The human genome project: approaches to the automation of sequence data input.
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Automation↗
Biomedical subjects
W J Martin
Publications and source records attributed to W J Martin.
Detection of human papillomavirus in formalin-fixed, invasive squamous carcinomas using the polymerase chain reaction.
We analyzed 88 formalin-fixed, paraffin-embedded invasive squamous carcinomas for human papillomavirus-related DNA sequences (HPV types 16 and 18) following in vitro gene amplification using the polymerase chain reaction. HPV DNA sequences were found in 35 of 50 (70%) carcinomas of the anogenital region, including four of four (100%) anal, six of eight (75%) vulvar, nine of 14 (64%) vaginal, two of five (40%) penile, and 14 of 19 (74%) cervical tumors. Nine of 25 (36%) oropharyngeal squamous carcinomas contained HPV DNA sequences, including four of 10 (40%) laryngeal, three of eight (38%) buccal, and two of seven (29%) glossal tumors. HPV DNA sequences were not found in 13 esophageal carcinomas. Of the 44 cases that contained viral DNA, HPV-16 was detected in 41 cases (93%) and HPV-18 in five cases (11%), while both types were found in two cases (one anal and one vulvar). HPV DNA sequences were found in 43 of 83 (52%) nonverrucous and in one of five (20%) verrucous carcinomas, but this difference was not significant. These findings demonstrate that HPV DNA sequences are more frequently associated with anogenital than oropharyngeal squamous carcinomas and can be readily detected in routinely processed tissues using the polymerase chain reaction.
Blotting, Southern↗
New technologies for large-genome sequencing.
Interest in DNA sequencing has increased in recent years and research into new instrument systems to optimize and automate the process has been intensified. A number of commercial devices, from Europe, Japan, and the United States, which instrument aspects of (mainly) the Sanger method, are now available and several new DNA sequencing techniques have been reported. This review briefly describes current methods of DNA sequencing and provides an account of systems presently available or under development for instrumenting the technology. The problems of automation for large-scale DNA sequencing are discussed.
Automation↗
Pneumocystis carinii pneumonia. Differences in lung parasite number and inflammation in patients with and without AIDS.
Pneumocystis carinii pneumonia has emerged as a significant cause of morbidity and mortality in immunocompromised patients with and without AIDS. To determine differences in P. carinii pneumonia in patients with and without AIDS, the P. carinii parasite numbers, lung inflammatory cell populations, gas exchange, and survival were assessed in a series of 75 consecutive patients with P. carinii pneumonia. Bronchoalveolar lavage was used to quantify the parasite and inflammatory cell numbers in these patients. The data from this study indicate: (1) patients with P. carinii pneumonia and AIDS have significantly greater numbers of P. carinii per ml of lavage compared to other immunocompromised patients with P. carinii pneumonia (p less than 0.0001); (2) patients with P. carinii pneumonia and AIDS also have significantly fewer neutrophils recovered in the lavage compared to other immunocompromised patients with P. carinii pneumonia (p = 0.0001); (3) patients with AIDS and P. carinii pneumonia have higher arterial oxygen tensions than those patients with P. carinii pneumonia in conditions other than AIDS (p = 0.008); and (4) increased lavage neutrophils (rather than parasite number) correlate with poorer oxygenation and poorer patient survival (p = 0.01). This investigation demonstrates substantial differences in lung inflammation and parasite number during P. carinii pneumonia in patients with and without AIDS. The data further suggest that lung inflammation contributes substantially to respiratory impairment in patients with P. carinii pneumonia.
Acquired Immunodeficiency Syndrome↗
Disparities in assessment of disease activity.
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Humans↗
Oxygen-mediated impairment of human pulmonary endothelial cell growth: evidence for a specific threshold of toxicity.
Oxygen toxicity to the lung is characterized by injury of the pulmonary capillary endothelium with progressive loss of functioning alveolar-capillary units. Current concepts suggest that the risk of O2 toxicity in human subjects is greatly increased with O2 concentrations exceeding 50% to 60%, although there are no data to support a cellular basis for this apparent threshold of toxicity. Our study suggests that a cellular threshold may exist in human pulmonary endothelial cells for O2 toxicity. Hyperoxia was directly toxic to cultured human pulmonary artery endothelial (HPAE) cells, with impairment of replicative function, expressed as growth impairment (GI) index, monitored by two independent parameters: cell number determination and tritiated thymidine incorporation. Impaired cell growth occurred as early as 8 hours after beginning exposure to 95% O2 and with concentrations as low as 60% during a 48-hour incubation. For example, 60% O2 resulted in an impairment of HPAE cell growth at 48 hours with a GI index (cell number) of 37.5 +/- 2.1 (p less than 0.001, comparison with control cells in normoxia). Furthermore, 95% O2 impaired cell growth, as monitored by tritiated thymidine incorporation, as early as 8 hours after exposure (GI index of 43.6 +/- 4.9) however, the injury was completely reversible when cells were reincubated in normoxia for 6 hours (GI index of 4.2 +/- 4.7), p less than 0.001. O2 toxicity was associated with an increase in cellular glutathione levels but was not associated with a detectable loss of antioxidant enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Catalase↗
Suppression of experimental uveitis in rats by anti-I-A antibodies.
I-region-associated (Ia) class II major histocompatibility complex (MHC) products are known to play a major role in autoimmunity. Effects of anti-I-A and anti-I-E monoclonal antibodies on development of experimental autoimmune uveitis (EAU) were investigated in Lewis rats. Prior to sensitization with S-antigen, seven groups of rats, six in each group, were injected intraperitoneally with one of the following agents. Groups 1, 2, and 3 (controls) received saline, RPMI and mouse immunoglobulin G (IgG), respectively. Groups 4 and 5 were injected with anti-I-E antibodies, 80 micrograms and 1000 micrograms, respectively. Similarly, groups 6 and 7 received anti-I-A, 100 micrograms and 750 micrograms, respectively. The treatments were repeated on days 1, 2, 5, 8, and 11 after S-antigen injection. All these animals were killed on day 18. In addition, two groups of rats sensitized with S-antigen were treated with 750 micrograms anti-I-A antibodies on days 5, 6, and 7 (group 8) and on days 7, 8 and 9 (group 9). An additional group (group 10) of Lewis rats was treated with 750 micrograms anti-I-A 1 day prior to and on days 1 and 2 after S-antigen injection. These group-10 animals were killed on day 31. Histopathologically, the enucleated globes of animals treated with high dose anti-I-A revealed marked suppression or inhibition of uveitis development. Such inhibition was virtually complete when the antibody was administered within a week of S-antigen injection, and the inhibitory effect lasted for at least 31 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Mechanism of phospholipidosis in amiodarone pulmonary toxicity.
Amiodarone therapy can be complicated by life-threatening pulmonary toxicity and is invariably associated with characteristic morphologic changes in the lung consistent with a severe phospholipidosis. To determine possible mechanisms, this study utilizes a unique in vitro cell culture model of amiodarone pulmonary toxicity and demonstrates that amiodarone can directly induce an accumulation of phospholipids within bovine pulmonary artery endothelial (BPAE) cells during the first 24 hr using amiodarone concentrations equivalent to concentrations found in the blood and lungs of human subjects. For example, amiodarone at 7.5 microM during a 24-hr incubation increases [32P]orthophosphate incorporation into phospholipids from 193 +/- 10 X 10(3) dpm/10(6) cells to 266 +/- 19 X 10(3) dpm/10(6) cells (P less than .01). A generalized increase in all phospholipids occurs after amiodarone treatment of the cultured cells; however, several specific phospholipids including phosphatidylinositol, phosphatidic acid and bis(monoacylglycerol) phosphate are all significantly increased to a greater extent than other phospholipids. Furthermore, the data indicate that amiodarone is a potent inhibitor of lysosomal phospholipase A1 and A2 activities derived from the BPAE cells; whereas, amiodarone has no effect on phospholipase A1 and A2 activities from the BPAE microsomal fraction. Thus, this study demonstrates phospholipids accumulate in cultured BPAE cells exposed to amiodarone and provides direct evidence that the drug initiates a specific and nearly complete inhibition of phospholipid degradation by lysosomal phospholipase A1 and A2 suggesting a similar process may occur in vivo in the lungs of subjects with amiodarone pulmonary toxicity.
Amiodarone↗
Molecular characterization of the C3HfB/HeN H-2Kkm2 mutation. Implications for the molecular basis of alloreactivity.
The C3HfB/HeN (C3Hf) mouse strain expresses an H-2Kk molecule, previously denoted H-2Kkv1, that is structurally and functionally distinct from H-2Kk of the parental C3H strain. By molecular genetic analysis, we demonstrate that the C3Hf H-2K gene carries a homozygous coding region mutation relative to the C3H allele, revealing that C3Hf meets the requirements for assignment of a mutant haplotype, H-2km2. C3Hf H-2Kkm2 bears a single clustered substitution of four nucleotides within 14 contiguous nucleotides in exon 3. Since this sequence also is present intact at the homologous position in H-2Dk of both C3H and C3Hf, the origin of the H-2Kkm2 mutation is consistent with a nonreciprocal sequence transfer from the H-2Dk donor gene, analogous to the mechanism proposed for generation of the H-2Kb mutations. The H-2Kkm2 mutation encodes three clustered amino acid substitutions, at positions 95, 98, and 99, that map to one of the large beta strands at the bottom of the peptide antigen binding cleft of the H-2Kkm2 molecule. The nature and location of these amino acid substitutions are unique relative to any other known H-2 mutant or HLA variant, and underscore the importance of the beta-pleated sheet in influencing CTL recognition. These results indicate that H-2Kkm2 alloantigenicity may derive largely from altered presentation of self cellular peptides.
Amino Acid Sequence↗
Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: a bridge between molecular biology and classical histology.
DNA sequences from human tissues paraffin embedded 40 yr ago were studied using the in vitro gene amplification technique known as the polymerase chain reaction. Although significant DNA degradation was observed, single copy genomic sequences and viral segments were readily detected from single 5- to 10-microns tissue sections. This demonstrates that the world-wide collection of archival paraffin-embedded tissues may be used to study the association of biological agents (viral, bacterial, or parasitic) or endogenous DNA lesions with disease over time and to carry out retrospective studies on material where the clinical outcome has already been established. This will be especially valuable in studying rare cancers and other rare diseases.
Base Sequence↗
Incomplete hydrolysis of the calcium indicator precursor fura-2 pentaacetoxymethyl ester (fura-2 AM) by cells.
Fura-2 AM is an esterified cell-permeant form of the Ca2+ indicator fura-2 (1-[2-(5-carboxyoxal-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2'-a mino-5'- methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid). Fura-2 AM has been reported to be completely cleaved by cellular esterases to fura-2 which is trapped within cells and is used to measure intracellular free Ca2+ concentration by a fluorescence ratio method. Successful application of the method requires that fura-2 be the major cellular fluorescent metabolite of fura-2 AM. We have used high-performance liquid chromatography to study fura-2 AM hydrolysis by cells. Murine N1E-115 neuroblastoma cells incubated with 10 microM fura-2 AM formed fura-2 at a rate of 9.7 pmol/min/10(6) cells. The concentration of fura-2 in the cells after 60 min, assuming uniform distribution, was 137 microM. Smaller amounts of at least four other metabolites were present, as well as large amounts of unhydrolyzed fura-2 AM. Washing the cells with medium containing 2% bovine serum albumin decreased the concentration of fura-2 to 40 microM and that of fura-2 AM to 90 microM. The half-time for loss of fura-2 from neuroblastoma cells after washing was 34 min. Human pulmonary artery endothelial (HPAE) cells formed fura-2 at a rate of 2.6 nmol/min/10(6) cells and the concentration of fura-2 after 60 min of incubation and washing with albumin containing medium was 130 microM, and the concentration of fura-2 AM was 58 microM. The half-time for loss of fura-2 from washed HPAE cells was 74 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction.
Human papilloma virus (HPV) DNA sequences have been detected in paraffin-embedded tissue using an enzymatic in vitro amplification technique known as the polymerase chain reaction. Amplification of a HPV DNA sequence before its detection with a cDNA probe significantly increases the rapidity as well as the sensitivity of detection such that a single 5-10-micron thick paraffin-embedded tissue section can be analyzed within 24 h. The assay specifically detected HPV 16 or 18 without crossreactivity with HPV 6 or 11. As few as 20 viral copies could be detected. The rapid and sensitive analysis of HPV in normal and pathological tissues using this technique may contribute significantly to identifying the role of HPV as a risk factor in carcinoma.
Carcinoma, Squamous Cell↗
Serum levels of interleukin-2 receptor and activity of rheumatic diseases characterized by immune system activation.
Levels of interleukin-2 receptors (IL-2R), as measured by a double-antibody "sandwich" enzyme-linked immunosorbent assay technique, were markedly elevated in the serum of patients with systemic lupus erythematosus, rheumatoid arthritis, and bacterial endocarditis, but not in patients with acute gout. Serum levels of IL-2R correlated strongly with clinical and laboratory indicators of disease activity in patients with lupus and in those with rheumatoid arthritis. This relationship was confirmed by sequential determinations in individual patients. Serum IL-2R values correlated with disease activity better than did the Westergren erythrocyte sedimentation rate. Our findings indicate that serum levels of IL-2R may serve as a reliable serologic indicator of disease activity in inflammatory diseases characterized by immune system activation.
Antiviral Agents↗
Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus.
Detection of cytomegalovirus (CMV) DNA can be facilitated by the polymerase chain reaction (PCR), an in vitro gene amplification technique. Twenty-eight CMV tissue culture isolates were examined by amplification of two separate CMV genes. All were found to contain CMV, although two of the isolates were positive for only one of the two genes. No detectable amplification occurred with human genomic or other viral DNA controls. The amplification products from as few as one CMV plaque-forming unit could be detected after the PCR. CMV DNA was detected in the blood of 14 of 27 patients with AIDS and one of six patients who were infected with human immunodeficiency virus but who did not have AIDS. Normal CMV-seropositive or -seronegative individuals did not have CMV DNA detected in their blood. The CMV PCR was more sensitive than the standard culture assay, can be completed in one to two days, uses only 20 microL of blood, and may be useful for rapidly detecting CMV in clinical specimens.
Acquired Immunodeficiency Syndrome↗
Clinical laboratory applications of monoclonal antibodies.
Monoclonal antibody (MAb) technology is well recognized as a significant development for producing specific serologic reagents to a wide variety of antigens in unlimited amounts. These reagents have provided the means for developing a number of highly specific and reproducible immunological assays for rapid and accurate diagnosis of an extensive list of diseases, including infectious diseases. The impact that MAbs have had in characterizing infectious disease pathogens, as well as their current and future applications for use in clinical microbiology laboratories, is reviewed. In addition, the advantages (and disadvantages) of the use of MAbs in a number of immunoassays, such as particle agglutination, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescent-antibody assays, and immunohistology, are explored, including the use of these reagents in novel test system assays. Also, nucleic acid probe technology is compared with the use of MAbs from the perspective of their respective applications in the diagnosis of infectious disease agents. There is no question that hybridoma technology has the potential to alter significantly the methods currently used in most clinical microbiology laboratories.
Antibodies, Monoclonal↗
The effects of sinus bacteria on human ciliated nasal epithelium in vitro.
The mechanisms by which bacteria colonize and damage ciliated epithelium are important in understanding the pathophysiology of rhinitis, sinusitis, and otitis. Bacteria that have the ability to impair mucociliary clearance would be at an advantage in establishing infection of ciliated surfaces. This study investigates the effect of Hemophilus Influenzae, Streptococcus pneumoniae, Branhamella catarrhalis, and Staphylococcus epidermidis on the ciliary activity of normal ciliated nasal epithelium in human beings. Ciliary activity of the nasal epithelium in the presence of each pathogen was assessed for more than 240 minutes with a photometric method of ciliary beat frequency (CBF) measurement. H. influenzae exerted significant effects on ciliary activity, with a 46% decrease in the CBF by 4 hours (with bacteria-containing broth) and a 32% decrease with bacteria-free filtrate. S. epidermidis decreased CBF by 44% with the bacterial broth. A sterile cell-free filtrate had no significant effect. S. pneumoniae and B. catarrhalis had no significant effect on CBF within a 240-minute period. H. influenzae and S. epidermidis disrupted normal synchronous ciliary motion, causing adjacent cilia to beat at different rates.
Bacterial Physiological Phenomena↗
Amiodarone pulmonary toxicity. Recognition and pathogenesis (Part I).
Amiodarone represents an important new approach in the treatment of serious cardiac rhythm disturbances and is associated with significant pulmonary toxicity in approximately 5 to 10 percent of patients. The recognition of APT in patients receiving the drug early in the course of the disease will likely preclude the development of a permanent loss of pulmonary function in these patients. It is important for the clinician to individualize both the diagnostic and therapeutic approach to the patient receiving amiodarone who is thought to have pulmonary toxicity secondary to the drug. Several diagnostic and therapeutic decisions are available, and it is important to constantly reevaluate the risk-benefit ratios of the decision making process. Future studies may improve insight into the assessment of APT and may permit the clinician to diagnose this toxicity at an earlier and potentially more reversible stage of the disease process.
Amiodarone↗
Amiodarone pulmonary toxicity. Recognition and pathogenesis (Part 2).
The pulmonary toxicity associated with amiodarone therapy is clinically complex and likely reflects underlying mechanisms of lung injury that result from direct toxic effects of the drug (or its metabolites) as well as indirect inflammatory and immunologic processes induced by the drug therapy (Fig 2). A role for the direct toxicity of the drug is likely because (a) toxicity in part is related to dosage and duration of therapy, (b) many patients with amiodarone pulmonary toxicity have no evidence of an inflammatory or immune response in the lung, (c) in vitro studies indicate that amiodarone can be directly toxic to cultured lung cells or perfused isolated lung tissue, and (d) recent studies suggest plausible biochemical mechanisms that may explain in part the mechanism(s) of direct toxicity of the drug. A role for indirect inflammatory or immune processes within the lung of some patients with APT is supported by: (a) variable relationship of pulmonary toxicity to amiodarone dosages and blood levels, (b) preliminary studies suggest altered immunologic markers in the blood and lungs of some patients with APT, and (c) the cellular findings of bronchoalveolar lavage indicating a CD8 lymphocytosis with or without influx of polymorphonuclear leukocytes, which is consistent with previous studies of hypersensitivity reactions. As our understanding of the biochemical and cellular mechanisms of APT improve, a number of key clinical issues may be clarified: (1) risk factor assessment for APT, (2) criteria for early diagnosis of APT, and (3) improved therapeutic approach to patients with APT.
Amiodarone↗