cells and their precursors from mouse bone marrow was maintained in culture for greater than 1 year. Feeder layers of adherent bone marrow cells, established in medium containing fetal calf serum and no exogenous steroids, were used to provide an in vitro environment that supported continuous growth and development of these cells. In such bone marrow cultures, the number of cells bearing membrane immunoglobulin increased gradually for 4 wk and then decreased. Between 10 and 14 wk, some of the cultures gave rise to continuously dividing B-cell populations that contained pre-B cells (producing mu heavy chains only and sensitive to transformation by Abelson murine leukemia virus) as well as mature B cells (synthesizing both light and heavy chains of IgM). Immunoglobulin molecules synthesized by cells in these populations were heterogeneous in two-dimensional gel analysis. This suggests that mature B cells arose via maturation of pre-B cells in the cultures that involved rearrangement and expression of different variable region gene segments.

The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-beta-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.

Cryostat sections from skin biopsies from 24-h allergen-induced late-phase cutaneous reactions (LPR) in 14 human atopic subjects were hybridized with 35S-labeled RNA probes for a number of cytokines. mRNA was detected for interleukin 3 (IL-3) (8/14), IL-4 (10/14), IL-5 (11/14), and granulocyte/macrophage colony-stimulating factor (GM-CSF) (13/14). Only 5 of 14 gave hybridization signals for IL-2, and 0 of 14 for interferon gamma. Biopsies from diluent controls gave only occasional weak signals. These results suggest that cells infiltrating the site of the 24-h LPR transcribe mRNA for the IL-3, IL-4, IL-5, and GM-CSF gene cluster and support the hypothesis that atopy is associated with preferential activation of cells having a similar cytokine profile to the murine T helper type 2 subset.

We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co-localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co-localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co-expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co-localization and interactions of proteins in a variety of experimental dicotyledonous hosts.

Here we describe the conditions which allow cultured human tracheal epithelial cells to retain the ion transport properties and ultrastructure of the original tissue. The order of potency of growth supports and media additives in elevating baseline short-circuit current (Isc) and responses to mediators were vitrogen gel (VIT) greater than extracellular matrix from bovine corneal endothelial cells (ECM) greater than human placental collagen (HPC), and 2% Ultroser G serum substitute (USG) greater than 5% fetal calf serum (FCS) greater than defined growth factors (GF). For all combinations of medium and growth supports, an air interface (AIR) gave better electrical properties than immersion feeding (IMM). As opposed to our earlier conditions (HPC/FCS/IMM), the best new combination (VIT/USG/AIR) produced higher baseline Isc (58.0 +/- 10.6 vs. 5.1 +/- 1.0 microA/cm2) and increased Isc responses to isoproterenol (6.1 +/- 1.5 vs. 0.8 +/- 0.3 microA/cm2) and bradykinin (9.6 +/- 2.0 vs. 1.0 +/- 0.2 microA/cm2), while retaining high transepithelial resistance (227 +/- 5 omega.cm2). VIT/USG/AIR led to the appearance of cilia, an increase in the depth of the cell sheets (50 vs. 10 microns), longer and more frequent apical microvilli, and increased interdigitations of the basolateral membrane. Protein and DNA content were also significantly increased. Secretory granules were present which stained with antibody to goblet cells, but not to serous or mucous gland cells. CF cells grown in VIT/USG/AIR showed high baseline Isc (69 +/- 18 microA/cm2) and a proportionately larger inhibition of Isc by amiloride (70 +/- 10 vs. 34 +/- 3%). Isc did not respond to isoproterenol, and the response to bradykinin was 22% normal.

CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. We demonstrate here that when antibody against CD40 is used to treat mice with syngeneic lymphoma, a rapid cytotoxic T-cell response independent of T-helper cells occurs, with tenfold expansion of CD8+ T cells over a period of 5 days. This response eradicates the lymphoma and provides protection against tumor rechallenge without further antibody treatment. Thus, it seems that by treating mice with monoclonal antibody against CD40, we are immunizing against syngeneic tumors. The phenomenon proved reproducible with two antibodies against CD40 (3/23 and FGK-45) in three CD40+ lymphomas (A20, A31 and BCL1) and gave partial protection in one of two CD40- lymphomas (EL4 and Ten1). Although the nature of the target antigens on these lymphomas is unknown, CD8+ T cells recovered from responding mice showed powerful cytotoxic activity against the target B-cell lymphoma in vitro.

BACKGROUND

The proportion of recurrent tuberculosis cases attributable to relapse or reinfection and the risk factors associated with these different mechanisms are poorly understood. We followed up a cohort of 326 South African mineworkers, who had successfully completed treatment for pulmonary tuberculosis in 1995, to determine the rate and mechanisms of recurrence.

METHODS

Patients were examined 3 and 6 months after cure, and then were monitored by the routine tuberculosis surveillance system until December, 1998. IS6110 DNA fingerprints from initial and subsequent episodes of tuberculosis were compared to determine whether recurrence was due to relapse or reinfection All patients gave consent for HIV-1 testing.

RESULTS

During follow-up (median 25.1 months, IQR 13.2-33.4), 65 patients (20%) had a recurrent episode of tuberculosis, a recurrence rate of 10.3 episodes per 100 person-years at risk (PYAR)-16.0 per 100 pyar in HIV-1-positive patients and 6.4 per 100 pyar in HIV-1-negative patients. Paired DNA fingerprints were available in 39 of 65 recurrences: 25 pairs were identical (relapse) and 14 were different (reinfection). 93% (13/14) of recurrences within the first 6 months were attributable to relapse compared with 48% (12/25) of later recurrences. HIV-1 infection was a risk factor for recurrence (hazard ratio 2.4, 95% CI 1.5-4.0), due to its strong association with disease caused by reinfection (18.7 2.4-143), but not relapse (0.58; 0.24-1.4). Residual cavitation and increasing years of employment at the mine were risk factors for relapse.

CONCLUSIONS

In a setting with a high risk of tuberculous infection, HIV-1 increases the risk of recurrent tuberculosis because of an increased risk of reinfection. Interventions to prevent recurrent disease, such as lifelong chemoprophylaxis in HIV-1-positive tuberculosis patients, should be further assessed.

We have used nuclear transplantation to test whether the reprogramming activity of oocytes can reestablish developmental pluripotency of malignant cancer cells. We show here that the nuclei of leukemia, lymphoma, and breast cancer cells could support normal preimplantation development to the blastocyst stage but failed to produce embryonic stem (ES) cells. However, a blastocyst cloned from a RAS-inducible melanoma nucleus gave rise to ES cells with the potential to differentiate into multiple cell types in vivo including melanocytes, lymphocytes, and fibroblasts. Chimeras produced from these ES cells developed cancer with higher penetrance, shorter latency, and an expanded tumor spectrum when compared with the donor mouse model. These results demonstrate that the secondary changes of a melanoma nucleus are compatible with a broad developmental potential but predispose mice to melanomas and other malignant tumors on reactivation of RAS. Our findings serve as a paradigm for studying the tumorigenic effect of a given cancer genome in the context of a whole animal.

A highly reproducible, commercial and nonlinear, wide-range immobilized pH gradient (IPG) was used to generate two-dimensional (2-D) gel maps of [35S]methionine-labeled proteins from noncultured, unfractionated normal human epidermal keratinocytes. Forty one proteins, common to most human cell types and recorded in the human keratinocyte 2-D gel protein database were identified in the 2-D gel maps and their isoelectric points (pI) were determined using narrow-range IPGs. The latter established a pH scale that allowed comparisons between 2-D gel maps generated either with other IPGs in the first dimension or with different human protein samples. Of the 41 proteins identified, a subset of 18 was defined as suitable to evaluate the correlation between calculated and experimental pI values for polypeptides with known composition. The variance calculated for the discrepancies between calculated and experimental pI values for these proteins was 0.001 pH units. Comparison of the values by the t-test for dependent samples (paired test) gave a p-level of 0.49, indicating that there is no significant difference between the calculated and experimental pI values. The precision of the calculated values depended on the buffer capacity of the proteins, and on average, it improved with increased buffer capacity. As shown here, the widely available information on protein sequences cannot, a priori, be assumed to be sufficient for calculating pI values because post-translational modifications, in particular N-terminal blockage, pose a major problem. Of the 36 proteins analyzed in this study, 18-20 were found to be N-terminally blocked and of these only 6 were indicated as such in databases. The probability of N-terminal blockage depended on the nature of the N-terminal group. Twenty six of the proteins had either M, S or A as N-terminal amino acids and of these 17-19 were blocked. Only 1 in 10 proteins containing other N-terminal groups were blocked.

A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

The aims of this paper were to establish the origin of cells producing IgA antibody to cholera toxoid in the lamina propria of the small intestine and to define the role of antigen in their distribution. The use of Thirty-Vella loops made it possible to restrict antigenic challenge to a defined segment of the intestine in rats which had been primed i.p. with toxoid in Freund's complete adjuvant. The anti-toxin-containing cells (ACC) which appeared in the draining thoracic duct lymph after challenge of a loop were almost all of IgA specificity and their numbers were proportional to the length of intestine exposed to antigen. The abolition of this cellular response which occurred when Peyer's patches (PP) were removed from a loop before challenge and the failure of mesenteric lymphadenectomy significantly to affect the response indicated that ACC originated exclusively from PP. Cell transfer studies showed that although nonrecirculating large lymphocytes gave rise to ACC in the lamina propria, some of the recirculating small lymphocytes also developed subsequently into ACC. Counts of ACC in the lamina propria of challenged loops were consistently greater than in nonchallenged loops although some ACC were always present in the latter. However, a time-course study on the appearance of ACC in the lamina propria of cannulated rats given a single dose of thoracic duct lymphocytes from immunized donors demonstrated that ACC continued to accumulate and persist in challenged loops but only appeared transiently in nonchallenged loops. These transients did not migrate from the lamina propria back into the lymph and they presumably died in situ. The increase in the number of ACC in loops which had been challenged with antigen was probably due both to cell division in the lamina propria and to the development of new ACC from recirculating lymphocytes which had been recruited into the loop. Thus, the cells which give rise to intestinal ACC can migrate into the lamina propria independently of antigen, but antigen has a profound effect on the location, magnitude, and persistence of the response.

1. In the monkey superior colliculus (SC), the activity of most saccade-related neurons studied so far consists of a burst of activity in a population of cells at one place on the SC movement map. In contrast, recent experiments in the cat have described saccade-related activity as a slow increase in discharge before saccades followed by a hill of activity moving across the SC map. In order to explore this striking difference in the distribution of activity across the SC, we recorded from all saccade-related neurons that we encountered in microelectrode penetrations through the monkey SC and placed them in categories according to their activity during the generation of saccades. 2. When we considered the activity preceding the onset of the saccade, we could divide the cells into two categories. Cells with burst activity had a high-frequency discharge just before saccade onset but little activity between the signal to make a saccade and saccade onset. About two thirds of the saccade-related cells had only a burst of activity. Cells with a buildup of activity began to discharge at a low frequency after the signal to make a saccade and the discharge continued until generation of the saccade. About one third of the saccade-related cells studied had a buildup of activity, and about three fourths of these cells also gave a burst of activity with the saccade in addition to the slow buildup of activity. 3. The buildup of activity seemed to be more closely related to preparation to make a saccade than to the generation of the saccade. The buildup developed even in cases when no saccade occurred. 4. The falling phase of the discharge of these saccade-related cells stopped with the end of the saccade (a clipped discharge), shortly after the end of the saccade (partially clipped), or long after the end of the saccade (unclipped). 5. Some cells had closed movement fields in which saccades that were substantially smaller or larger than the optimal amplitude were not associated with increased activity. Other cells tended to have open-ended movement fields without any peripheral border; they were active for all saccades of optimal direction whose amplitudes were equal to or greater than a given amplitude.(ABSTRACT TRUNCATED AT 400 WORDS)

OBJECTIVE

To prospectively compare the sensitivity and specificity of magnetic resonance (MR) elastography with those of the routinely available aspartate aminotransferase-to-platelet ratio index (APRI) test for staging hepatic fibrosis in patients who have undergone liver biopsy for suspicion of chronic liver disease, with histopathologic examination as the reference standard.

METHODS

The study was approved by the ethics committee. All patients gave written informed consent. Eighty-eight patients (37 men, 51 women; mean age, 54.0 years +/- 13.1 [standard deviation]) who underwent liver biopsy for suspicion of chronic liver disease underwent MR elastography and APRI testing within 2 days after liver biopsy. At histopathologic examination, the fibrosis stage was assessed according to METAVIR scores (fibrosis scores F0 [no fibrosis] to F4 [cirrhosis]). MR elastography was performed by transmitting mechanical waves within the liver and measuring the small cyclic displacement of the liver spins with a phase-contrast spin-echo sequence. The performances of MR elastography and APRI testing were assessed, and the optimal cutoff values for fibrosis stage were determined with receiver operating characteristic (ROC) curve analysis.

RESULTS

At MR elastography, areas under the ROC curves (A(z)) for elasticity and viscosity, respectively, were 0.999 and 0.863 at fibrosis scores greater than or equal to F2, 0.997 and 0.962 at scores greater than or equal to F3, and 1.000 and 0.986 at score F4. A(z) values for elasticity at MR were significantly larger than those for the APRI (0.854 at scores>> or = F2, P < .001; 0.886 at scores>> or = F3, P = .003; and 0.851 at score F4, P = .004). Optimal cutoff values of elasticity were 2.5 kPa for fibrosis scores greater than or equal to F2, 3.1 kPa for scores greater than or equal to F3, and 4.3 kPa for score F4.

CONCLUSIONS

Large A(z) values for elasticity (>0.990 for scores>> or = F2,>> or = F3, and F4) show that MR elastography was accurate in liver fibrosis staging and superior to biochemical testing with APRIs.