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J T Merrill

Publications and source records attributed to J T Merrill.

31 records · Page 2Linked to original sources
PubMed1996

The antiphospholipid syndrome and SLE: is there a clue in the link between complement and coagulation?

The heterogenous immunoglobulins known as antiphospholipid antibodies (APLA) or lupus "anticoagulants" (LA) are prevalent in lupus patients and have been implicated in life-threatening thromboembolic events. Unfortunately, observing the presence of these antibodies in an individual does not predict the likelihood of an event nor does it predict when it may occur. A pathogenic role for these antibodies is supported by the observation that high titers and IgG isotype confer an increased risk of thromboembolism. Additionally, numerous reports indicate that isolated patient antibodies interfere with various elements of the coagulation cascade. Nevertheless, attempts to correlate specific antibody characteristics with the future likelihood of a hypercoagulable event in an individual patient have been unsuccessful to date. Given such uncertainty combined with the potential complications of anticoagulant medications, patients are generally not treated until significant morbidity has occurred. Finally, because of the apparent high rate of reoccurrence most patients must remain on anticoagulant therapy indefinitely, regardless of need.

Antiphospholipid Syndrome↗
PubMed1995

Selection of a gene for apolipoprotein A1 using autoantibodies from a patient with systemic lupus erythematosus.

OBJECTIVE: To investigate immunoreactivity of systemic lupus erythematosus (SLE) sera with apolipoprotein A1, (Apo A1), the major lipid-binding protein of high-density lipoprotein (HDL). METHODS: Since early attempts to identify Apo A1 autoantibodies using standard enzyme-linked immunosorbent assay (ELISA) and immunoblotting techniques had been unsuccessful, a mouse complementary DNA lambda phage expression library was screened. RESULTS: A selected clone (MA1) was found to have 82% DNA sequence homology to a segment of human Apo A1. Since there were nonconservative substitutions in the MA1 protein and lack of a complete sequence, it was possible that the SLE patient's antibodies were binding MA1 epitopes that were shared by the complete human protein but had not been conformationally accessible using the earlier techniques. Thus, gamma-irradiated ELISA plates were used as an alternative antigen-binding surface for intact human Apo A1, and high-titer anti-human Apo A1 autoantibodies were then identified in the sera of 5 more SLE patients. CONCLUSION: These findings show that Apo A1 is immunogenic. Apo A1 antibodies may play a role in the decreased HDL levels and Apo A1:Apo B ratios previously reported to occur in subgroups of SLE patients.

Amino Acid Sequence↗
PubMed1994

Dynamic state of beta 2 integrin phosphorylation: regulation of neutrophil aggregation involves a phosphatase-dependent pathway.

The role of phosphorylation and dephosphorylation events in homotypic neutrophil aggregation mediated by CD11b/CD18 molecules was investigated using okadaic acid, an inhibitor of serine and threonine phosphatases. In the absence of exogenous stimuli the addition of okadaic acid to neutrophils resulted in a dose-dependent increase in phosphorylation of the CD18 beta chain that was further augmented by PMA but unaffected by FMLP. Phosphorylation induced by okadaic acid was reversed by staurosporine and minimally decreased by the less selective PKA/PKC inhibitors, H-7 and H-8. This suggests the existence of constitutive phosphatase and kinase activity emphasizing the dynamic state of phosphorylation and dephosphorylation of the beta 2 integrins. Unlike PMA, okadaic acid did not promote homotypic neutrophil aggregation. Furthermore, both the PMA-induced pathway of irreversible aggregation, blocked by staurosporine, as well as the FMLP-induced pathway of reversible aggregation, augmented by staurosporine, were inhibited by okadaic acid in a dose- and time-dependent manner. These results provide evidence that a phosphatase-dependent step is involved in each of these two distinct pathways that regulate neutrophil aggregation mediated by beta 2 integrin activation.

Adult↗
PubMed1991

Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18).

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.

Alkaloids↗
PubMed1990

Two pathways of CD11b/CD18-mediated neutrophil aggregation with different involvement of protein kinase C-dependent phosphorylation.

The characteristics of homotypic neutrophil aggregation, mediated by the adhesion molecule CD11b/CD18, differ according to whether activation takes place via intracellular protein kinase C(PKC) inducers or chemoattractants. In response to diacylglycerol (DAG) analogues such as PMA and 1,2-dioctanoyl-sn-glycerol, a prolonged cellular aggregation occurs that is associated with intense phosphorylation of the CD18 beta-chain. In response to the chemoattractant FMLP, a more transient aggregation event results that is associated with minimal beta-chain phosphorylation. By using the PKC inhibitor staurosporine, we now show that these differences are likely to reflect two different pathways of activation. Both aggregation and phosphorylation induced by DAG analogues are completely abolished by staurosporine in a parallel dose-dependent manner. Conversely, FMLP-induced aggregation is enhanced and prolonged by staurosporine whereas the associated minimal phosphorylation event is further diminished by staurosporine. Accordingly, activation of neutrophil aggregation by DAG analogues is associated with and presumably due to phosphorylation of the CD18 beta-chain. This intense phosphorylation occurs via a staurosporine-sensitive kinase such as PKC. FMLP, on the other hand, appears to activate CD11b/CD18 by a distinct mechanism. This latter mechanism does not seem to be dependent on what may be a minor PKC-induced phosphorylation of the beta-chain, and indeed is enhanced by inhibition of PKC. Of note, staurosporine was also found to cause selective release of specific granules with concomitant increase in surface display of CD11b/CD18. These data further support previous observations that up-regulation of this adhesive molecule is not the primary event in the induction of cellular adhesiveness.

Alkaloids↗
PubMed1982

Multi-parameter, computer controlled operation of a FACS II cell sorter.

The ease of operation and versatility of a FACS II cell sorter were improved by replacing the original pulse height analyzer (PHA) with a computer-based dual parameter PHA and a single channel analyzer (SCA). An ND620 LSI-11 based PHA is now used to generate the sorting decisions. Sorting windows are created by intensifying one or more regions of any shape on the PHA single or dual parameter histograms. For three parameter operation, the third parameter SCA contols the dual parameter information sent to the analyzer. The SCA can also trigger an abort circuit during the PHA 15 microsecond analyzing dead time, improving the purity of the sorted fraction. The electronics that interfaces the new PHA to the sorter replaces all of the original FACS II analog signal processing circuitry with an interchangeable circuit board. The principles involved should apply to any cell sorter.

Cell Separation↗
PubMed1981

Evaluation of selected aerosol-control measures on flow sorters.

Flow sorters produce microdroplets as part of their normal operation, and if these microdroplets escape into the room, they are potentially hazardous. We have tested several aerosol-control measures on a commercial flow sorter. To accomplish this, T-4 phages were introduced into the sorter's liquid jet through the sample injection tube, and culture plates containing lawns of T-4-sensitive Escherichia coli bacteria were exposed around the sorter for each operational configuration. After the exposure, the plates were incubated and then scored for plaques. A single phage-containing microdroplet landing on a plate was sufficient to cause a plaque of lysed bacteria to form. The number of plaques was thus an indicator of how much aerosol was released for each configuration. Aerosols were controlled most effectively by catching the central, undeflected stream in a vacuum-exhausted tube; this technique, coupled with the manufacturer's vacuum-exhaustion of the air around the sorting location, produced no plaques. Several failure modes were tested, including having the central stream hit the outside of the catch-tube or deflection plates, and loss the manufacturer's vacuum-exhaustion system. Many flow sorters allow the underflected stream to splash into a flask or beaker, an approach that produces the most plaques if instrument-failure modes are excluded. The simple addition of a catch-tube removed this major contributor to the aerosol production.

Accident Prevention↗
PubMed1979

Investigations in high-precision sorting.

We have investigated the accuracy with which droplets containing cells can be sorted individually onto known and thus relocatable positions. The presence and random arrival of cells and particles in the sorter jet disturbs the orderly production and deflection of droplets, causing a dispersion of sorted droplet trajectories. The magnitude of this dispersion is a function of the phase relationship between the arrival of a cell at the end of the jet and the droplet formation. Using a modified Becton Dickinson Fluorescence-Activated Cell Sorter, we selected for sorting only those droplets that formed with a cell near the center of the droplet. We used this technique to sort Lewis lung tumor cells. The dispersion of droplet positions was reduced from over 3% to about 1% of an average deflection of typically 15 mm for a nozzle with a 50-micron diameter orifice. Sorting onto a surface such as magnetic tape or a microscope slide introduces another uncertainty in position because the cell may be located anywhere within the wetted radius of the droplet on the slide. Sorting onto less-wettable surfaces reduces the wetted radius and thus the variation in cell position.

Animals↗
PubMed1979

Slit-scan flow cytometry of mammalian chromosomes.

A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded values represents the distribution of fluorescence along the chromosome and is stored in the memory of a waveform recorder. The total fluorescence of each chromosome is also measured and recorded. Preliminary studies show that doublets of 1.83 micrometers diameter microspheres flow with their long axes parallel to the direction of flow and that the two microspheres are resolved in the slit-scan profile. Ethidium bromide stained Muntjac and Chinese hamster chromosomes have also been slit-scanned. Centromeres were resolved in many of the Nos. 1 and 2 Chinese hamster chromosomes and the Nos. 1 and X + 3 Muntjac chromosomes.

Animals↗
PubMedDate not supplied

Cytomorphology of gynecologic specimens analyzed and sorted by two-parameter flow cytometry.

The purpose of this study was to define the fluorescence and light scatter properties of the different cell types encountered in gynecologic specimens. Samples stained with Chromomycins A3 were analyzed on a laser-activated electronic cell sorter, and two-parameter histograms of the cell populations were generated from measurements of nuclear fluorescence and small angle light scatter. Cell populations with defined scatter and fluorescence ranges were sorted, stained by a modified Papanicolaou technique, and evaluated using conventional cytomorphologic criteria. We focused on determining which regions of the histogram corresponded with abnormal cells and cells defining specimen adequacy (columnar and metaplastic cells). Sorted cells corresponding with the main fluorescence region are single, benign, mononuclear cells. Relative proportions of the different types of cells in this region vary with light scatter. Leukocytes predominate at low scatter, mature squamous cells predominate at high scatter, and metaplastic and endocervical cells occur throughout this range. Cells sorted with higher values of nuclear fluorescence include dysplastic and malignant cells as well as benign cells occurring as groups, multinucleates, or with adherent leukocytes or bacteria. This study demonstrates that cytomorphology is a useful method for monitoring and interpreting flow cytometry data.

Chromomycin A3↗