Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity.

Coagulation Factor V is an essential component of the prothrombinase complex, which activates the zymogen prothrombin to thrombin. A patient was described who developed a Factor V inhibitor that neutralized the procoagulant activity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we have found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant activity of Factor Va, implying that the neutralization resulted from specific binding to the C2 domain. We have previously demonstrated that deletion of the C2 domain results in loss of procoagulant activity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserine-specific binding of Factor V. Conversely, preincubation of Factor Va with procoagulant phospholipids protected the cofactor from inactivation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant activity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex.

Human cytotoxic T cells specific for autologous melanoma cells: successful generation from lymph node cells in seven consecutive cases.

Human T-cell populations specifically cytotoxic for autologous melanoma cells have been successfully generated from lymph node cells obtained from seven consecutive patients. The lymph node cells were stimulated in vitro with autologous irradiated melanoma cells; stimulation was repeated every 10-15 days at a tumor cell-to-lymphocyte ratio of approximately 1:20. Cytotoxic activity was assessed by a 4-hour 51Cr release assay. Mean lysis of autologous tumor cells was 47% at an effector-to-target cell ratio of 20:1, while mean lyses of the human myeloid leukemia cell line K562, allogeneic melanoma cells, and an osteosarcoma cell were 20%, 13%, and 11%, respectively. There was no lysis of autologous fibroblasts, fresh lymphocytes, or phytohemagglutinin-stimulated blasts. Three grades of specificity developed sequentially. In grade I, lysis of autologous tumor cells exceeded lysis of allogeneic tumor cells but did not exceed lysis of K562 cells. In grade II, lysis of autologous tumor cells exceeded lysis of K562 cells and all allogeneic tumor cells tested. In grade III, potent lysis of autologous tumor cells (greater than 40%) exceeded lysis of K562 cells and of all allogeneic tumor cells tested. All seven lymphocyte populations reached or exceeded grade I. Six reached or exceeded grade II. Two progressed to grade III. The generated cells were T cells, as determined by phenotypic analysis with flow cytometry. CD4+ cells and CD8+ cells accounted for 83%-100% of the cells. CD8+ T cells were separated from CD4+ T cells by panning with OKT8 and OKT4 antibodies. The resulting CD8-enriched and CD4-enriched populations were compared as effectors in cytotoxicity assays. The results suggest that the cell responsible for lysis of autologous tumor cells is CD8+. The methods used in this study have repeatedly resulted in the successful generation of cytotoxic T lymphocytes specifically cytotoxic for autologous melanoma cells; it is suggested that these cells have potential application for adoptive immunotherapy of melanoma.

Mutation of hydrophobic residues in the factor Va C1 and C2 domains blocks membrane-dependent prothrombin activation.

The binding of factor (FVa) to phosphatidylserine (PS) membranes regulates assembly of the prothrombinase complex. Two pairs of solvent-exposed amino acids, Tyr(1956)/Leu(1957) in the C1 domain and Trp(2063)/Trp(2064) in the C2 domain, each make significant contributions to the affinity of FVa for PS membranes, but individually neither pair of amino acids is required for prothrombinase assembly on 25% PS membranes. In this study we characterize a FVa mutant with alanine substitutions in both the C1 and C2 domains: (Y1956,L1957,W2063,W2064)A. We conclude that: (i) prothrombinase assembly on PS membranes requires Trp(2063)/Trp(2064) and/or Tyr(1956)/Leu(1957); (ii) combined mutation of Trp(2063)/Trp(2064) and Tyr(1956)/Leu(1957) results in only a modest 4-fold decrease in the rate of thrombin generation in the absence of membranes; (iii) the present data provide experimental support for the joint participation of the C1 and C2 domains in the binding of FVa to phospholipid membranes as suggested by the recently solved structure for FVai (A1/A3-C1-C2).

Effects of bis(2-ethylhexyl) phthalate on chromosomes of human leukocytes and human fetal lung cells.

Blood from two male and two female donors was exposed at 37degrees for 4 hr to concentrations of 60.0, 6.0, 0.6, and 0.06 mug of a widely used plasticizer, bis (2-ethylhexyl) phthalate, per milliliter of blood. The bis(2-ethylhexyl) phthalate was solubilized with polysorbate 80. Appropriate polysorbate and nonpolysorbate controls also were established. Following the 4 hr of incubation, phytohemagglutinin was added and tissue cultures were established. In addition, human fetal lung cells were exposed in tissue culture to a medium containing 6.0 mug/ml of bis(2-ethylhexyl) phthalate in polysorbate 80 for 5 days. Similar controls also were established for these experiments. Analysis of chromosome preparations from all cultures obtained failed to show any increased evidence of isochromatid and chromatid breaks or gaps or abnormal forms at any studied concentration when compared to the control cultures. In addition, analysis of fetal lung cell preparations for aneuploidy failed to reveal any differences between cells from study and control cultures. This study involved a short-term exposure to bis(2-ethylhexyl) phthalate in various concentrations which did not cause damage in leukocytes or fetal lung cells.

Tuberculosis: the other epidemic.

In 1990, the number of cases of tuberculosis (TB) reported in the United States rose 6%. This is a noticeable change since, in prior years, TB had been declining. The increased incidence of TB is largely related to the HIV epidemic. TB is of particular interest in HIV-related illnesses, since it is preventable and treatable. The presentation of HIV-associated TB is different from "standard TB." Nurses need to be informed about the clinical presentation of the disease, the procedures to diagnose it, the treatments, and their potential side effects.

Melioidosis in imported non-human primates.

In 1969, five cases of melioidosis in three separate outbreaks were diagnosed in nonhuman primates in the United States. In the first outbreak, two stump-tailed macaque monkeys (Macaca arctoides) developed signs of the disease approximately 6 months after purchase. A third animal, a chimpanzee (Pan troglodytes), probably acquired its infection from one of these monkeys. Two other unrelated cases involving a pig-tailed monkey (Macaca nemestrina) and a rhesus monkey (Macaca mulatta) were diagnosed. These monkeys had been imported 3 years and 6 months, respectively, prior to the recognized onset of their disease. These cases represent the first known occurrences of spontaneous melioidosis in nonhuman primates in the United States.

The passive axial compression (PAC) test: a new adjunctive provocative maneuver for the clinical diagnosis of hallucal sesamoiditis.

The authors describe a previously unreported adjunctive passive provocative maneuver that has been found to clinically reproduce the intensity of symptoms in patients diagnosed with disorders of the sesamoids. This test is useful for the initial diagnosis as well as monitoring response to treatment.

CA 125 as a tumour marker in epithelial ovarian cancer.

CA 125, a high molecular weight glycoprotein, was measured in sera from patients with epithelial ovarian cancer, patients with benign gynaecological disease and in patients with non-ovarian adenocarcinomas. High levels (greater than 35 U/ml) were found in 48/50 patients with active ovarian cancer but in only 3/26 patients who had an ovarian cancer previously diagnosed but who were apparently disease free. 6/23 patients with non-ovarian adenocarcinomas as well as 4/18 patients with benign gynaecological disease also had elevated levels. CA 125 levels were higher in serious than non-serous ovarian cancers and tended to increase with increasing stage. In all of 19 patients with ovarian cancer who responded to treatment CA 125 levels fell while 17/20 with progressive disease showed a rise. In 7/8 patients, serial determination of CA 125 showed a rise before the clinical detection of recurrence, the median lead-time being 3.5 months. We conclude that CA 125 is an excellent marker in the management of patients with epithelial ovarian cancers.

Bacterial, archaeal and eukaryotic diversity of smooth and pustular microbial mat communities in the hypersaline lagoon of Shark Bay.

The bacterial, archaeal and eukaryotic populations of nonlithifying mats with pustular and smooth morphology from Hamelin Pool, Shark Bay were characterised using small subunit rRNA gene analysis and microbial isolation. A highly diverse bacterial population was detected for each mat, with 16S rDNA clones related to Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonas, Planctomycetes, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia and candidate division TM6 present in each mat. Spirochaetes were detected in the smooth mat only, whereas candidate division OP11 was only detected in the pustular mat. Targeting populations with specific primers revealed additional cyanobacterial diversity. The archaeal population of the pustular mat was comprised purely of Halobacteriales, whereas the smooth mat contained 16S rDNA clones from the Halobacteriales, two groups of Euryarchaea with no close characterised matches, and the Thaumarchaea. Nematodes and fungi were present in each mat type, with diatom 18S rDNA clones only obtained from the smooth mat, and tardigrade and microalgae clones only retrieved from the pustular mat. Cultured isolates belonged to the Firmicutes, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Halobacteriales. The mat populations were significantly more diverse than those previously reported for Hamelin Pool stromatolites, suggesting specific microbial populations may be associated with the nonlithifying and lithifying microbial communities of Hamelin Pool.

Analysis of intergenic spacer region length polymorphisms to investigate the halophilic archaeal diversity of stromatolites and microbial mats.

Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel intergenic spacer profiles. ARISA-PCR, performed directly on extracted DNA from different sample sites, provided significant insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in this environment.

Experiences with anaerobes.

Anaerobiology has long been an underdeveloped entity in the routine clinical microbiology laboratory. The difficulties and successes in setting up this specialist department in such a laboratory are outlined. It is concluded that specimen quality, technical expertise, taxonomy and identification remain the areas which need most attention.

In vitro recordings from area postrema neurons demonstrate responsiveness to adrenomedullin.

Adrenomedullin (ADM) is a recently discovered 52-amino acid peptide that exerts potent vasodilatory effects in the periphery and influences the control of body fluid balance when injected centrally. In this study extracellular single-unit recordings were obtained from 94 AP neurons in rat brain slices. Bath application of ADM (10(-7) M) excited 47% (32 of 68) of cells tested, and these effects were found to be dose dependent from 10(-7) to 10(-9) M. Excitation was maintained during synaptic blockade in a low-Ca2+ artificial cerebrospinal fluid solution, demonstrating direct actions of ADM on these neurons. The remaining cells were either unaffected (n = 25) or inhibited (n = 11) by ADM. ADM (10(-7) M) also influenced the spontaneous activity of 9 (7 inhibited, 2 excited) of 16 neurons located in the nucleus tractus solitarii (NTS). However, these effects could be eliminated during synaptic blockade, suggesting indirect actions of the peptide on NTS neurons. These data demonstrate that a specific population of CNS neurons within the AP are directly influenced by ADM and suggest that ADM may exert its effects on the central control of fluid balance through direct actions at this circumventricular organ.

Chemical stability of prostacyclin (PGI2) in aqueous solutions.

The rate constant for the hydrolysis of prostacyclin (PGI2) to 6-keto-PGF1alpha was measured by monitoring the UV spectral change, over a pH range 6 to 10 at 25 degrees C and the total ionic strength of 0.5 M. The first-order rate constant (kdegreesobs) extrapolated to zero buffer concentration follows an expression, kdegreesobs = kH+ (H+), where kH+ is a second-order rate constant for the specific acid catalyzed hydrolysis. The value of kH+ obtained (3.71 x 10(4) sec-1 M-1) Is estimated approximately 700-fold greater than a kH+ value expected from the hydrolysis of other vinyl ethers. Such an unusually high reactivity of PGI2 even for a vinyl ether is attributed to a possible ring strain release that would occur upon the rate controlling protonation of C5. A Brønsted slope (alpha) of 0.71 was obtained for the acid (including H3O+) catalytic constants, from which a pH independent first-order rate constant for the spontaneous hydrolysis (catalyzed by H2O as a general acid) was estimated to be 1.3 x 10(-6) sec-1. An apparent activation energy (Ea) of 11.85 Kcal/mole was obtained for the hydrolysis at pH 7.48, from which a half-life of PGI2 at 4 degrees C was estimated to be approximately 14.5 min. when the total phosphate concentration is 0.165 M (cf. 3.5 min. at 25 degrees C).

A study of the mechanism of transport of benzylpenicillin in the rat submaxillary gland.

In vitro studies performed on slices of rat submaxillary gland to evaluate uptake and efflux of 14C-benzylpenicillin (10(-6)M, 10(-5)M) showed that uptake of 14C-benzylpenicillin was not significantly altered by aeration with N2, addition of 10(-5)M CN- or 10(-3) M probenecid, or substitution of K2SO4 in place of NaCl in the KRT buffer to produce a depolarizing solution. Lowering the extracellular pH (pHe) resulted in increases rates of uptake and efflux, whereas increasing the pHe resulted in decreased rates. The in vivo uptake of penicillin into whole glands excised from rates treated in vivo was very similar to that seen in the slice preparation. However, addition of probenecid to salivary gland slices in vitro decreased the rate of effux. These data suggest that the movement of penicillin in the rat salivary gland may occur by a two-step mechanism: (1) passive exchange of penicillin into the gland dependent on the degree of dissociation of penicillin, and (2) active excretion of the drug from the gland by a transport mechanism similar to that of the kidney densitive to probenecid.

Adrenomedullin microinjection into the area postrema increases blood pressure.

Adrenomedullin (ADM) circulates in the blood at concentrations comparable to other vasoactive peptides with established roles in cardiovascular regulation. Intravenously administered ADM produces a clear hypotensive effect, whereas intracerebroventricular microinjections result in increases in blood pressure (BP). Recently, we demonstrated that ADM influences neurons of the area postrema (AP), a central nervous system site implicated in cardiovascular control. However, to address directly the physiological significance of the actions of ADM at the AP, an in vivo microinjection study was undertaken. ADM, at two concentrations (1 and 10 microM), in volumes of 50, 100, and 200 nl, was microinjected into the AP or NTS of 21 urethan-anesthetized male Sprague-Dawley rats. Microinjection of 10 microM ADM (100 nl) resulted in significant transient (2-5 min) increases in BP [120 s area under the curve (AUC): 684.3 +/- 268.6 mmHg/s (P < 0.05)], and heart rate (HR) [AUC: 12.5 +/- 4.5 beats/min (P < 0.05)]. The lower concentration of ADM (1 microM) had no effect on either BP (179.1 +/- 143.6 mmHg/s) or HR (0.8 +/- 2.6 beats/min). ADM was also microinjected into the immediately adjacent nucleus of the solitary tract, where it was found to be without effect on either BP or HR. This study demonstrates, for the first time, a physiological role for ADM acting at a specific brain site, the AP, to produce significant cardiovascular responses.

A study of the mechanism of transport of diphenylhydantoin in the rat submaxillary gland in vitro.

In vitro studies performed on rat submaxillary gland slices with diphenylhydantoin (6 X 10(-6) M) showed that variations of extracellular pH (pHe) had no significant effects on uptake and only slight effects on efflux (lowering the pHe slightly decreased efflux rates, whereas increasing pHe slightly increased efflux rates). Increasing diphenylhydantoin concentration significantly decreased uptake and slightly increased the rate of efflux. Uptake of 14C-diphenylhydantoin was significantly decreased when compared to control conditions (pH 7.40, 37 degrees C, 100% O2 aeration, 6 X 10(-6) M) by the following alterations: aeration of the incubation medium with N2 instead of O2, decrease of bath temperature from 37 degrees C to 5 degrees C and addition of the following metabolic inhibitors: iodoacetic acid (10(-3)M), 2,4-dinitrophenol (10(-3)M) and cyanide (10(-3)M). Probenecid (10(-3)M) had no significant effect on diphenylhydantoin uptake when compared to control values. No evidence of salivary biotransformation of diphenylhydantoin was seen in the in vitro system using thin-layer chromatography. These in vitro data suggest an active transport process that is concentration-dependent with a possible saturable binding site on the membrane or in the interior of the cell.

MD@: a physician-friendly decision analysis tool.

The cost-effective use of medical resources is increasingly important in justifying strategies for medical diagnosis and management. Although some software is available to help with decision analysis, it can be difficult to use these tools for medical applications. We have developed a prototype package for modeling various medical decision strategies, which can be used with a Macintosh or Windows-based personal computer. The system is graphically based, intuitive, and user-friendly. The user constructs decision trees for comparing alternative strategies for diagnosis and management. Selecting blocks from a library, the user sets mean values for variables such as prevalence, sensitivity, specificity, cost, morbidity, and mortality. The system then generates the probabilities of various pathways, using Bayesian analysis, without requiring the user to enter equations. It displays the best strategy in terms of a particular criterion and, when appropriate, performs sensitivity analysis.