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Biomedical subjects

G E Austin

Publications and source records attributed to G E Austin.

At least 37 records · Page 2Linked to original sources
PubMed1994

Myeloperoxidase gene expression in normal granulopoiesis and acute leukemias.

Myeloperoxidase (MPO) is an abundant heme protein found in granulocytes and monocytes, which plays an important role in host defense against infection. MPO enzyme activity as determined by light microscopic cytochemistry has long been an important marker used in the diagnosis of acute leukemias and other hematopoietic disorders. Recently, MPO expression has been studied at the electron microscopic level, and monoclonal antibodies (mAbs) against MPO protein have been developed. Furthermore, techniques and probes for analysing MPO expression at the RNA level are now available. This has made possible more extensive studies of MPO expression in a wide range of neoplastic and preneoplastic blood disorders. This review will discuss the fundamental biology of MPO as well as recent developments in our understanding of MPO expression in leukemic cells and cell lines of various lineages.

Acute Disease↗
PubMed1993

Detection of leukemic blasts in peripheral blood specimens by reverse transcriptase polymerase chain reaction assay for myeloperoxidase mRNA.

The enzyme myeloperoxidase (MPO), an important constituent of granulocytes, is used clinically in the diagnosis and classification of acute leukemia. Whereas MPO protein or enzyme activity is detectable in mature granulocytes, MPO RNA is present only in myeloblasts or promyelocytes. Some undifferentiated, biphenotypic, or lymphoblastic leukemias, although lacking MPO enzyme activity, express low levels of MPO RNA, a fact that may be of prognostic and therapeutic importance. However, current methods are inadequate for reliably detecting low levels of MPO RNA in leukemic cells containing few copies of the message. We have developed a new and highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detecting low levels of MPO RNA in peripheral blood specimens of leukemic patients. This report describes the assay, and demonstrates its potential applicability to the diagnosis and classification of acute leukemias.

Base Sequence↗
PubMed1993

Determination of regional myocardial blood flow using fluorescent microspheres.

Determination of blood flow in tissues at risk for infarction is necessary for in vivo studies of the pathologic effects of vascular occlusion. Such blood flow measurements are traditionally carried out using radioactive microspheres. These are expensive and may pose a significant hazard to laboratory personnel. In addition, disposal of radioactive wastes, especially radioactive carcasses, is an increasingly expensive and inconvenient process. To avoid these problems, we have developed a modified procedure using fluorescent microspheres. To measure blood flow, spheres are injected intravenously and a blood sample is taken. Following cardiectomy, appropriate segments of left ventricular myocardium are digested with proteases to release the spheres, which are then purified through sucrose gradients. The spheres may be counted manually using an epifluorescence microscope, or by flow cytometry. Comparison of this method with the traditional radioactive microsphere procedure reveals similar results for the two methods. Fluorescent microspheres should provide a useful and accurate alternative method for measuring blood flow in studies of vascular disease.

Animals↗
PubMed1993

Sequence comparison of putative regulatory DNA of the 5' flanking region of the myeloperoxidase gene in normal and leukemic bone marrow cells.

Myeloperoxidase (MPO) is an enzyme which is exclusively expressed in immature myeloid cells with downregulation of gene expression occurring during granulocytic maturation. Levels of MPO RNA, protein, and enzyme activity differ, usually in a concordant fashion, among the various classes of acute leukemia and among different cases within a particular class. One portion of the gene thought to be involved in regulation of MPO expression is the proximal 5' flanking region. To determine if mutations in this putatively regulatory region of the MPO gene might be responsible for some of the differences in level of MPO expression among different cases or classes of acute leukemia, we compared the nucleotide sequence of this part of the gene from 16 patients with acute leukemia, with DNA from normal human bone marrow cells and selected other neoplasms and cell lines. The sequence of this regulatory region was found to be identical in cases of acute myeloid leukemia (AML) with tha of normal DNA except for a dA to dG transition in the Alu region, 463 bases upstream from the transcription start site. This base substitution was seen in almost all cases of AML studied, regardless of the level of MPO which they expressed. It was absent from normal human DNA obtained from various tissues, and cases of acute and chronic lymphocytic leukemia, carcinoma of lung, and most cell lines examined. The base substitution was also absent in a remission blood sample from one of the cases which showed the dA to dG transition in leukemic marrow, suggesting that the base substitution is a mutation rather than a polymorphism. Our results suggest that mutations in promoter or enhancer DNA are not an important cause of the differences in level of MPO gene expression seen among different cases or different classes of AML. However, the base substitution we have detected could potentially serve as a useful marker for detection of residual disease in patients with AML following treatment.

Base Sequence↗
PubMed1992

Effect of education on the appropriateness of serum drug concentration determination.

The purpose of this study was to document the effect of education on the appropriateness of serum drug concentration (SDC) collection. This study included a period of education for hospital personnel involved in the acquisition of drug levels. These included nursing staff, lab personnel, house-staff (residents/interns), ward clerks, and pharmacists. This study included patients receiving aminoglycoside and/or vancomycin while on the general medicine service during the specified study periods. Patients receiving less than 72 h of antimicrobial therapy and patients transferred to or from the surgery service were excluded from the study. A retrospective pre- and posteducation comparison using Chi-square analysis was made to determine whether the percentage of appropriate and useful SDCs was improved through education. There was a significant difference (p less than 0.005) between the percentage of appropriate SDCs in the pre- and posteducation period. As a result of the study, a change in procedure for ordering SDCs was initiated, using an educational program, in order to continue the trend of appropriate SDCs at the Veterans Administration.

Aminoglycosides↗
PubMed1991

Reduction of myocardial infarct size by poloxamer 188 and mannitol in a canine model.

Poloxamer 188 has been reported to inhibit thrombosis, decrease whole blood viscosity, and improve perfusion of damaged tissue. Mannitol has free radical scavenging capabilities that might contribute to myocardial salvage after ischemia. Because these agents appear to work in different ways, we studied their cardioprotective properties when they were used separately and in combination. After 75 minutes of left anterior descending coronary artery (LAD) occlusion, dogs received poloxamer 188 (48 mg/kg), mannitol (0.5 gm/kg), or both intravenously during an additional 15 minutes of LAD occlusion and for 45 minutes of reperfusion, whereas control dogs received an equal volume of saline solution. After surgery the animals were maintained for 24 hours and then killed. Areas of myocardial infarction (MI) and risk of infarction (R) were calculated by means of planimetric analysis of slices of myocardium stained with 1.5% triphenyltetrazolium and 0.5% Evans blue dye. The ratio of MI/R (mean +/- standard error of the mean) were: control, 25.6 +/- 1.8% (n = 10); poloxamer 188, 12.7 +/- 2.0% (n = 10); mannitol, 10.6 +/- 2.5% (n = 11); and poloxamer 188 plus mannitol, 8.0 +/- 4.1% (n = 10). Measurement of microvascular blood flow indicated a similar 86% to 91% reduction of blood flow to the area at risk in all treatment groups. Consequently both poloxamer 188 and mannitol appear to increase salvage of ischemic myocardium and a combination of the two may be more effective than either agent alone.

Animals↗
PubMed1991

A comparison of home glucose monitoring with determinations of hemoglobin A1c, total glycated hemoglobin, fructosamine, and random serum glucose in diabetic patients.

We compared four objective measures of glycemic control (fructosamine, total glycated hemoglobin, hemoglobin A1c, and random serum glucose) with home glucose monitoring records in 17 diabetic patients followed up prospectively for 4 months. There was good overall correlation between all of these objective measures and weekly mean capillary glucose values. However, considerable scatter was seen in the data such that none of the glycated protein measurements was an ideal predictor of home glucose values. For example, all markedly elevated home glucose levels (greater than 11.1 mmol/L) were associated with elevated glycated protein levels, but moderately high blood glucose levels (8.3 to 11.1 mmol/L) were associated with one or more normal glycated protein values in some patients. Similar correlations were obtained whether glycemia was estimated by 1-week or 6-week home averages. Random serum glucose level also correlated with average home glucose level; however, there was wide fluctuation within individual subjects. All three glycated protein measurements (hemoglobin A1c, glycated hemoglobin, and fructosamine) appear equally useful as a supplement to home glucose monitoring in the assessment of glycemic control. Of the three types of glycated protein assays, fructosamine, with its advantage of speed and simplicity, may offer a more cost-effective alternative.

Adolescent↗
PubMed1990

Chromosomal localization of the human myeloperoxidase gene by in situ hybridization using oligonucleotide probes.

Oligonucleotide probes have been used to map the myeloperoxidase (MPO) gene locus to chromosome bands 17q21-22. This is in agreement with results reported using conventional cDNA probes. No evidence for the existence of a second MPO gene locus was obtained. Six synthetic 72-base oligonucleotides, corresponding to different exon regions of the MPO gene, were tritium-labeled and used as in situ hybridization probes. Synthetic oligonucleotide probes offer a useful alternative to conventional DNA probes for gene mapping.

Chromosome Banding↗
PubMed1990

Morphologic characterization of early prostatic carcinomas in the ACI rat: a light and electron microscopic study.

The ACI rat constitutes a unique model for human prostatic carcinogenesis. A high percentage of these animals spontaneously develop prostatic carcinomas in the ventral lobe as they age. The light microscopic appearance of these tumors is similar to the cribriform pattern of adenocarcinoma in human prostate. In order to further characterize this useful model, we carried out light and electron microscopy studies of the morphology of carcinomatous lesions developing in these animals. Sixteen rats ranging in age from 25 to 43 months were examined histologically, and ultrastructural studies were performed on eight of these cases. The neoplastic cells showed features of well-developed secretory epithelium including prominent Golgi apparatus, abundant rough endoplasmic reticulum, and numerous secretory vacuoles. Microvilli were numerous in some cells and focal apocrine secretory activity was present. Intraluminal crystals similar to those associated with human prostate carcinoma were observed in one of our cases. Prostate carcinomas developing in the ACI rat share many of the ultrastructural features of human prostatic carcinoma.

Adenocarcinoma↗
PubMed1990

Studies of myeloperoxidase gene expression at the cellular level by in situ hybridization.

Recent studies have demonstrated myeloperoxidase (MPO) gene expression during granulocytic differentiation. Since these studies have been done exclusively by Northern and dot blot analysis and frequently with mixed populations of cells, quantitative changes in gene expression for particular populations of cells are difficult to assess. We therefore examined MPO expression at the cellular level in various normal and malignant hematopoietic cells by the in situ hybridization (ISH) technique. Using this approach, we demonstrated that inducing the promyelocytic HL-60 cell line to differentiate along either monocytic or granulocytic pathways decreases MPO mRNA expression. Similarly, when ISH was performed on normal bone marrow, relatively high levels of MPO mRNA were detected in myeloblasts, promyelocytes, and early eosinophilic precursors, whereas the expression was markedly decreased in more advanced stages of myeloid differentiation. These findings agree with the known decrease in MPO protein synthesis observed during granulocytic differentiation and suggest that regulation of MPO protein synthesis occurs at the level of MPO mRNA expression. We conclude by showing that ISH can detect MPO mRNA in myeloblasts of patients with acute leukemia and can be a potentially useful technique in the study of myeloid differentiation in acute leukemias.

Adolescent↗
PubMed1990

Latissimus dorsi dynamic cardiomyoplasty of the right ventricle. Potential for use as a partial myocardial substitute.

Full-thickness right ventricular latissimus dorsi dynamic cardiomyoplasty with the Medtronic Cardiomyostimulator (Medtronic, Inc., Minneapolis, Minn.) was performed in a chronic canine model. In one group (n = 2) the latissimus dorsi was electrically preconditioned before cardiomyoplasty. In a second group (n = 3) cardiomyoplasty was performed and the muscle was progressively stimulated, with conditioning accomplished while the latissimus dorsi was functioning on the ventricle. The contribution of the stimulated latissimus dorsi to global ventricular function was assessed, and the effects of varying muscle stimulation parameters on latissimus dorsi function and hemodynamics were examined. Right ventricular systolic pressure increased 8%, from 23.2 +/- 0.95 to 25.1 +/- 1.5 mm Hg. The rate of pressure rise increased 37% from 226 +/- 13 to 309 +/- 12 mm Hg/sec. Right ventricular ejection fraction was measured in two dogs and increased 29% with latissimus dorsi stimulation, from 51.5% +/- 13.5% to 66.5% +/- 14.5%. Although the sample size was small, there was no difference observed between the preconditioned and nonpreconditioned groups. Right ventricular systolic pressure, rate of pressure rise, and percent latissimus dorsi fiber shortening increased as voltage and burst frequency of the muscle stimulus increased, whereas increasing the burst duration had little effect in two dogs so studied. Latissimus dorsi dynamic cardiomyoplasty can function as a partial myocardial replacement in a chronic canine model, apparently without preconditioning of the muscle. The degree of cardiac assist obtained with cardiomyoplasty appears to be influenced by the voltage and frequency of the stimulus applied to the muscle. Although it is unclear whether these results can be extrapolated to the left ventricle, this technique may find application in the treatment of ventricular aneurysm or ventricular tumor.

Animals↗
PubMed1989

Human myeloperoxidase gene expression in acute leukemia.

To evaluate the relationship between myeloperoxidase (MPO) gene expression and specific lineages of hematopoietic differentiation, we analyzed expression of the MPO gene in a variety of normal and leukemic human cells. As a first step, we synthesized several oligonucleotide probes and isolated cDNA clones for the MPO gene. MPO expression was detected in most acute myeloid leukemias, and the level of expression correlated well with cytochemical enzymatic activity. No MPO message was detected in pure lymphoid leukemias, whereas very low basal levels of MPO transcripts were found in normal bone marrows. In four cases of acute undifferentiated leukemias cytochemically negative for MPO, significant levels of MPO transcripts were detected, suggesting a myeloid origin for these cases. These results indicate that MPO gene expression may serve as an additional marker for subclassification of acute leukemias and may be used to identify leukemic cells arrested at an early stage of the myeloid differentiation pathway.

Blotting, Northern↗
PubMed1989

Production and characterization of monoclonal antibodies to human myeloperoxidase.

Monoclonal antibodies (mAbs) against human myeloperoxidase (MPO) have been derived for immunopurifying MPO and immunophenotyping acute leukemias. Eight antibodies were obtained from a fusion of the P3 plasmacytoma cell line with splenic lymphocytes from mice immunized with purified human MPO. Hybridoma supernatant culture fluids were screened for antibody to MPO by an enzyme-linked immunosorbent assay. The specificity of the mAbs was characterized by immunoblotting, immunoprecipitation, and reactivity against various hematopoietic cells, cell lines, and leukemic blast cells. The eight mAbs generated were of the IgG1 isotype. One of these mAbs was successfully used to develop a one-step immunoaffinity chromatographic purification procedure for MPO. Immunoblotting and immunoprecipitation experiments suggested that the eight mAbs were reactive with at least three antigenic determinants on the MPO molecule. Immunofluorescent studies showed that these mAbs reacted specifically with cells of granulocytic-monocytic lineage and were negative with lymphoid cells. Our results suggest that these mAbs should be useful reagents for immunophenotyping hematopoietic cells.

Animals↗
PubMed1989

Precision and reliability of paraprotein determinations by high-resolution agarose gel electrophoresis.

Agarose gel electrophoresis has recently replaced cellulose acetate electrophoresis as the preferred technique for monitoring paraprotein levels in patients with plasma cell dyscrasias. The authors studied the accuracy and precision of this method for paraprotein determination. Twenty-seven serum samples with paraprotein concentrations ranging from 5 to 73 g/L were aliquotted and assayed on 20 separate occasions, and the mean and standard deviation for the paraprotein concentration in each serum was established. Linear regression analysis showed that the standard deviation of paraprotein concentration (SD) increased as a function of paraprotein concentration (PC). For IgG paraproteins, the regression equation was SD = 0.041 (PC) + 1.06; R = 0.942; standard error = 0.32. For non-IgG paraproteins the equation was SD = 0.101 (PC) - 0.04; R = 0.851; standard error = 0.5. The accuracy of paraprotein determinations by the agarose gel electrophoretic technique was assessed by comparison with values obtained with the use of a previously validated enzyme-linked immunosorbent assay (ELISA) method for quantitation of IgG subclasses. Results obtained by the two methods were similar and highly correlated: (concentration by electrophoresis) = 0.921 (concentration by ELISA) + 0.46; R = 0.988; standard error = 0.34. The laser densitometric scanning procedure showed a loss of linearity above 60 g/L, indicating the need to dilute sera with very high paraprotein concentrations in order to obtain accurate results. A table is presented that should help pathologists who interpret such scans to determine whether small changes in paraprotein measurements occurring over time represent true changes in paraprotein concentration or merely reflect the analytic variability inherent in the technique.

Densitometry↗
PubMed1989

Antibodies to ras oncogene p21 proteins lack immunohistochemical specificity for neoplastic epithelium in human prostate tissue.

Increased expression of ras oncogene protein p21 has been claimed to be associated with neoplastic transformation in histochemical studies of prostate and other tissues. Therefore, we evaluated the expression of p21 in formaldehyde-fixed, paraffin-embedded prostatic tissue using a commercially available polyclonal antiserum prepared in sheep. We found no consistent difference in p21 expression between benign and malignant epithelium and no correlation between tumor grade and intensity of p21 staining. Our results do not support the contention that neoplastic transformation of human prostate is associated with a histochemically demonstrable increase in p21 expression.

Antibodies↗
PubMed1989

Nonenzymic glycation of human immunoglobulins does not impair their immunoreactivity.

Diabetic patients have an increased proportion of their immunoglobulins nonenzymically glycated. To investigate the possibility that this may contribute to increased susceptibility to infection, we compared the immunoreactivity of glycated and nonglycated human immunoglobulins against rubella and hepatitis; streptococcal exoenzyme and infectious mononucleosis; human lymphocytotoxic antigens (HLA); and Varicella zoster in terms of antigen-antibody binding, cell agglutination, cytotoxicity, and complement-fixation properties, respectively. We found no evidence to support the supposition that glycated immunoglobulins are functionally impaired.

Antibodies, Viral↗
PubMed1988

Capacity related characteristics of Glyco-Gel affinity chromatographic support.

We examined capacity related properties of "Glyco-Gel" (Pierce), a boronate agarose gel for separating and measuring glycated proteins by affinity chromatography. Our data indicate linear capacity to as much as 20 mg as applied hemoglobin or almost 10 mg as bound hemoglobin and 26 mg as applied serum proteins or a minimum of 2.5 mg as bound serum protein for each mL of gel. The capacity and affinity of the support for glycated proteins becomes optimum only after four regeneration cycles. The support matrix appears to have a small concentration of nonspecific binding sites equivalent to 0.09 to 0.18 mg as serum protein for each mL of gel. These sites do not bind hemoglobin. They lead to an overestimation of glycated protein that can cause large errors when the proportion of glycated protein is determined with small column loads. If near capacity loads are applied, the samples must be dialyzed or diluted to avoid decreased analytical recovery resulting from competitive and eluting properties of endogenous sugars.

Boronic Acids↗