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Biomedical subjects

R Knowles

Publications and source records attributed to R Knowles.

At least 19 recordsLinked to original sources
PubMed1995

Hypoxemia up-regulates interleukin-8 stimulated phagocytosis of polymorphonuclear leukocytes by differential regulation of CD32w and CD35 messenger RNA expression.

BACKGROUND: The purpose of these studies was to investigate the effect of hypoxemia on interleukin-8 (IL-8) regulation of phagocytosis and surface opsonic receptor expression of whole blood polymorphonuclear leukocytes (PMNs). METHODS: Whole blood PMNs were rendered hypoxemic (PvO2 less than 15 mm Hg) or normoxic (PvO2 equals 60 to 80 mm Hg) and incubated with IL-8 (50 ng/ml) before the sequential addition of serum-opsonized fluorescent microspheres (emission wavelength, 560 nm) and fluorescein isothiocyanate-conjugated mouse anti-human CD32w or CD35 antibodies (emission wavelength, 513 nm). Concomitant two color flow cytometric analysis was then performed to measure the mean channel fluorescence (MCF) and the percentage of positive PMNs. Steady-state messenger RNA levels for CD32w and CD35 were quantitated by means of Northern blot analysis by using total RNA extracted from purified PMNs from each study group. RESULTS: Hypoxemia increased IL-8 stimulated PMN phagocytosis (percentage of positive PMNs, 38 +/- 1.5 versus 27 +/- 2.0, MCF, 5491 +/- 182 versus 4060 +/- 121 for hypoxemia plus IL-8 versus normoxia plus IL-8, respectively; p < 0.05). Hypoxemia increased IL-8 stimulated PMN expression of CD32w and CD35 (MCF CD32w, 791 +/- 105 versus 336 +/- 81; MCF CD35, 542 +/- 87 versus 254 +/- 41; p < 0.05). Under normoxic conditions IL-8 decreased messenger RNA levels for both CD32w and CD35, but under hypoxemic conditions IL-8 increased messenger RNA levels only for CD32w. CONCLUSIONS: Hypoxemia directly regulates IL-8 control of PMNs by increasing phagocytosis, receptor expression, and messenger RNA for these receptors. Studies investigating cytokine regulation of PMN function need to take into account oxygen tension when establishing the fundamental effects of cytokines on PMN physiology.

Cytokines↗
PubMed1995

Performance on three-point vernier acuity targets as a function of age.

With certain (but not all) vernier acuity (VeA) stimulus displays observers exhibit no or slight performance decrements with aging. Because three-point VeA test displays are much more easily applied experimentally and clinically, we examined visual performance on this task as a function of age. Ages of groups tested ranged from 20 to 70+ years (some individuals were 90+ years old). Eyes were examined monocularly. We tested three- and two-point sharply defined VeA arrays and a three-point ground-glass-degraded display (simulating a 20/100 nuclear cataract). Gap or feature separations were varied. Results obtained with the three-point display showed little alteration with age. Image degradation resulted in reduction in performance when small gap separations were used between the test points, but only slight decrements for large gap separations were recorded. The finding that performance with certain VeA targets is largely unaffected by age is confirmed and is expanded.

Adult↗
PubMed1994

Organization of actin and microtubules during process formation in tau-expressing Sf9 cells.

Insect Sf9 cells usually elaborate a highly characteristic single process when infected with a baculovirus encoding recombinant human tau. The processes are unbranched, of uniform caliber, and contain bundles of microtubules. Because taxol treatment alone does not induce process outgrowth in these cells, it is believed that tau confers properties on microtubules that permits the conversion of microtubule assembly into the formation of processes. Here we have analyzed the reorganization of both actin filaments and microtubules during process initiation. A zone of organelle exclusion representing the focal reorganization of actin at one pole of the cell anticipated process emergence. A relationship between actin organization and process emergence was also suggested by a shift from single to multiple process formation after treatment with cytochalasin D. The rate of process elongation doubled after cytochalasin treatment of tau-expressing cells. The increase in rate was due to the inhibition of the growth arrest phases which occur in the absence of cytochalasin. In contrast, Sf9 cells treated with cytochalasin after more than 20 h of tau expression were relatively resistant to the drug's effects. We conclude that actin and microtubules are specifically reorganized during tau-induced process outgrowth and that a dynamic relationship between actin filaments and microtubules effects process formation.

Actin Cytoskeleton↗
PubMed1994

Cellular regulation of nitrate uptake in denitrifying Flexibacter canadensis.

Nitrate uptake and its regulation were investigated using an ion-specific nitrate electrode for denitrifying Flexibacter canadensis under anaerobic conditions. Glucose supported a greater rate of nitrate uptake than did glycerol, glutamate, lactose, cellobiose, or ethanol. Nitrate uptake closely approximated Michaelis--Menten kinetics; the estimated Ks(glucose) and apparent Km(nitrate) for nitrate uptake were 21 and 44 microM, respectively. Nitrate disappearance was correlated with nitrite accumulation, and nitrate had an inhibitory effect on nitrite reduction. Oxygen inhibition of nitrate uptake increased as the percent air saturation increased, and reversed readily as the percent air saturation decreased. The minimal air saturation showing inhibition of nitrate uptake was about 2-4%. Azide and cyanide completely inhibited nitrate uptake. No nitrate uptake was observed in cells grown in the presence of 1 or 5 mM tungstate (no added molybdate). When molybdate (100-200 microM) was present in the medium, nitrate uptake was exhibited by organisms grown with 1 mM, but not with 5 mM, tungstate, indicating that nitrate uptake was dependent on the presence of an active nitrate reductase, and that competition between tungsten and molybdenum occurred during the formation of nitrate reductase. Nitrite production from nitrate by whole cells but not cell-free extracts was inhibited by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, indicating that nitrate and (or) nitrite transport depended upon the electrochemical proton gradient.

Bacteroidetes↗
PubMed1993

Chlorpromazine inhibits both the constitutive nitric oxide synthase and the induction of nitric oxide synthase after LPS challenge.

The effects of chlorpromazine on either the activity of mouse brain nitric oxide synthase or the induction of lung nitric oxide synthase in mice and rats were studied. Chlorpromazine inhibited the nitric oxide synthase activity in mouse brain cytosol. This effect could be reversed by adding an excess of calmodulin. In addition, chlorpromazine was able to inhibit the induction of lung nitric oxide synthase, in both species, after LPS administration. Furthermore, chlorpromazine also inhibited arginase activity in mouse lung cytosol.

Amino Acid Oxidoreductases↗
PubMed1992

Effect of a single amino acid mutation on the activating and immunosuppressive properties of a "humanized" OKT3 monoclonal antibody.

The binding specificity of the murine OKT3 has been transferred into a human antibody framework to reduce its immunogenicity. This "humanized" anti-CD3 mAb (gOKT3-5) was previously shown to retain, in vitro, all the properties of native OKT3, including T cell activation, which has been correlated, in vivo, with the severe side effects observed in transplant recipients after the first administration of the mAb. T cell activation is thought to be triggered by the cross-linking mediated by the antibodies between T cells and Fc receptor-bearing cells. In this study, we introduced a single amino acid mutation from a leucine to a glutamic acid at position 235 in the Fc receptor binding segment of the gOKT3-5 mAb to produce Glu-235 mAb. This mutation generated a 100-fold decrease in the affinity of the antibody for the Fc receptor on U937 cells, without affecting Ag binding. In parallel, we observed a marked reduction in the T cell activation triggered by the mAb (proliferation, cell surface expression of early activation markers including Leu 23 and IL-2R, and release of TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF). In contrast, the mutated mAb retained suppressive properties similar to the gOKT3-5 mAb, as assessed by significant modulation of the T cell receptor complex and suppression of Ag-specific CTL activity. We conclude that this anti-CD3 mAb bearing a single amino acid mutation in its Fc portion retains important immunosuppressive properties, while exhibiting significantly less T cell activation than OKT3 in vitro. This drug might achieve potent immunosuppression while minimizing acute toxicity in vivo and thus be useful in transplantation as well as in autoimmune diseases.

Amino Acid Sequence↗
PubMed1992

Monitoring body fluid distribution in microgravity using impedance tomography (APT (applied potential tomography)).

For an astronaut, the excitement of going into orbit is accompanied by a shift of 1 to 1.5 l of fluid from the legs into the upper body. Information on the way the redistributed fluid is handled by the body is very useful to space physiologists studying the process of adaptation to zero-gravity. Applied potential tomography (APT) can be used to image changes in fluid distribution. To ensure that the technique was capable of measuring fluid shifts induced by changing gravitational forces on the body, a standard Sheffield APT system was used to study several subjects during the eight ESA parabolic flight campaign. The results clearly demonstrated the feasibility of using APT for monitoring fluid redistribution during space flight. A battery-powered, body-worn APT system has now been developed for use in space. The equipment was tested on the eleventh parabolic flight campaign. The data collected with the miniaturised system was comparable to that obtained in the earlier experiment. Ergonomic tests indicated that the equipment is no more difficult to operate and maintain under weightless conditions than on earth. The system is undergoing space qualification tests in Munich. If no problems arise it will be used by German astronauts on missions to MIR and Skylab.

Body Fluids↗
PubMed1991

A high-resolution networked computer system for radiologic instruction of medical students.

A network of 18 Macintosh II-based workstations (Apple Computer Corp, Cupertino, CA) having high-resolution 1,024-line monitors is used for the radiologic instruction of medical students in our institution. Although this is an expensive system, it is time-shared with the Departments of Pathology, Physiology, Neuroscience, Biochemistry, Parasitology, and Microanatomy. The number of workstations available allows the entire class to access the system simultaneously by working in small groups. Because of its high resolution, medical imaging studies and text may be displayed on the same monitor side by side. We have chosen a question-and-answer format (Carousel). Forty-one radiologists in our affiliated medical institutions contributed cases. These cases in question-and-answer form supplement the lectures and film-based tutorials given as part of our mandatory third year radiology clerkship.

Computer Communication Networks↗
PubMed1991

Total parenteral nutrition increases mortality after hemorrhage.

OBJECTIVES: To determine the effect of total parenteral nutrition (TPN) and different enteral feeding formulas on survival and liver function following hemorrhage in rats. DESIGN: Prospective randomized controlled study. SETTING: Laboratory of a large university-affiliated medical school. SUBJECTS: Sixty-seven male Sprague-Dawley rats weighing 350 to 450 g. INTERVENTIONS: Jugular and gastroduodenal feeding catheters were inserted in animals 1 day before hemorrhage, and animals were started on one of six different fluid or nutritional regimens: TPN, iv saline, an enteral amino acid-based formula (AA) (Vivonex-TEN), an enteral peptide-based formula (PEP) (Reabilan-HN), an enteral intact-protein based formula (PRO) (Osmolite-HN), or enteral saline. A catheter was inserted in the tail artery and animals were hemorrhaged 5 mL/kg at baseline and 1 hr later. Animals were returned to their cages and observed for survival. Liver function was determined by measuring circulating bile acid levels at baseline and 24 hr after hemorrhage. MEASUREMENTS AND MAIN RESULTS: Mortality was significantly increased in animals receiving TPN (63%) and AA (24%). Mortality was 13% in animals receiving PRO and 0% in animals receiving PEP and saline. Liver function deteriorated in all animals after hemorrhage except the PEP group. CONCLUSIONS: TPN and AA increased mortality in animals after hemorrhage. PEP was associated with zero mortality and protection of liver function after hemorrhage.

Amino Acids↗
PubMed1991

Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil.

The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction. Most-probable-number estimates of naturally occurring F. canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F. canadensis (Is-11). Our survey suggests that F. canadensis is common in the natural environment.

Bacteriophages↗
PubMed1989

Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers.

Ammonia oxidizers (family Nitrobacteraceae) and methanotrophs (family Methylococcaceae) oxidize CO and CH4 to CO2 and NH4+ to NO2-. However, the relative contributions of the two groups of organisms to the metabolism of CO, CH4, and NH4+ in various environments are not known. In the ammonia oxidizers, ammonia monooxygenase, the enzyme responsible for the conversion of NH4+ to NH2OH, also catalyzes the oxidation of CH4 to CH3OH. Ammonia monooxygenase also mediates the transformation of CH3OH to CO2 and cell carbon, but the pathway by which this is done is not known. At least one species of ammonia oxidizer, Nitrosococcus oceanus, exhibits a Km for CH4 oxidation similar to that of methanotrophs. However, the highest rate of CH4 oxidation recorded in an ammonia oxidizer is still five times lower than rates in methanotrophs, and ammonia oxidizers are apparently unable to grow on CH4. Methanotrophs oxidize NH4+ to NH2OH via methane monooxygenase and NH4+ to NH2OH via methane monooxygenase and NH2OH to NO2- via an NH2OH oxidase which may resemble the enzyme found in ammonia oxidizers. Maximum rates of NH4+ oxidation are considerably lower than in ammonia oxidizers, and the affinity for NH4+ is generally lower than in ammonia oxidizers. NH4+ does not apparently support growth in methanotrophs. Both ammonia monooxygenase and methane monooxygenase oxidize CO to CO2, but CO cannot support growth in either ammonia oxidizers or methanotrophs. These organisms have affinities for CO which are comparable to those for their growth substrates and often higher than those in carboxydobacteria. The methane monooxygenases of methanotrophs exist in two forms: a soluble form and a particulate form. The soluble form is well characterized and appears unrelated to the particulate. Ammonia monooxygenase and the particulate methane monooxygenase share a number of similarities. Both enzymes contain copper and are membrane bound. They oxidize a variety of inorganic and organic compounds, and their inhibitor profiles are similar. Inhibitors thought to be specific to ammonia oxidizers have been used in environmental studies of nitrification. However, almost all of the numerous compounds found to inhibit ammonia oxidizers also inhibit methanotrophs, and most of the inhibitors act upon the monooxygenases. Many probably exert their effect by chelating copper, which is essential to the proper functioning of some monooxygenases. The lack of inhibitors specific for one or the other of the two groups of bacteria hampers the determination of their relative roles in nature.

Ammonia↗
PubMed1989

Immunophenotypic demonstration of two natural killer surface markers, H25 and H366, on fresh human leukemic cells.

Using a modified alkaline-phosphatase/antialkaline-phosphatase method for phenotyping fresh human leukemias, we could demonstrate peripheral blood and bone marrow-derived blast cells to specifically react with two monoclonal antibodies (MoAbs), H25 and H366, previously shown to recognize natural killer cells, activated T lymphocytes and a proportion of normal hematopoietic precursor cells. MoAbs H25 and H366 were found to identify the majority of leukemic cells in patients presenting with T-ALL, LGL leukemia, pre-B-ALL, CML, and AML, respectively.

Animals↗
PubMed1989

Analysis of the individual and combined reactivities of monoclonal antibodies H25, H366, and MY9 with normal progenitor cells and blast cells from patients with acute myeloblastic leukemia.

Recently we reported that two monoclonal antibodies (MoAbs), H25 and H366, which react with human natural killer cells and monocytes, also react with normal in vitro colony-forming cells including granulocyte-monocyte colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and erythroid colony-forming units and with leukemic blasts in preliminary testing of cells from patients with myeloid leukemias and T cell acute lymphocytic leukemia. In the present studies we examined the reactivities of MoAbs H25, H366, and MY9 (singly or combined) with the total leukemic cell population and the leukemic clonogenic cells (L-CFC) from 28 patients with acute myeloblastic leukemia. Using cytofluorography, we found the extent of expression of antigen H25 comparable to MY9 in the majority of patients, and both were more highly expressed than antigen H366. Incubation with H25 and H366 MoAbs simultaneously did not increase the number of positive cells over that seen when stained with H25 alone; however, the amount of antibody fluorescence intensity (FI) was increased. Leukemic cells simultaneously stained with MoAbs H25, H366, and MY9 displayed the highest number of positive cells and FI. Using magnetic beads coated with sheep anti-mouse IgG for depleting antibody-binding cells, greater than or equal to 90% of L-CFC were depleted by a combination of H25 and H366 MoAbs in 76% of AML cases tested as compared to 41% of the cases with MoAb MY9. Using a MoAb cocktail of H25, H366, and MY9, greater than or equal to 90% of L-CFC were depleted in 94% of cases tested, and greater than or equal to 99% of L-CFC were removed in 76% of the cases. Using the same depletion methods for normal bone marrow cells, a combination of anti-H25 and anti-H366 removed 90%, 98%, and 84% of CFU-GM, BFU-E, and multipotent colony-forming units (CFU-GEM), respectively, whereas the cocktail of H25, H366, and MY9 MoAbs removed 98%, 99.5%, and 97% of CFU-GM, BFU-E, and CFU-GEM, respectively. Incubation of H25 and H366-depleted bone marrow cells for 2 weeks in the presence of irradiated adherent cell layers from long-term marrow cultures generated CFU-GM and some BFU-E, as did H25, H366, and MY9-depleted marrow cells, although to a much lesser extent. Based on the overall data, combinations of H25, H366, and MY9 MoAbs and immunomagnetic beads conceivably might have therapeutic potential for ex vivo elimination of leukemic cells from AML remission marrows prior to autologous transplantation.

Adult↗
PubMed1988

Metabolism of acetylene and acetaldehyde by Rhodococcus rhodochrous.

We studied the ability of a soil bacterium, identified as Rhodococcus rhodochrous, to grow on acetylene and to accumulate acetaldehyde. Its maximum growth rate on acetylene was obtained at about 30 degrees C (mu = 0.11 h-1) and was independent of the concentration of this gas in air from 0.14 to 16% (v/v). During growth, acetylene was quantitatively transformed to acetaldehyde, ethanol, acetate, CO2, and biomass in proportions which varied with culture age and temperature. Growth was completely inhibited by acetaldehyde at a concentration of 10 mM. The inhibitory effect was relieved by addition of acetate. Growth on ethanol up to 140 mM did not result in acetaldehyde accumulation. Acetylene consumption was constitutive with apparent Km and Vmax equal to 250 microM and 800 nmol.min-1.(mg protein)-1, respectively. In resting cell suspensions, acetylene consumption rates decreased more rapidly under air than under nitrogen. The inhibitory effect of acetaldehyde was enhanced in the presence of oxygen. Acetaldehyde accumulation in aerobic resting cell conditions did not exceed 10 mM (440 mg/L), but under anaerobic conditions it attained more than 70 mM (3.08 g/L).

Acetaldehyde↗
PubMed1987

Expression of two natural killer cell antigens, H-25 and H-366, by human immature myeloid cells and by erythroid and granulocytic/monocytic colony-forming units.

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony-forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.

Antibodies, Monoclonal↗
PubMed1987

Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures.

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.

Antigens, Differentiation, T-Lymphocyte↗