CTA Evaluation of Basilar Septations: An Entity Better Characterized as Aberrant Basilar Fenestrations.

BACKGROUND AND PURPOSE: A basilar artery intraluminal septation is an exceedingly rarely reported, presumed congenital abnormality. In our clinical practice, we have occasionally noticed an intraluminal band within the inferior aspect of the basilar artery on CTA. Furthermore, we have noticed, at times, the presence of a punctate calcification associated with this finding. We hypothesized that what previous studies have called "basilar septations" in fact represent miniature and thus aberrant basilar fenestrations. MATERIALS AND METHODS: We retrospectively reviewed CTA studies obtained between January 1, 2017, and August 31, 2019. Identified intraluminal basilar abnormalities were classified as either basilar septations or basilar fenestrations. Association with other posterior circulation abnormalities was documented. RESULTS: A total of 3509 studies were examined. A basilar intraluminal abnormality was evident in 80 patients (2.3%). Of these 80 patients, 59 were classified as having a basilar fenestration (1.7%) and 21 were classified as having basilar septations (0.6%). Associated calcification was evident in 3 of the basilar fenestration cases and 13 of the basilar septation cases. CONCLUSIONS: Basilar septations most likely represent and should be referred to as aberrant basilar fenestrations. They should be interpreted as benign congenital incidental findings and should not be misinterpreted as focal dissections or arterial webs. Important variations in the morphology of aberrant basilar fenestrations exist, including areas of thinning, varying thickness, and nodularity. Therefore, when associated with calcification or nodularity, aberrant basilar fenestrations should not be confused with focal intraluminal thrombi or calcified or noncalcified emboli.

Simulating microbial processes in extraterrestrial, aqueous environments.

Finding evidence of life elsewhere in the Solar System is dependent on understanding biotic processes that could occur within potentially habitable environments. Here, we describe a suite of high-pressure flow-through reactors that have been developed to investigate biotic and abiotic processes within simulated sub-surface martian and icy moon environments.

The identification of sulfide oxidation as a potential metabolism driving primary production on late Noachian Mars.

The transition of the martian climate from the wet Noachian era to the dry Hesperian (4.1-3.0 Gya) likely resulted in saline surface waters that were rich in sulfur species. Terrestrial analogue environments that possess a similar chemistry to these proposed waters can be used to develop an understanding of the diversity of microorganisms that could have persisted on Mars under such conditions. Here, we report on the chemistry and microbial community of the highly reducing sediment of Colour Peak springs, a sulfidic and saline spring system located within the Canadian High Arctic. DNA and cDNA 16S rRNA gene profiling demonstrated that the microbial community was dominated by sulfur oxidising bacteria, suggesting that primary production in the sediment was driven by chemolithoautotrophic sulfur oxidation. It is possible that the sulfur oxidising bacteria also supported the persistence of the additional taxa. Gibbs energy values calculated for the brines, based on the chemistry of Gale crater, suggested that the oxidation of reduced sulfur species was an energetically viable metabolism for life on early Mars.

Draft Genome Sequences of the Nitrate-Dependent Iron-Oxidizing Proteobacteria Acidovorax sp. Strain BoFeN1 and Paracoccus pantotrophus Strain KS1.

The draft genomes of the nitrate-dependent iron-oxidizing bacteria Acidovorax sp. strain BoFeN1 and Paracoccus pantotrophus strain KS1 are presented. These genomes supply supporting data to investigations of the mechanisms underlying this anaerobic form of microbial biogeochemical iron cycling.

Analysis of the platelet-type thrombin receptor in 20 cases of large granular lymphocyte proliferations.

The platelet-type thrombin receptor was expressed by large granular lymphocytes (LGLs) in a variety of proliferative diseases. Twenty patients with LGL proliferative disease were examined, including five T cell clones and a variety of polyclonal proliferations, some secondary to rheumatoid arthritis and Felty's syndrome; 17/20 showed high number of CD3+, CD8+, and CD57+ lymphocytes and 9/20 also had high numbers of CD16+ or CD 56+ positive lymphocytes. The thrombin receptor was present on more than 20% of the LGLs in 13/20 patients. The clonal T cell expansions showed the highest receptor expression with greater than 75% cells positive. Regression analysis of all 20 cases showed striking and highly statistically significant positive Spearman rank correlation between the proportion of thrombin receptor and CD57-positive LGLs (r = 0.56, P = 0.009). A negative correlation with CD56 was also found (r = -0.46, P= 0.043). Dual antibody flow cytometry showed the receptor was more often co-expressed with CD57 (64%) than with CD16 (19%) or CD56 (11%). The expression of the platelet-type thrombin receptor by LGLs of this phenotype raises the possibility of a functional role for thrombin in the pathogenesis of LGL proliferative diseases.

The ex vivo function and expression of function-associated antigens of peripheral blood neutrophils and monocytes.

The availability of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors (rhG-CSF and rhGM-CSF) has prompted many studies of the analysis of antigen expression and function of monocytes and neutrophils from patients receiving these factors as therapeutic agents. Preparatory procedures for leukocytes are known to alter antigen expression and so function. We therefore investigated the use of a novel procedure in which live leukocytes are analyzed by flow cytometry without isolation from blood. The expression levels of CD11b, CD13, CD14, CD16, and CD18 antigens and L-selectin (TQ1 and Leu-8 epitopes) on neutrophils and monocytes from 15 normal individuals were determined and compared with a previously used method in which the leukocytes were fixed and the erythrocytes lysed before analysis. Significant differences for the apparent expression of CD11b, CD18, and L-selectin were observed between the two methods. The reasons for this were investigated. Since the new method allowed analysis of live cells, we also investigated whether modulation of antigen expression could be determined following receptor agonist interaction. This was found to be easily achievable, and we advocate using the new procedure where possible for the ex vivo analysis of function and function-associated antigens on monocytes and neutrophils.

Induction of surface tumor necrosis factor (TNF) expression and possible facilitation of surface TNF release from human monocytic cells by granulocyte-macrophage colony-stimulating factor or gamma interferon in combination with 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can regulate monocyte maturation and activation. Using the human monocytoid cell line U937, we have shown that these agents increase surface tumor necrosis factor (TNF) expression without directly affecting TNF release. GM-CSF and IFN-gamma combined with 1,25(OH)2D3 increased cellular TNF secretion to levels not seen with these agents alone. Ability to express and secrete TNF in part depended on degree of monocytic maturation. The combination of 1,25(OH)2D3 and GM-CSF, however, facilitated lipopolysaccharide (LPS)-mediated release of surface TNF from U937 cells, an effect that was temporally independent of maximal maturation. 1,25(OH)2D3 plus IFN-gamma was less effective than 1,25(OH)2D3 plus GM-CSF at facilitating TNF secretion. We postulate that 1,25(OH)2D3 and GM-CSF are required together to prime a specific mechanism, probably a protease, which cleaves TNF from the surface of monocytic cells. This protease, once primed, can be activated by a secondary stimulus such as LPS.

Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the detection of minimal residual disease (MRD) correlates with outcome in the trial setting. However, MRD assessment does not guide routine clinical management and its assessment remains complex. We incorporated detection of the B cell, tumor-specific antigen CD160 to develop a single-tube, flow cytometry assay (CD160FCA) for CLL MRD to a threshold of 10(-4) to 10(-5). One hundred and eighty-seven patients treated for CLL were enrolled. Utilizing the CD160FCA methodology, there was a high level of comparison between blood and bone marrow (R=0.87, P<0.001). In a validation cohort, CD160FCA and the international standardised approach of the European Research Initiative on CLL group demonstrated high concordance (R=0.91, P<0.01). Patients in complete remission (CR) and CD160FCA negative had longer event-free survival (EFS) (63 vs 16 months, P<0.01) and prolonged time to next treatment (60 vs 15 months, P<0.001) vs MRD positive patients; with a median time to MRD positivity of 36 months. In multivariate analysis, CD160FCA MRD detection was independently predictive of EFS in patients in CR and even predicted EFS in the good-risk cytogenetic subgroup. CD160FCA offers a simple assay for MRD detection in CLL and gives prognostic information across different CLL risk groups.

Differential effect of hypophysectomy on the synthesis of beta-glucuronidase and other androgen-inducible enzymes in mouse kidney.

The levels of several androgen responsive enzymes including beta-glucuronidase, alcohol dehydrogenase, D-amino acid oxidase and arginase, were compared in kidneys of normal and hypophysectomized female mice after treatment with testosterone. While hypophysectomy did not alter the basal level of glucuronidase, the androgen-mediated accumulation of kidney beta-glucuronidase was greatly decreased in hypophysectomized mice. Measurements of the rate of synthesis of glucuronidase showed that after androgen treatment the enzyme was synthesized in kidney of hypophysectomized mice at only 5% the normal rate. Glucuronidase activity in seven other organs was not appreciably affected by treatment with androgens or by hypophysectomy. Unlike the effect of hypophysectomy on kidney glucuronidase, there was no reduction in the accumulation of alcohol dehydrogenase or D-amino acid oxidase in kidney of hypophysectomized mice after androgen treatment. Hypophysectomy caused a large reduction in kidney arginase activity. However, subsequent administration of testosterone restored much of this activity. It is concluded that there are at least two mechanisms by which androgens increase enzyme activity in kidney. The normal increase in activity or rate of synthesis of beta-glucuronidase following androgen administration requires pituitary hormones and/or products of these hormones, while the increase in activity of enzymes like alcohol dehydrogenase and D-amino acid oxidase does not require pituitary hormones.

Comparative expression of major histocompatibility complex (MHC) antigens on CD5+ and CD5- B cells in patients with chronic lymphocytic leukaemia (CLL)

The aim of this study was to investigate the expression of major histocompatibility complex (MHC) antigens on CD5+ and CD5- B cells of 13 patients with chronic lymphocytic leukaemia (CLL). This was carried out using a series of monoclonal antibodies (MAbs) against polymorphic and monomorphic class I and class II antigens, as well as to the transferrin receptor and assessed by flow cytometry and direct and indirect immunofluorescence. The expression of these molecules was assessed as mean fluorescent intensity (MFI). The results showed that cells from all 13 individuals expressed monomorphic class I antigens. The number of cases expressing polymorphic HLA-Bw6, -Bw4, -B7, -B27 and -A2 class I antigens on CD5- B cells was 11 (85%), 6(46%), 2(15%), 1(8%), 3 (23%), respectively, which was consistent with the expected population frequency distributions of these antigens. For each of the class I antigens on CD5+ and CD5- B cells, the ratio of the MFI was greater than 1 in 12 of 13 cases. For the transferrin receptor (CD71), this ratio was also almost always greater than 1. These results indicate that, unlike solid tumours where the loss or abnormal expression of class I and II antigens is a frequent event, the expression of class I antigens in CLL patients seems to be normal. This indicates that the loss of these antigens cannot provide the leukaemic cells with a selective advantage to escape immunological detection.

A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood.

A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for 5 min at 4 degrees C, diluted and analysed in a five-parameter flow cytometer. The three major leucocyte subpopulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining intensity with LDS-751 in the FL3 channel (erythrocytes and platelets stain very weakly), whilst the fluorescence intensity due to bound fluorescein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives, erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitation of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of those leucocyte surface antigens whose expression is not subject to rapid modulation.

Hoechst 33258: a fluorescent nuclear counterstain suitable for double-labelling immunofluorescence.

Counterstaining with the chromosomal dye Hoechst 33258 is a simple procedure that provides an excellent general purpose nuclear counterstain for immunofluorescent work. Benefits from its use include the ready identification and orientation of structures, differentiation of lymphoid and non-lymphoid cells and easy assessment of frequency in specifically labelled cellular subpopulations. It can be used at the same time as FITC and TRITC double-labelling immunofluorescence.

Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes.

This study investigated the effects of commonly used procedures for the isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesion molecules which are known to be increased and decreased respectively by cell activation). Washing of granulocytes or monocytes with Hanks' buffered salt solution after separation by either dextran sedimentation or density gradient centrifugation, increased surface expression of CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient centrifugation (Histopaque). The increase in CD11b expression on granulocytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treated with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004). The expression of L-selectin was reduced on granulocytes that had been isolated by dextran sedimentation followed by hypotonic lysis of contaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect was less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L-selectin on lymphocytes. We conclude that isolation of lymphocytes by density gradient centrifugation and of granulocytes and monocytes by dextran-sedimentation and centrifugation using Histopaque gradients, but avoiding washing and the use of hypotonic erythrocyte lysis, are appropriate techniques for studying the expression and function of adhesion molecules.

Expression of functional antigens on neutrophils. Effects of preparation.

We have studied how different conditions of cell labelling and isolation affect the expression of five functional antigens on neutrophils from healthy subjects. Fluorescein isothiocyanate conjugated (FITC) antisera specific for the C3bi receptor CR3 (CD11b), aminopeptidase N (CD13), the LPS:LPS binding protein receptor (CD14) and the receptors for human IgG (Fc gamma RII CDw32 and Fc gamma RIII CD16) were incubated with (i) unfixed whole blood at 4 degrees C and at room temperature (RT, approximately 20 degrees C), and the leukocytes prepared for analysis using the Coulter Q-Prep system, (ii) leukocytes which had obtained following the removal of erythrocytes from whole blood by dextran sedimentation and which had been washed or left unwashed at RT, and (iii) leukocytes which had been prepared from whole blood that had been formaldehyde fixed immediately following venesection. The amount of fluorescence associated with the cells was determined by flow cytometry. The expression of CD14 was low under all conditions. However the expression of CD11b, CD16 and CDw32 was significantly higher (p less than 0.05) on neutrophils obtained by dextran sedimentation (n = 4) than on cells which had been fixed with formaldehyde ex vivo; the increase in expression was even greater if the cells had been washed. In contrast, the expression of CD13 on formaldehyde fixed cells was higher than on cells which had been labelled at 4 degrees C or at room temperature and was similar to or slightly lower than that on cells obtained by dextran sedimentation. Increasing the time between 10 and 60 min for which the whole blood was incubated with antisera at RT or at 4 degrees C, resulted in progressive increases in the expression of CD11b and CD13. When neutrophils which had been obtained by dextran sedimentation were incubated with unlabelled antibodies to CD16 or CDw32 and FITC labelled antibodies to CD11b there was a marked increase in the expression of CD11b. Altogether these findings indicate that the analysis of functional molecules on neutrophils (which may be rapidly up-regulated during activation) should be performed under clearly defined and controlled conditions. Dual fluorescence studies may, in some circumstances, produce misleading results.

How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures.

For patients undergoing stem cell transplantation after intensive marrow ablative therapy it is important to enumerate the CD34+ stem cells in peripheral blood so that the harvest can be timed in order to maximize the number of cells collected by leucophoresis for subsequent haematopoietic reconstitution. The use of rapid flow cytometric techniques for the determination CD34+ leucocyte numbers has been advocated, although there is no consensus as to the best method. In this study, we have examined the effects of preparation procedures for flow cytometry on the binding of four CD34 antibodies (Immu-133, QBEND-10, HPCA2 and BIRMA-K3) to the three classes of epitopes on leucocytes. Whole blood, bone marrow and leucophoresis samples were analysed either directly after labelling with a vital nuclear dye (LDS-751) and fluorochrome-conjugated antibodies or after additional erythrocyte lysis and leucocyte fixation using four commercially available reagents (Q-Prep, OptiLyse B, OptiLyse C and FACS Lysing Solution). By comparison with the results obtained from viable leucocytes in unmanipulated samples, it was found that the binding of all four antibodies could be affected by lysis and fixation procedures and that the binding of the class I antibody Immu-133 was most markedly decreased. We conclude that CD34+ cells are best analysed using a whole blood procedure in which nucleated cells are identified by their side light scatter and the fluorescence associated with a vital nuclear dye (in this instance LDS-751) and the CD34+ cells are detected with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies.

von Willebrand factor release and P-selectin expression is stimulated by thrombin and trypsin but not IL-1 in cultured human endothelial cells.

The stimulated release of von Willebrand factor (vWF) from endothelial cells by secretagogues such as thrombin is associated with the translocation of Weibel-Palade bodies to the cell membrane and the surface expression of P-selectin (also known as GMP 140, PADGEM and CD 62). P-selectin, which is stored in Weibel-Palade bodies, is a neutrophil and monocyte adhesion molecule important in the initiation of inflammation. We have developed a simple assay for the detection of P-selectin on endothelial cells using indirect immunofluorescence and flow cytometry and have confirmed that this is temporally related to vWF release. The assay has been used to demonstrate that IL-1 does not cause Weibel-Palade body degranulation but that trypsin does. This has implications for the use of passaged endothelial cells in the study of vWF release and the assay has numerous possible applications in study of mechanisms of stimulated vWF release.

The anti-leukaemic effects and the mechanism of sodium selenite.

The anti-proliferative effects of selenium were studied both in vivo and in vitro. At a selenium concentration of 0.6 micrograms/ml, cells from patients with ALL-L1, L2 and AML-M1, M3 and M5 were more sensitive than cells from patients with CML. Cells from patients with AML-M2, CLL and leukaemic lymphoma were least sensitive. Normal bone marrow or peripheral blood cells were not sensitive to selenium at this concentration. In the mouse leukaemia models (L797, L615, L7712), the sensitivity of leukaemic cells were: L797 (93% cytotoxicity) greater than L615 (49.7% cytotoxicity) greater than L7712 (4.4% cytotoxicity). Sodium selenite injected i.p. increased the longevity of L797-inoculated mice. Administration of 40 micrograms selenium daily for 7 days resulted in a significant increase in the longevity of mice inoculated with 10(5) L797 cells. However, no remarkable increase of the longevity was observed in either L615- or L7712-inoculated mice after treatment with sodium selenite for 7 days. Treatment of the HL-60 leukaemic cell line with selenium caused a dose- and time-related decrease in DNA, RNA and protein syntheses as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. The inhibitory effect of selenium on DNA synthesis was reversed when selenium was removed from the medium, demonstrating that selenium-induced inhibition of DNA synthesis was due to interference with DNA biosynthesis rather than DNA template damage. These results suggest that the anti-leukaemic effect of sodium selenite is associated with inhibition of DNA replication, transcription and translation.

Gamma interferon augments functional and phenotypic characteristics of vitamin D3-induced monocytoid differentiation in the U937 human leukaemic cell line.

We studied the effect of gamma interferon on 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced differentiation of the human leukaemic cell line U937. Both phenotypic and functional aspects of 1,25(OH)2D3-induced differentiation were significantly augmented by gamma interferon (IFN). Gamma interferon had little effect alone but increased butyrate esterase staining and expression of CD14 antigen and the 40 kD Fc receptor (FcRII) in response to 1,25(OH)2D3. Ability to phagocytose IgG-opsonized bacteria, and superoxide burst in response to both IgG-opsonized bacteria and phorbol ester, was greater after incubation with both IFN and 1,25(OH)2D3 than with either agent alone. The degree of functional activation of cells showed a positive correlation with FcRII expression. In addition, IgG-induced generation of superoxide by differentiated cells was considerably reduced by pre-incubation with the anti-Fc receptor antibody IV3. We conclude that gamma interferon augments 1,25(OH)2D3-induced differentiation and functional activation of the U937 cell line. Increased functional activation may, in part, be due to up-regulation of surface FcR11.

Involvement of antioxidant enzymes in multiple-drug resistance in a human T-lymphoblastic leukemia-cell line which over-expresses p-glycoprotein.

We have compared the specific activities of Cu/Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in a vinblastine sensitive human T-lymphoblastic cell line (CCRF-CEM) and its multiple drug resistant (MDR) counterpart cell line (CEM/VLB100), which over-expresses P-glycoprotein (PGP). We have found that the specific activity Cu/Zn SOD was consistently 38% increased in CEM/VLB100 cells compared with CCRF-CEM cells. In contrast, the activities of CAT and GSH-Px were similar in the two cell lines. These results suggest that MDR in CEM/VLB100 is a complicated phenotype which not only involves a PGP mechanism, but also a SOD protection mechanism against drug-mediated O2.- cytotoxicity.