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R M Donovan

Publications and source records attributed to R M Donovan.

35 records · Page 2Linked to original sources
PubMed1987

Comparison of non-radioactive DNA hybridization probes to detect human immunodeficiency virus nucleic acid.

Simple and sensitive methods to directly detect the human immunodeficiency virus (HIV) are needed for routine use in the clinical laboratory. In this study, we compared DNA probes prepared by: (1) nick translation with biotinylated dATP; (2) direct covalent biotinylation with photobiotin; (3) direct covalent reaction with 2-acetylaminofluorene (AAF); and (4) a standard radioactive (32P) nick translation procedure. These four DNA probes were hybridized with dilutions of purified target HIV DNA blotted onto nitrocellulose strips. Hybridization was detected using a complex of strepavidin-alkaline phosphatase [for (1) and (2)], alkaline phosphatase-tagged antibodies [for (3)] and by autoradiography [for (4)]. Alkaline phosphatase was detected colorimetrically using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, AAF probes were most sensitive (amount detected less than 5 pg), followed by biotin (10 pg), photobiotinylated probes (20 pg) and the radioactive probe (10 pg). The AAF probes were then used to detect HIV DNA in infected CEM cells. We conclude that non-radioactive DNA labelling methods can be used to directly detect HIV DNA under conditions compatible with present clinical laboratory procedures.

2-Acetylaminofluorene↗
PubMed1987

A computer-assisted image-analysis system for analyzing polymorphonuclear leukocyte chemotaxis in patients with diabetes mellitus.

A computer-assisted image-analysis system that precisely tracks the cell movements of up to 50 polymorphonuclear leukocytes (PMNLs) was developed and used to quantitatively measure cellular chemokinesis and chemotaxis in normal individuals and in diabetic patients with hyperglycemia. The PMNLs were tested in Zigmond chambers with or without a gradient of 10(-7) M n-formylmethionylleucylphenylalanine (f-Met-Leu-Phe). Cellular movement was recorded on videotape by using a videocamera mounted on the microscope. The videotapes were analyzed by computer programs to calculate the speed and direction of each PMNL at 10-sec intervals. Average rates of chemokinesis were 19.6 microns/min without and 25.3 microns/min with f-Met-Leu-Phe. McCutcheon indices, which measure chemotaxis, were 0.01 without and 0.48 with f-Met-Leu-Phe. Similar values were observed in diabetic patients after fasting (average glucose, 217 mg/100 ml) and 2 hr after glucose challenge (average glucose, 309 mg/100 ml). These values demonstrate that PMNLs from diabetic patients with hyperglycemia move at normal rates and respond appropriately to f-Met-Leu-Phe.

Adolescent↗
PubMed1987

Activation and long-term proliferation of human cord blood T cells with human recombinant interleukin-2.

We report here the results of in vitro studies that demonstrate that purified human recombinant interleukin-2 (hrIL-2) is a potent stimulant for freshly isolated cord blood lymphocytes of healthy full-term infants. Different culture parameters were studied to define the optimal conditions for eliciting maximal levels of activation. Highest levels of hrIL-2-induced TdR[3H] uptake were recorded on day 7 for cultures containing 100 U hrIL-2/ml. Results of comparative studies demonstrated that the reactivity of cord blood lymphocytes to hrIL-2 was equal to, if not greater than, that of healthy adult lymphocytes. Cord blood cells that had been activated with hrIL-2 could be propagated in long-term (greater than 30 days) cultures as hrIL-2-dependent lines, and these lines could be initiated with a high degree of success. Phenotypic analysis was performed using different monoclonal antibodies and cytofluorometry, and studies characterizing cells of the long-term lines have shown that they consisted of a heterogenous population of T4 helper and T8 cytotoxic/suppressor cells; in some instances, natural killer (NK) cells were also present. Other experiments demonstrated that hrIL-2-activated and hrIL-2-propagated T cells expressed the IL-2 receptor (IL-2R; defined by monoclonal antibody anti-Tac) and the number of IL-2-R-positive cells could be increased two-fold or more by exposing the cells to a phorbol ester. This report provides additional information to support the hypothesis that hrIL-2 not only sustains T cell proliferation (i.e., second signal) but also induces T cell activation (i.e., first signal).(ABSTRACT TRUNCATED AT 250 WORDS)

Culture Media↗
PubMed1986

Effect of human recombinant tumor necrosis factor on the growth of different human and mouse long-term hematopoietic cell lines.

Previous studies have demonstrated that one of the most salient features of tumor necrosis factor (TNF) is its ability to induce tumor necrosis in vivo, and the specificity of its cytotoxic/cytostatic activity for tumor cells has been demonstrated in in vitro studies in which this lymphokine has been shown to kill cultured cells of malignant lines and to have no effect on cells of normal diploid lines. Studies described herein defined the effect of highly purified human recombinant TNF on cells of 34 different human and murine hematopoietic cell lines, particularly human leukemic T and B cells of long-term lymphoblastoid cultures. Results of these studies demonstrated that TNF at concentrations of 3,600 U/ml had no significant effect on the growth of these cells as defined by cytotoxicity, measured with the use of the trypan blue dye-exclusion assay and as defined by cytostasis, assayed by the enumeration of cells and the uptake of [3H]-thymidine and -uridine. In contrast, positive control cultures of TNF-sensitive cells from a murine tumor (L-M/clone L-929, connective tissue) displayed at 50% (LD50) reduction in growth by TNF at approximately 5 U/ml. Likewise, human tumors (MCF-7, breast, and HT-29, colon) were also highly sensitive (LD50 less than 100 U/ml). These studies demonstrate that T and B cells of lymphoblastoid lines as well as cells of other hematopoietic lines display little or no sensitivity to TNF.

Animals↗
PubMed1986

A quantitative method for the analysis of cell shape and locomotion.

A rapid, semiautomated system to quantitate and analyze leukocyte shape and locomotion was developed. Video images of moving leukocytes were obtained using a Vidicon camera mounted on a Nikon phase microscope. The video signal was either inputted directly, or indirectly via a video cassette recorder, to a Datacube video analog-digital, digital-analog converter. A Digital Equipment Corporation LSI 11/23 computer using the RT-11/TSX-Plus operating system and computer programs written in FORTRAN and MARCO assembly language permitted image segmentation, image display, and calculation of position, speed, direction of movement and orientation of each leukocyte at 10 s intervals. These data were stored on a winchester disk for subsequent evaluation of the leukocyte orientation, speed and direction of movement using statistical and graphical methods. The reproducibility of measurements made with the video system was tested by comparison with manual measurements; a correlation coefficient of 0.998 was obtained for the two methods. Rates of chemokinesis were then determined for unstimulated and chemokinetically stimulated polymorphonuclear leukocytes (PMNs) and found to average 12.8 micron/min and 18.1 micron/min, respectively. The high speed, ease of data analysis, and potential for multiparameter evaluation makes this system useful for directly evaluating leukocyte locomotion.

Cell Movement↗
PubMed1986

Acidification of phagosomes in murine macrophages: blockage by Nocardia asteroides.

Most strains of Nocardia asteroides are susceptible to the detrimental effects of pH 5 when grown in buffered brain-heart infusion broth. Preventing phagosomal acidification may be a mechanism by which this organism survives the microbicidal activity of macrophages. Fluorescein isothiocyanate was conjugated to the surface of Nocardia and Saccharomyces to form pH-sensitive fluorescent probes. The fluorescent emission, and thus the pH, of this probe was quantitated within individual phagosomes by using a computerized cytospectrophotometer. When either live or dead cells of virulent N. asteroides strain GUH-2 were ingested, the phagosomal pH remained above pH 7 for 2 hr. A nonpathogenic soil isolate, N. asteroides strain 19247, only partially blocked acidification. In contrast, when Saccharomyces was used as a control for normal response, the pH decreased to approximately pH 5. Therefore, virulent N. asteroides blocks phagosomal acidification. Because killed Nocardia act in the same manner, this inhibition of acidification appears to be associated with cellular components. This capacity to prevent phagosomal acidification may be prerequisite to the survival of intracellular pathogens.

Animals↗
PubMed1986

Modulation of lysosomal protease-esterase and lysozyme in Kupffer cells and peritoneal macrophages infected with Nocardia asteroides.

Virulent Nocardia asteroides reduces lysosomal acid phosphatase activity in murine macrophages. A computer-assisted imaging photometry system was used to quantitate lysozyme and nonspecific esterase-neutral protease levels within individual macrophages following ingestion of nocardiae. In contrast to acid phosphatase, lysozyme and esterase-neutral protease activity was either unchanged or increased following infection by increasing numbers of nocardial cells.

Acid Phosphatase↗
PubMed1985

Reactivation of tritonated models of human polymorphonuclear leukocytes (PMNs): a computer-assisted analysis.

The orientation (chemotaxis) and locomotion (chemokinesis) of human polymorphonuclear leukocytes (PMNs) are generated by an internal movement mechanism that involves active cytoplasmic movement; they are influenced by external environmental and ionic conditions. We have studied the degree to which the orientation and movement mechanisms of PMNs are self-contained within the cell and the degree to which they are under membrane control. PMNs were partially and selectively demembranated by treatment with the non-ionic detergent, octyl-phenoxyl-polyethoxyethanol (commercially known as Triton X-100) under controlled conditions. The tritonated PMNs (referred to in the literature as models) were non-motile and non-locomotory. Addition of ATP/Mg++ with a trace amount of Ca++ to the medium was followed by reactivation of the tritonated PMN models to move again as motile cells. Although these reactivated PMN models actively locomoted, they could no longer orient to chemoattractants. Thus, the reactivation process restored the physical self-contained movement parameters but could not reestablish the orientation capacity (chemotactic responsiveness) that was characteristic of live PMNs. The demembranation process apparently destroyed the chemotactic receptors and/or eradicated the coordination function of the membrane. Videotapes of normal (control) as well as reactivated PMN movement were analyzed for movement characteristics. These characteristics were objectively analyzed with a newly designed computer-assisted micro-image-processing technique whereby the videotapes were digitized and quantified and the actual PMN movement printed out in computer-graphics and tracings (Freeman codes) for confirmation of orientation and movement arising as a result of reactivation.

Adenosine Triphosphate↗
PubMed1985

Monocyte function in rhesus monkeys with simian acquired immune deficiency syndrome.

Monocyte function in rhesus monkeys with simian acquired immune deficiency syndrome (SAIDS) was compared with that in age-matched normal juvenile rhesus monkeys. The functional tests were 1) chemotaxis, 2) phagocytosis of opsonized Candida albicans, 3) killing and/or growth inhibition of Candida albicans, 4) generation of respiratory burst, and 5) monocyte-derived macrophage response (morphology and/or respiratory burst) to stimulating agents such as lymphokines, gamma interferon, endotoxin, and phorbol myristate acetate. The monkeys tested had either clinical SAIDS (alive with lymphadenopathy, splenomegaly, and lymphopenia or neutropenia) or had terminal SAIDS (moribund due to the disease). Responses of monocytes from 14 monkeys with clinical SAIDS were indistinguishable from those of 9 normal juvenile rhesus monkeys, whereas monocytes from 3 monkeys with terminal SAIDS had enhanced phagocytosis and respiratory burst capacity. Chemotaxis, candidacidal/stasis activity, and response to stimulating agents were normal in these terminal cases. Plasma from the SAIDS monkeys was as capable of opsonizing yeasts and of being able to generate chemotactic factors by endotoxin as was control plasma. SAIDS retrovirus (SRV) was detected by co-cultivation of pure monocyte-derived macrophage cultures with Raji cells, an indicator cell line which forms syncytia in the presence of SRV. Four terminal SAIDS cases and one late-stage clinical SAIDS case were virus-positive when the number of macrophages in the cultures ranged from less than 50 to about 500. Terminal SAIDS monocyte-derived macrophages in culture as long as 17 days produced SRV. These data show that in monkeys with SAIDS the major effector functions of monocytes and macrophages involved in host defense are intact (even up until death). Additionally, some of the monocytes are productively infected, and these infected monocytes are viable and adherent in culture.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Intracellular acid phosphatase content and ability of different macrophage populations to kill Nocardia asteroides.

It has been reported that the activity of lysosomal acid phosphatase decreases inversely with numbers of ingested virulent Nocardia spp. in normal murine peritoneal and alveolar macrophages. These studies suggested that this relationship correlated with the effectiveness of these macrophage populations in killing Nocardia asteroides. Experiments were designed to determine if acid phosphatase activity is affected by infection with N. asteroides in four different macrophage populations isolated from normal and nocardia-immunized mice. Macrophages were also tested simultaneously for their ability to kill N. asteroides. Peritoneal, alveolar, and splenic macrophages and Kupffer cells were infected in vitro with strains of N. asteroides of differing virulence. Uptake and killing assays were performed. Acid phosphatase levels and numbers of intracellular nocardiae were quantitated in the same macrophages, using a computer-assisted cytophotometry system. Acid phosphatase activity decreased inversely with numbers of intracellular nocardiae in macrophages that could not kill or inhibit this pathogen. Acid phosphatase activity was not significantly changed in macrophages that inhibited growth of, but did not kill, N. asteroides, whereas activity was increased or enhanced in macrophages that killed most of the ingested nocardiae. The order of nocardicidal effectiveness (and resistance to enzyme activity reduction with infection) for normal macrophages was splenic greater than peritoneal greater than alveolar greater than Kupffer. In contrast, the order of these two parameters for macrophages isolated from immunized mice was Kupffer greater than peritoneal greater than alveolar greater than splenic. These results demonstrate that lysosomal acid phosphatase activity is an effective marker of the ability of macrophages to inhibit growth of and kill N. asteroides and that macrophages isolated from different anatomical sites differ functionally from each other with respect to nocardicidal and acid phosphatase activities.

Acid Phosphatase↗
PubMed1985

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry.

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.

Animals↗
PubMed1984

Comparison of lavaged and intrapulmonary alveolar macrophages in respect to lysozyme content and size in the rat.

Lavaged and in situ rat alveolar macrophages were compared with respect to lysozyme content and size in order to assess the extent to which macrophages from pulmonary lavages reflect the in situ cell population. This relationship was studied in normal rats and in rats with pulmonary granulomas induced by glucan stimulation (10 mg/kg given intravenously on Days 5, 3, and 1 before being killed). Alveolar macrophages in pulmonary lavages and histologic sections were stained for lysozyme by the immunoperoxidase method using rabbit antiserum to rat lysozyme. Enzyme content and cell size were measured with a conventional scanning cytospectrophotometer and an automated image analysis system (LEYTAS). Scanning cytospectrophotometry measurements showed that 26% of in situ alveolar macrophages from glucan-treated rats contained more lysozyme than did control cells and that 31% possessed larger areas. Fewer large alveolar macrophages containing increased amounts of lysozyme were detected in lavages of glucan-treated rats. Frequency histograms of lysozyme content and cell size were similar for lavaged and in situ macrophages from control rats. Measurements with LEYTAS confirmed the results. These experiments demonstrate that alveolar macrophages obtained by lavage are representative of their in situ counterparts in normal but not in glucan-treated rats.

Animals↗
PubMed1983

Effect of virulent and less virulent strains of Nocardia asteroides on acid-phosphatase activity in alveolar and peritoneal macrophages maintained in vitro.

Since virulent strains of Nocardia asteroides grow within macrophages, experiments were designed to determine whether this intracellular pathogen affects lysosomal enzyme levels. Peritoneal and alveolar macrophages from mice were infected in vitro with live or killed N asteroides of the virulent strain GUH-2 or with live nocardiae of the less virulent strain 10905, which is killed by macrophages. The activity of acid phosphatase in individual macrophages was quantitated by means of a computer-assisted cytospectrophotometry system. Slide preparations were Gram stained for quantitation of ingested nocardiae in the same macrophages. The level of acid phosphatase activity in alveolar and peritoneal macrophages was decreased by infection with live cells of the virulent strain; the level of activity was inversely proportional to the number of nocardiae ingested. This effect was not seen with killed bacteria of this strain or with live bacteria of the less virulent strain. These results demonstrate a relation between the virulence of N asteroides strains and the extent of reductions in lysosomal acid-phosphatase activity of macrophages.

Acid Phosphatase↗
PubMed1982

Unusual sites of acute osteomyelitis in childhood.

In children osteomyelitis is common in the long hours of femur, tibia and humerus. This study reports 16 children, aged 2-13 years, with osteomyelitis at unusual sites: in the bones of the thoracic cage including three involving the clavicle; in the spine, foot and elsewhere. In one case, multifocal involvement of the vertebral body and the knee occurred. In two large series reported previously, the incidence of osteomyelitis was 1-3% in the clavicle, 3-8% in the calcaneus and less than 1% in the ribs. Four out of 16 cases (two involving clavicles, one rib and one with multifocal sites of the lesion) required open biopsies and histological examination to achieve the final diagnosis of osteomyelitis; in three of these patients the causative agent was not identified on culture. Staphylococcus aureus was the infective organism in 50% of cases where cultures were obtained. In five cases there was no growth on culture and specific search for less common organisms, including mycobacteria tuberculosis (AAFB), proved negative. It is suggested that in such situations diagnostic problems may present as the clinical and radiological findings may not be specific or conclusive. In such cases early biopsy is mandatory.

Acute Disease↗
PubMed1980

Microphotometric differentiation of human T and B cells tagged with monospecific immunoadsorbent beads.

A comparative and statistical study was done of the classification of peripheral blood lymphocytes (PBLs) as T cells, B cells and monocytes by various immunologic procedures and computerized microphotometric analysis. PBLs from 15 healthy male subjects were examined by immunofluorescence, E rosettes, and immunoadsorbent beads (IAB) for T cells and B cells, by phase contrast microscopy and as fixed slide preparations. Cells tagged with IAB for T cells and B cells were fixed, stained with Papanicolaou stain and analyzed. Evaluation of immunologic data shows 50% to 57% T cells, 9.7% to 24.1% B cells, 13.4% to 16.1% monocytes and about 20% unmarked cells. Analysis of T cells shows significant correlations between E and T cell-IAB rosettes, but neither rosetting procedure reveals a positive correlation with immunofluorescence-labelled T cells. Comparison of B cells shows an insignificant correlation between B-cell IAB and immunofluorescence. Results showed that permanent slides of rosetted cells can be made without alteration in relative numbers of rosetted cells. Assessment of immunologically tagged cell samples by image analysis correctly classified 80% to 90% of cells as T and B cells. Evaluation of homogeneity of the T and B cell populations shows the existence of four subsets of B cells and five subsets of T cells.

B-Lymphocytes↗
PubMedDate not supplied

Applications of computerized microscopic image analysis in infectious diseases.

Advances in computerized microscopy have resulted in image analysis systems that rapidly and precisely measure various aspects of cellular morphology and physiology. These systems-composed of a microscope and attached photomultiplier tube or camera, an image processor, and a computer-have been used to measure lysosomal enzymes, pH, and calcium within phagocytes; to detect viral nucleic acids in in situ hybridization preparations; and to quantitate rates of cellular movement. These experiments have shown that (1) the intracellular proliferation of virulent microorganisms is associated with reductions in acid phosphatase, beta-glucuronidase, and lysozyme activity; (2) virulent Toxoplasma gondii, Legionella pneumophila, and Nocardia asteroides inhibit phagosomal acidification; and (3) changes in intracellular calcium movement affect phagocytic function. These methods have also been used to detect the AIDS virus within cultured lymphocytes and to measure cellular chemotaxis and chemokinesis. Further advances in technology should produce improved microscopic image analysis systems with wider applications for the investigation of infectious diseases.

Antibodies↗