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Biomedical subjects

M Reed

Publications and source records attributed to M Reed.

At least 19 recordsLinked to original sources
PubMed2000

Severe acro-renal-uterine-mandibular syndrome.

Although limb and renal defects occur together in a variety of patterns of multiple malformations, familial cases of acro-renal disorders are rare. In 1980, Halal et al. ¿Am J Med Genet 5:277-284 described two sisters with unusual limb deficiencies, renal anomalies, and mandibular hypoplasia and termed this condition acro-renal-mandibular syndrome. A girl reported earlier by Fitch and Lachance ¿1972; Can Med Assoc J 107:653-656 had similarly limb and renal findings, but an apparently normal jaw. We document three sibs with unusual limb deficiencies, renal agenesis, uterine anomalies in the two females, and orofacial defects, who clearly have a similar but more severe type of acrorenal disorder, apparently inherited as an autosomal recessive condition. The sibs with limb deficiencies and renal agenesis reported by Hennekam et al. ¿1994; Am J Med Genet 53:102-107 appear to be additional cases of this very rare disorder, the pathogenesis of which may be related to abnormal epithelial-mesenchymal interactions.

Abnormalities, Multiple↗
PubMed2000

C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding.

We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.

Alkylating Agents↗
PubMed2000

Malignancy in HIV/AIDs: a single hospital experience.

BACKGROUND AND OBJECTIVES: Our hospital serves an area with a significant number of patients seropositive for the human immunodeficiency virus (HIV). Intravenous drug abuse and heterosexual exposure are by far the predominant risk factors for HIV and acquired immunodeficiency syndrome (AIDS). Seven percent of these patients develop malignancies. Our aim was to study the types of tumor, their distribution, and to evaluate the patients' outcome. METHODS: Of 3,578 patients with HIV infection or AIDS treated between 1993 and 1998, 245 had 1 or more malignancies. Information was collected on age, sex, race, predisposing risk factors for AIDS, malignancies, symptoms at presentation, the time of the onset of AIDS, CD4 cell counts, pathology findings, and mortality. RESULTS: Although aspects of our patients resembled those of previously studied groups of patients with AIDS, there also were ways in which our patients differed from those other groups. Of our patients, 21. 6% had non-AIDS-defining (NAD) invasive malignancies. This was considerably higher than the rate in most studies. Twenty-seven patients with such malignancies died during the study. Forty-two other patients had pre-invasive cancers. Among patients having AIDS-defining (AD) malignancies, 55.9% died, a fact that was related to patients' low CD4 cell counts and late presentation. Our 97 patients with Kaposi sarcoma included 22 women, a relatively high number that may be related to the fact that most of our patients were intravenous drug abusers or had become infected by heterosexual transmission of HIV. CONCLUSIONS: AIDS is associated with a high risk of malignancy and an unusual spectrum of tumors. Patients with invasive tumors have advanced disease at the time of initial presentation. Those with AD tumors have a worse prognosis than patients with NAD tumors. The impact of highly active antiretroviral therapy on both AD and NAD tumors needs to be further evaluated.

AIDS-Related Complex↗
PubMed2000

Oligonucleotide scanning of native mRNAs in extracts predicts intracellular ribozyme efficiency: ribozyme-mediated reduction of the murine DNA methyltransferase.

Modulation of gene expression by catalytic RNA requires accessible ribozyme cleavage sites in the target mRNA, and accessibility is determined by the secondary and tertiary structure of the target RNA, as affected by its interactions with cellular proteins. As we previously reported, an oligonucleotide-scanning approach using antisense oligonucleotides can be used to determine RNA accessibility in cell extracts. To test whether this method can be used to improve selection of ribozyme target sites, we designed ribozymes corresponding to the sites identified by oligonucleotide scanning and have evaluated their catalytic activities, first in cell extracts and then in transduced cell lines. As a target we used the mRNA of murine DNA (cytosine-5)-methyltransferase 1 (MTase). For intracellular studies, the ribozyme genes were inserted downstream of a Pol III tRNAVAL promoter, which in turn was cloned in the U3 region of a retroviral vector. We find that the efficiency of the ribozymes both in cell extracts and in vivo corresponds with the relative effectiveness predicted by the oligonucleotide-scanning assay. The best ribozyme causes a 70-80% reduction in the MTase mRNA levels in NIH 3T3 cells that are stably transduced with the retroviral constructs. This reduction in mRNA levels is accompanied by a small decrease in the methylation of repetitive intercisternal A particle DNA elements. Ribozyme expression also increased several-fold the reactivation frequency of a methylation-silenced green fluorescent protein (GFP) transgene. Both the reduction in methylation and reactivation of GFP were roughly equivalent to the effects obtained by treating NIH 3T3 cells with 2.5 microM 5-azacytidine, which gives an effect of about 10% of maximum. These results confirm the validity of the cell extract approach for ribozyme site selection and provide a potentially useful ribozyme for future study of DNA methyltransferase function.

3T3 Cells↗
PubMed2000

Molecular breeding of viruses.

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.

3T3 Cells↗
PubMed2000

Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis.

UNLABELLED: Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis. BACKGROUND: SPARC has been implicated as a counteradhesive and antiproliferative protein associated with deposits of extracellular matrix in renal disease. METHOD: We have examined the effect of recombinant SPARC containing a C-terminal His tag (rSPARC) in an acute model of mesangial cell injury that is induced in the rat by an antibody against the Thy1 antigen on the mesangial cell membrane. The recombinant protein was administered 24 hours after the induction of nephritis and was infused through day 4. RESULTS: rSPARC was localized to the renal glomeruli of rats treated with anti-Thy1 antibody. Type I collagen and fibronectin, as well as transforming growth factor-beta1 (TGF-beta1), were increased at day 5 in rats treated with rSPARC (N = 4, P < 0.05 vs. delivery buffer), but only minimal effects were seen on mesangial cell and endothelial cell proliferation. In primary cultures of rat mesangial cells, infusion of rSPARC was associated with increases in TGF-beta1 mRNA and in total, secreted TGF-beta1 protein. CONCLUSIONS: rSPARC stimulates expression of TGF-beta1 both in vitro and in vivo. Given the closely regulated expression of SPARC, TGF-beta1, and type I collagen in several animal models of glomerulonephritis, we propose that SPARC could be one of the major mediators of the induction of TGF-beta1 in renal disease.

Animals↗
PubMed2000

The role of vital tissue staining in the marginal control of oral squamous cell carcinoma.

Since vital staining has been advocated as a sensitive method of displaying epithelial atypia, 14 oral squamous cell carcinomas were stained immediately preoperatively with toluidine blue and then resected 1 cm outside clinically abnormal or positively staining tissue. The integrity of the entire tissue margin was histologically examined in each case. Whilst vital staining delineated all 14 invasive carcinomas at the centre of each resected specimen, 10 foci of carcinoma-in-situ or severe dysplasia were identified not to have stained at the resection margins. Toluidine blue may, therefore, be an adjunct in identifying invasive tumour at mucosal resection margins. However, it would appear to be of no benefit in delineating positive resection margins due to carcinoma-in-situ or severe dysplasia, and hence it may be of little value in reducing the incidence of local recurrences.

Aged↗
PubMed1999

Expression of SPARC (secreted protein, acidic and rich in cysteine) in healing intestinal anastomoses and short bowel syndrome in rats.

Due to the proposed functions in soft tissue repair, we evaluated the spatial and temporal distribution of SPARC, a counteradhesive, matricellular glycoprotein in healing intestinal anastomoses and short bowel syndrome (SBS) in rats. Intestinal anastomoses were performed in the jejunum of male Wistar rats. SBS was induced by resecting 70% of the small bowel. In situ hybridization was performed to localize SPARC mRNA and immunohistochemical studies for locating the SPARC protein. The granulation tissue in the anastomotic area exhibited immunoreactivity for SPARC at all time points. The level of expression was maximal at seven to nine days. Endothelial cells of capillaries, smooth muscle cells, fibroblastic cells, and macrophages, as well as mesothelial cells on the serosal surface, were stained. The immunoreactivity was mostly intracellular. SPARC mRNA transcripts were localized to the edges of the anastomotic area at days 1 and 4 and on the newly formed granulation tissue later. The expression of SPARC mRNA was maximal at seven days and decreased thereafter. Both in normal controls and in SBS, SPARC was expressed in endothelial cells of submucosal capillaries and in smooth muscle cells but not in epithelium. Based on the restricted temporal and spatial distribution during the healing of intestinal anastomoses and in SBS we propose that SPARC plays a significant role in intestinal repair and adaptation.

Anastomosis, Surgical↗
PubMed1999

Validation of the Ottawa Ankle Rules in children with ankle injuries.

OBJECTIVES: The Ottawa Ankle Rules (OAR) have been found to be 100% sensitive in adult patients with ankle injuries, and application of the OAR has resulted in a 28% reduction in the number of x-rays ordered. The objectives of this study were to determine the sensitivity and specificity of the OAR in children and to determine the potential change in x-ray utilization. METHODS: Children, aged 2-16 years, presenting to the EDs of two children's hospitals, with an ankle injury in the previous 48 hours, were enrolled. All patients were assessed by either staff physicians or fellows. X-rays were ordered according to standard clinical practice. Prior to reviewing x-rays, the physical examination was recorded on a standardized form. Positive outcomes (clinically significant) were defined as fractures with fragments > or =3 mm. Patients not x-rayed and asymptomatic at five to seven days postinjury were considered to have no significant fracture. RESULTS: Six hundred seventy patients were enrolled. The OAR were 100% sensitive (95% CI = 95% to 100%) for significant ankle fractures, with a specificity of 24% (95% CI = 20% to 28%). The OAR were 100% sensitive (95% CI = 82% to 100%) for the midfoot, with a specificity of 36% (95% CI = 29% to 43%). If the OAR had been followed, there would have been a reduction of ankle x-rays by 16% and foot x-rays by 29% without missing any clinically significant fracture. However, analysis of the two hospitals showed that if the rules had been applied, one would have a reduction in x-rays, while the other center would have an increase. CONCLUSIONS: This study demonstrates the OAR to be sensitive for detecting clinically significant (> or =3 mm) ankle and midfoot fractures in children. The application of these rules may reduce the number of x-rays ordered. A further study is required to determine the effect of using the OAR in clinical practice.

Adolescent↗
PubMed1999

Observer variability in interpretation of abdominal radiographs of infants with suspected necrotizing enterocolitis.

We examined (1) the observer variability (both interobserver and intraobserver) in interpretation of abdominal radiographs of infants with suspected necrotizing enterocolitis (NEC), (2) the interobserver variability for individual radiologic signs used to diagnose NEC, and (3) the influence of experience in determining the extent of observer variability. Our hypotheses were (1) there would be considerable observer variability in interpretation of abdominal radiographs of infants with suspected NEC; (2) the extent of observer variability would differ for individual radiologic signs of NEC; and (3) the extent of observer variability would be determined by the observer's experience. The participants included 12 observers: two pediatric radiologists, four attending neonatologists, three neonatal fellows, and three pediatric residents. The participating observers under similar interpretation conditions, twice independently, interpreted the same 40 pairs of abdominal radiographs from infants with suspected NEC. The interval between the two interpretations was 3 to 6 months. Intraobserver and interobserver variability was assessed by applying the Kappa statistic to the radiologic signs of NEC for the two separate interpretations. The observers were blinded to patient's identity and the clinical course. Each observer recorded the absence, suspicion, or presence of (1) intestinal distention, (2) air fluid levels, (3) bowel wall thickening, (4) pneumatosis intestinalis, (5) portal venous gas, (6) pneumoperitoneum, and (7) NEC. We found low intraobserver and interobserver agreements. There was considerable variation in observer variability for individual radiologic signs. Trained observers performed better than intraining observers. We conclude that the radiologic signs in isolation should not be considered reliable. We recommend studies to formulate more objective criteria for many of the radiographic features of NEC. Standardization and periodic enforcement of these criteria among observers could reduce observer variability. We suggest that, to decrease both false-negative and false-positive interpretation, an experienced observer should always review the radiographs of infants with suspected NEC.

Diagnosis, Differential↗
PubMed1999

5-Aminolevulinic acid photosensitization of dysplastic Barrett's esophagus: a pharmacokinetic study.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) may have a role in the treatment of dysplastic Barrett's esophagus. Before ALA-induced PDT can be used clinically, optimum treatment parameters must be established. In this study of 35 patients, the issues of drug dosage, time interval between drug and light delivery and side effects of oral ALA administration are addressed. Spectrofluorometric analysis of tissue samples demonstrates that oral ALA administration induces porphyrin accumulation in esophageal tissues, with maximum levels at 4-6 h. High-performance liquid chromatography confirms the identity of this porphyrin as PpIX, and fluorescence microscopy analysis demonstrates that it preferentially accumulates in the esophageal mucosa, rather than in the underlying stroma. Side effects of ALA administration included malaise, headache, photosensitivity, alopecia, transient derangement of liver function, nausea and vomiting. Fewer side effects and less hepatic toxicity was seen with 30 mg/kg than 50 mg/kg ALA. In conclusion, oral ALA administration induces preferential PpIX accumulation in the esophageal mucosa, with peak PpIX fluorescence noted at 4 h and minimal systemic toxicity at a dose of 30 mg/kg.

Aminolevulinic Acid↗
PubMed1998

Synergistic transcriptional activation of the MCK promoter by p53: tetramers link separated DNA response elements by DNA looping.

The WAF1, Cyclin G and muscle creatine kinase (MCK) genes, all contain multiple copies of the consensus p53-binding element within their regulatory regions. We examined the role of these elements in transactivation of the muscle creatine kinase (MCK) gene by p53. The MCK promoter possesses distal (-3182 to -3133) and proximal (-177 to -81) p53-binding elements within which residues -3182 to -3151 (distal) and -176 to -149 (proximal) show homology to the consensus p53-binding site. Using promoter deletion studies, we find that both proximal and distal elements are required for high level, synergistic transcriptional activation in vivo. Electron microscopy indicates that p53-p53 interactions link proximal and distal p53-binding elements and cause looping out of intervening DNA, suggesting that this DNA sequence may be dispensable for synergy. This idea was confirmed by progressive deletion of the DNA between p53-binding elements. Synergism persisted with spacing reduced to only 150 bp. Tetramerization-deficient p53 mutants were defective for transcriptional activation but still capable of synergy. Our results provide evidence for a model by which high level transcriptional activation of promoters with multiple p53 response elements is achieved.

Animals↗
PubMed1998

The application of toluidine blue as a diagnostic adjunct in the detection of epithelial dysplasia.

The detection of premalignant lesions of the oral mucosa allows for treatment that may be sufficiently early to prevent their progression to invasive carcinoma. Although a number of techniques for supplementing clinical examination have been described, on the whole they rely on laboratory-based investigations with variable false-negative rates. Toluidine blue vital staining, on the other hand, has been advocated as a simple, inexpensive, and sensitive chairside test. Most of the clinical series investigating its efficacy, however, have relied on clinically abnormal mucosa or tissue that stained. For the purpose of quantifying the true sensitivity of toluidine blue, the present study involved the examination of clinically normal mucosa as well as abnormal tissue and compared it with the presence or absence of staining. With this technique the false-negative rates of 42% and 58% obtained for carcinoma-in-situ and mild-to-moderate dysplasia, respectively, suggest restricting the use of vital staining to selective cases.

Aged↗
PubMed1998

HLA associations of seropositive rheumatoid arthritis in a Cree and Ojibway population.

OBJECTIVE: To determine the HLA associations of seropositive rheumatoid arthritis (RA) in a Cree and Ojibway population; to determine whether specific alleles distinguish juvenile or adult onset. METHODS: HLA-A, B, C, and DRB1 alleles were analyzed in 23 Ojibway and Cree patients with RA seen in a single tertiary care center. Comparisons were made with published results of controls and with results of 18 patients with rheumatoid factor (RF) positive polyarticular juvenile rheumatoid arthritis (JRA) from the same population. RESULTS: Comparisons among patients with RA, patients with RF positive polyarticular JRA, and controls showed increased frequencies of the RA shared epitope in patients with RA and of DRB1*0901 in patients with seropositive polyarticular JRA, while the frequency of DRB1*08 alleles was decreased in patients with RF positive polyarticular JRA. CONCLUSION: In this population, DRB1*0901 may promote while DRB1*08 alleles may protect against a juvenile onset of RA specifically. In contrast, the RA shared epitope may have a greater effect on the risk of adult onset seropositive RA. Due to the small patient numbers, these results require confirmation.

Adult↗
PubMed1997

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Animals↗
PubMed1997

FHIT gene expression in human ovarian, endometrial, and cervical cancer cell lines.

The fragile histidine triad (FHIT) gene, located at 3p14.2, has been shown to be altered in numerous epithelial cancers. Because previous studies have shown a loss of heterozygosity and cytogenetic abnormalities at the 3p region in ovarian, endometrial, and cervical carcinomas, we examined the status of the FHIT gene in 14 ovarian, 8 cervical, and 4 endometrial human cancer cell lines. RNA was isolated and subjected to reverse transcription-PCR to amplify the FHIT gene transcript. Sixty-three % (5 of 8) of cervical cell lines, 14% (2 of 14) of ovarian cell lines, and none (0 of 4) of the endometrial cell lines displayed aberrantly migrating FHIT transcripts. DNA sequencing demonstrated that the aberrantly migrating bands primarily lacked exons 5, 6, and 7 (with other exon losses also observed), resulting in shorter mRNA transcripts. Southern blot analysis of DNA from five of the cervical carcinomas demonstrated alterations in four of them, three of which had exhibited no normally sized FHIT transcripts. The results suggest that the expression of the FHIT gene may be altered in cervical tumor tissue, potentially implicating this gene in cervical tumorigenesis, whereas the involvement of this gene appears to be less important in the development of ovarian and endometrial cancer.

Acid Anhydride Hydrolases↗