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Biomedical subjects

P Han

Publications and source records attributed to P Han.

At least 19 recordsLinked to original sources
PubMed2002

Immune escape mechanisms of childhood ALL and a potential countering role for DC-like leukemia cells.

BACKGROUND: Pre-B ALL cells generally elicit a weak immune host response, due to poor expression of co-stimulatory molecules and/or suppression of immune function. A possible way to enhance immunogenicity of pre-B ALL cells is to convert them to DC-like cells. METHODS: To study the effect of ALL cells on T-cell function, ALL cells were incubated with T adult cells activated by OKT3 MAb. Liquid culture of de novo pre-B ALL cells for 7 days, in a medium containing IL-1alpha, IL-3, IL-7, Flt 3 ligand (L) and tumor-necrosis factor alpha (TNF-alpha) produced DC-like cells. These were evaluated for morphology, viability, phenotype, as measured by flow cytometry, and function, including MLR. RESULTS: Pre-B ALL cell-lines NALM-6, BALM and de novo pre-B ALL cells failed to stimulate T cells, but suppressed stimulated T cells. The DC-like cells displayed characteristic features of DCs: filiform cytoplasmic projections, and phenotypic expression of co-stimulatory molecules CD80/86, MHC Class I and II molecules, CD83 and CD1a. Genetic monoclonality study confirmed their leukemic origin. In a 5-day MLR culture, the DC-like cells potently activated allogeneic adult and cord CD4+ and CD8+ T cells. Furthermore, both CD4+ and CD8+ T cells were primed towards a Type I. No such effect was seen with unmanipulated de novo pre-B ALL cells. DISCUSSION: DC-like cells can be generated from childhood pre-B ALL cells and are potent stimulators of adult and naïve cord CD8+ T cells via CD4+ cells. These cells may form part of an immunotherapy strategy to overcome tolerance to ALL cells.

B-Lymphocytes↗
PubMed2002

Enhanced expansion and maturation of megakaryocytic progenitors by fibronectin.

BACKGROUND: Megakaryopoiesis occurs as a result of a complex interaction between hemopoietic cells, stromal cells, hemopoietic growth factors and extracellular matrix (ECM). Megakaryocyte growth and development factor (MGDF) or thrombopoietin is the main growth factor that induces megakaryocyte commitment. The role of adhesion proteins in the generation of megakaryocytic progenitors has not been well studied. METHODS: We isolated CD34(+) cells from umbilical cord (UC) blood and cultured them in serum-free medium containing IL-3, IL-6, SCF and MGDF, with or without fibronectin. Cultures were sampled at Days 4 and 8 for cell count, immunophenotyping by flow cytometry, and megakaryocyte colony-forming units (CFU-Mk) assay. RESULTS: Fibronectin in synergy with MGDF increased both total and CD34(+) cell count. Immunophenotyping of the CD34(+) and CD34(-) cells showed that the percentage expression of CD61 and CD41a on CD34(-) cells was increased by fibronectin compared with CD34(+) cells by Day 8 of culture. The CFU-Mk assay confirmed that fibronectin increased generation of megakaryocytic progenitors by 2.4-fold by Day 8 of culture, but the absolute number was less than that of Day 4 culture. The cells in the CFU-Mk colonies derived from culture containing fibronectin were larger than those from cytokine-only culture. CONCLUSION: It was concluded that the addition of fibronectin to the culture system enhanced MGDF, induced ex vivo expansion of megakaryocytic progenitors from CD34(+) cells, as well as increasing their maturation towards later stages of megakaryocyte differentiation.

Antibodies, Monoclonal↗
PubMed2001

The transmembrane domain of the Alzheimer's beta-secretase (BACE1) determines its late Golgi localization and access to beta -amyloid precursor protein (APP) substrate.

Release of Abeta peptides from beta-amyloid precursor protein (APP) requires sequential cleavage by two endopeptidases, beta- and gamma-secretases. beta-Secretase was recently identified as a novel membrane-bound aspartyl protease, named BACE1, Asp2, or memapsin 2. Employing confocal microscopy and subcellular fractionation, we have found that BACE1 is largely situated in the distal Golgi membrane with a minor presence in the endoplasmic reticulum, endosomes, and plasma membrane in human neuroblastoma SHEP cells and in mouse Neuro-2a cell lines expressing either endogenous mouse BACE1 or additional exogenous human BACE1. The major cellular beta-secretase activity is located in the late Golgi apparatus, consistent with its cellular localization. Furthermore, we demonstrate that the single transmembrane domain of BACE1 alone determines the retention of BACE1 to the Golgi compartments, through examination of recombinant proteins of various BACE1 fragments fused to a reporter green fluorescence protein. In addition, we show that the transmembrane domain of BACE1 is required for the access of BACE1 enzymatic activity to the cellular APP substrate and hence for the optimal generation of the C-terminal fragment of APP (CTF99). The results suggest a molecular and cell biological mechanism for the regulation of beta-secretase activity in vivo.

Alzheimer Disease↗
PubMed2001

Tuned-aperture computed tomography for detection of occlusal caries.

OBJECTIVES: To evaluate the use of tuned-aperture computed tomography (TACT) for the detection of primary occlusal caries. MATERIALS AND METHODS: The material comprised 76 extracted posterior teeth, 51 with caries. Radiographs were recorded both with the Sens-A-Ray (Regam Medical Systems Int. AB, Sundsvall, Sweden) digital radiographic system and with Kodak EktaSpeed Plus film (Eastman-Kodak Co., Rochester NY, USA) employing an Orthopantomograph OP 100 (Instrumentarium Imaging, Tuusula, Finland) and a Prostyle Intra (Planmeca, Helsinki, Finland) dental unit respectively. The Sens-A-Ray radiographs were used to construct TACT slices and TACT iterative slices. The teeth were subsequently sectioned in 300 microm thick slices. Microradiographs were exposed and analysed with respect to the true presence of lesions. Seven observers subjectively evaluated the radiographs and ROC analyses performed. Conventional radiographs were compared with TACT images by means of the area under the ROC curves, Az. Paired t-test was used to compare Az values. Interobserver agreement was evaluated using the Kendall coefficient and the Friedman Anova. RESULTS: TACT radiographs were significantly better than conventional radiographs for diagnosing all types of occlusal caries combined (P(TACT) (slices)=0.02. P(TACT) (iterative slices)=0.01). However, neither TACT system was significantly better than film for enamel and dentinal caries separately. Observer agreement was moderate. However, observers demonstrated significant systematic differences in their readings (P<0.001). CONCLUSION: TACT may be a feasible method for diagnosing primary occlusal caries.

Analysis of Variance↗
PubMed2001

Effect of intermediate-purity factor VIII (FVIII) concentrate on lymphocyte proliferation and apoptosis: transforming growth factor-beta is a significant immunomodulatory component of FVIII.

Factor concentrates have been shown to have a variety of immunomodulatory effects in vitro. The presence of plasma-derived factor VIII (pdFVIII) has been shown to diminish lymphocyte proliferative response to mitogens. Recently, we have shown the presence of transforming growth factor-beta (TGF-beta) as an immunomodulatory component present in plasma-derived FVIII concentrate. However, the addition of neutralizing antibody to TGF-beta did not abrogate the inhibitory effect of pdFVIII on monocyte cytokine production, suggesting the presence of other, as yet undetermined, immunomodulatory agent/s in pdFVIII. To further characterize the immunomodulatory effects of pdFVIII, the in vitro effect of pdFVIII concentrate on proliferation and apoptosis of mitogen-stimulated T cells was studied using whole blood and purified T cells. The presence of pdFVIII increased the apoptosis of phytohaemagglutinn (PHA) -stimulated CD4 and CD8 T-cell subsets as determined by Annexin V binding and DNA fragmentation. T-cell subsets showed a pdFVIII dose-dependent inhibition of entry into S-phase and G(1) arrest. Addition of neutralizing anti-TGF-beta reduced some of these changes. To determine the physiological relevance of these findings, blood samples from five patients receiving FVIII prophylaxis were similarly studied ex vivo and showed significantly increased apoptosis of T-cell subsets as determined by Annexin V staining. TGF-beta has been reported to be a potent inhibitor of T-cell proliferation, arresting the cell cycle in G(1) phase and causing apoptosis. Together, these findings suggest that TGF-beta is a significant immunomodulatory component of pdFVIII concentrates.

Adult↗
PubMed2001

Factor VIII concentrate inhibits T helper type 2 cytokine production in vitro: relevance to inhibitor antibody formation.

Inhibitor antibody formation in patients with haemophilia receiving factor VIII (FVIII) concentrate is a serious problem. T helper type 2 (Th2) cytokines are necessary for antibody production by B cells and have been shown to be produced predominantly by CD30(+)/CD45RO(+)/CD3(+) cells. We have previously shown that the Th2 cytokine, interleukin (IL)-6, is inhibited but IL-10 is upregulated, in the presence of plasma-derived FVIII (pdFVIII). To clarify further the overall effect of FVIII on Th2 cytokine production, the percentage of T cells expressing the CD30(+)/CD45RO(+)/CD3(+) Th2 phenotype was studied over 72 h and the production of the Th2 cytokines, IL-4 and IL-5, determined at 24 h in the presence of FVIII following whole-blood stimulation using multiparameter flow cytometry. The production of IL-4 and IL-5 by T cells was significantly inhibited in the presence of pdFVIII. The percentage of CD30(+)/CD45RO(+)/CD3(+) increased with stimulation of whole blood cultures over 72 h but was significantly inhibited by the presence of pdFVIII or TGF-beta at 72 h. The combined inhibitory effect of prednisolone (a commonly used immunosuppressive agent used to treat patients with inhibitors) with pdFVIII on T-cell CD30(+)/CD45RO(+) upregulation, was additive. There was no significant alteration in Th2 cytokine production or phenotype noted in the presence of recombinant FVIII (rFVIII) concentrate. Neutralizing antibody to TGF-beta significantly abrogated the inhibitory effects of pdFVIII on Th2 upregulation, indicating TGF-beta to be a major inhibitory component of pdFVIII on Th2 cytokine production. We now provide evidence that pdFVIII, by inhibiting Th2 cytokine production, may result in decreased antibody formation and may be more appropriate than rFVIII at reducing inhibitor formation. A clinical study needs to be undertaken to determine the significance of these in vitro findings.

Cell Culture Techniques↗
PubMed2001

Cord blood leucocyte expression of functionally significant molecules involved in the regulation of cellular immunity.

The cellular immune system of the newborn infant is immature and hypo-responsive when compared with adults. The extent to which immaturity of the leucocyte function underlies hyporesponsiveness in the newborn is incompletely understood. In this study flow cytometric techniques were applied to investigate the concurrent expression of a range of surface and intracellular leucocyte functional molecules and cytokines in resting and stimulated cord and adult blood. Production of interleukin (IL)-2 and expression of the components of its receptor, IL-2R alpha/beta/gamma, were investigated. No differences in the proportion of leucocytes producing IL-2R alpha and IL-2R gamma were observed for newborns and adults. A lower proportion of T cells and natural killer (NK) cells from newborns expressed IL-2R beta and upregulation of expression was slower. We hypothesize that reduced IL-2R beta may curtail early autocrine IL-2 activation of immune responses in the newborn. This hypothesis was supported by the observation that an increased proportion of stimulated T cells from newborns produced IL-2 at 4 h poststimulation, but at 24 h the proportion was lower than for adult T cells. The very low levels of interferon (IFN)-gamma produced by neonatal T cells and NK cells may also be partly explained by a curtailment of early autocrine activation of T cells. Expression and kinetics of upregulation for other functional molecules were studied. CD71, HLA-DR, tissue factor and CD152 levels were not significantly different for adults and newborns, suggesting that cord blood leucocytes, in some respects, may demonstrate functional maturity. IL-6 secretion by stimulated monocytes was also comparable in cord and adult blood. However, IL-1 alpha and IL-1 beta were produced by a lower proportion of monocytes from newborns than adults. Similarly, tumour necrosis factor (TNF)-alpha production for monocytes and T cells was lower in cord blood. The mean fluorescence intensity for IL-1 alpha, IL-1 beta and TNF-alpha was also lower for leucocytes from cord blood. These findings are significant in relation to the inability of newborn infants to mount a febrile response to infection. The findings of lower expression of IL-2R beta and lower production of inflammatory cytokines IL-1 alpha, IL-1 beta and TNF-alpha is a basis for improved understanding of the immunological immaturity of leucocytes in the newborn.

Adult↗
PubMed2001

Acquired haemophilia in South Australia: a case series.

Of eight cases of acquired haemophilia presenting over an 8-year period, six received immunosuppressive treatment, five with cyclophosphamide, vincristine and prednisolone (CVP). Five patients (four on immunosuppressive treatment) entered remission, two patients died and one was lost to follow up. Initially, the remissions were only partial. The median duration until partial remission was 10 weeks (range 1-55 weeks) and until complete remission was 35 weeks (range 2-59 weeks). Partial remission may proceed to complete remission without further chemotherapy.

Adult↗
PubMed2000

Increased levels of apoptosis of leukocyte subsets in cultured PBMCs compared to whole blood as shown by Annexin V binding: relevance to cytokine production.

Most of the investigatory studies of cytokine production by cells have been performed on purified cells or cell lines by measuring the secreted cytokine levels in the bulk culture supernatant. However, results of cytokine production from isolated peripheral blood mononuclear cells (PBMCs) cultivated in synthetic media, have been reported to be inaccurate and of low reproducibility. Isolation procedures have been shown to be toxic to certain cells. We hypothesised that purified cell culture techniques may result in increased levels of apoptosis of cells compared with whole blood culture techniques. To compare the effects on cell viability between PBMCs and whole blood techniques, an Annexin V binding assay was utilised. The effect of different cell concentration and serum/plasma concentrations on apoptosis levels in the various leukocyte subsets in PBMC and whole blood cultures following stimulation was investigated. There were significantly increased levels of apoptosis of cells in PBMC compared to whole culture at similar plasma concentrations, suggesting that cell viability was plasma concentration-dependent. There were significantly increased levels of apoptosis in PBMC cultures at the same cell concentration to whole blood techniques, suggesting that interaction between all cellular elements (as in whole blood techniques) is important in maintaining cell viability. These results suggest that whole blood culture techniques provide the best conditions for study of leukocyte cytokine production. If PBMC culture is performed, similar plasma and cell concentration to whole blood will best preserve cell viability.

Adult↗
PubMed2000

[CT virtual endoscopy: A study of the capability to display the structures and abnormalities in nasal cavity].

To evaluate displaying ability of virtual endoscopy and its clinical application in comparison with fiberoptic nasal endoscopy, 11 patients (22 nasal cavities) were examined by virtual endoscopy after axial spiral CT scanning was performed. Virtual endoscopy was performed by Explorer software package in a computer workstation. 9 patients (18 nasal cavities) underwent fiberoptic endoscopy. Results showed virtual endoscopy could clearly demonstrate the anatomical structures in nasal cavity, septal deviation, nasal meatus narrowing and obstruction, turbinate hyperplasia, and pathological masses larger than 3 mm in diameter. However, the "false adhesions" may appear with virtual endoscopy. The main limitation of virtual endoscopy was inability to evaluate mucosa and lack of histological diagnosis. Virtual endoscopy is a new and non-invasive method for demonstrating anatomical structures and diseases in nasal cavity. Its displaying ability is comparable with fiberoptic nasal endoscopy. It can serve as a supplementary method of fiberoptic nasal endoscopy.

Adult↗
PubMed2000

Virtual endoscopy of the nasal cavity in comparison with fiberoptic endoscopy.

PURPOSE: To apply virtual-endoscopic mode to display the nasal cavity and to evaluate clinical application of virtual endoscopy in comparison with nasal fiberoptic endoscopy. MATERIALS AND METHODS: Eleven patients were examined by virtual endoscopy after axial spiral computed tomography (CT) scanning was performed. In addition, 9 out of these 11 patients underwent fiberoptic endoscopy. Spiral CT scanning was performed with 1-mm collimation, 0.5- to 1-mm increment, 25-250 mA s, and pitch 1-2. Virtual endoscopy was performed by Explorer software package. RESULTS: Virtual endoscopy could clearly demonstrate anatomic structures in the nasal cavity, septal deviation, stenosis and obstruction of the middle meatus, turbinate hyperplasia, and pathological masses larger than 3 mm in diameter. However, "false adhesions" may appear in virtual endoscopy. The main limitation of virtual endoscopy was the inability to evaluate mucosa and its surface. CONCLUSION: Virtual endoscopy of the nasal cavity is a new and noninvasive method. It can demonstrate normal and pathological structures in the nasal cavity. Its ability to visualize is comparable with fiberoptic endoscopy except for evaluating mucosal surface and secretions. In the future, this method will probably be a basic instrument of computer-assisted surgery in the midfacial region.

Adult↗
PubMed2000

Effect of factor VIII concentrate on antigen-presenting cell (APC)/T-cell interactions in vitro: relevance to inhibitor formation and tolerance induction.

Inhibitor formation in patients with haemophilia receiving factor VIII (FVIII) concentrate is a common problem requiring tolerance induction therapy. Immune tolerance is dependent on defective T cell/antigen-presenting cell (APC) interactions and inhibitor antibody formation is associated with effective T-cell/B-cell interaction. We studied the expression of the cell-surface molecules involved with these interactions using multiparameter flow cytometry and a whole blood stimulation assay-phytohaemaglutinin (PHA) to activate T cells and Escherichia coli lipopolysaccharide (LPS) to activate monocytes and B cells. Up-regulation of T-cell co-stimulatory receptors CD11a, CD40 ligand (CD40L) and CTLA4 were inhibited in a dose-dependent manner by plasma-derived (pd)FVIII, but CD28 was unchanged. Up-regulation of monocyte and B-cell co-stimulatory ligands CD4O, B7-1 (CD80) and B7-2 (CD86) were also inhibited in a dose-dependent manner by pdFVIII, but LFA-3 (CD58) was unchanged. The combined inhibitory effect of prednisolone, an immunosuppressive agent used in several tolerance induction protocols, with pdFVIII on co-stimulatory molecules, was additive. There was no significant alteration in T-cell/APC adhesion or co-stimulatory molecules noted in the presence of recombinant (rh)FVIII concentrate. The inhibitory effect of pdFVIII on molecules involved in interaction between T cells and APCs may result in immune tolerance in recipients of pdFVIII concentrate. The inhibitory effect of pdFVIII on CD40/CD40L up-regulation may result in defective antibody formation. We now provide evidence that the use of pdFVIII, through interfering with APC/T-cell interactions, may be more appropriate than rhFVIII for tolerance induction.

Antigen-Presenting Cells↗
PubMed2000

Surface and intracellular interleukin-2 receptor expression on various resting and activated populations involved in cell-mediated immunity in human peripheral blood.

The kinetics of assembly of the high-affinity interleukin-2 receptor (IL-2R)alpha/beta/gamma were investigated by studying intracellular and surface expression of IL-2Ralpha, beta and gamma by T cells, monocytes and natural killer (NK) cells. IL-2Ralpha and IL-2Rgamma were expressed by small numbers of resting T cells. These numbers increased following stimulation, to maximal expression at 48 h and 72 h, respectively. This observation was consistent with de novo synthesis of the receptor protein in response to the stimulus. The proportion of T cells producing IL-2Rbeta was smaller and up-regulated later than the proportion of cells producing IL-2Ralpha or IL-2Rgamma. IL-2Rbeta may therefore slow the assembly of the high-affinity IL-2R on T cells. A small number of resting NK cells expressed IL-2Ralpha, both on the cell surface and intracellularly, but this increased over 72 h on stimulated NK cells. IL-2Rbeta was constitutively expressed, both on the cell surface and intracellularly, by monocytes and NK cells. An increased proportion of NK cells and monocytes produced IL-2Rbeta, 24 h and 4 h post-stimulation, respectively. Maximal or plateau expression occurred at 72 h and 24 h post-stimulation, for NK cells and monocytes, respectively. The early up-regulation of intracellular IL-2Rbeta for monocytes may facilitate the up-regulation of surface IL-2Rbeta, and early assembly of the high-affinity IL-2R, accelerating monocyte activation and function. High constitutive intracellular IL-2Rgamma expression (> 80%) in all types of leucocyte investigated, decreased over the 72 h following stimulation with a concurrent increase in surface expression. IL-2Rgamma was expressed by increased proportions of T cells, monocytes and NK cells, 4 h following stimulation. The intracellular storage of IL-2Rgamma may accelerate translocation to the cell surface after stimulation. The early translocation of IL-2Rgamma may reflect its usage as a signal transduction molecule by other cytokine receptors - IL-4, IL-7, IL-9 and IL-15. This study delineated the potential expression of the high-affinity IL-2Ralpha/beta/gamma on various stimulated leucocytes. The differential kinetics of assembly of the high-affinity IL-2Ralpha/beta/gamma on different leucocyte subsets suggests that IL-2 may regulate the inflammatory cellular responses in a sequential manner, paralleling the timed expression of IL-2Ralpha/beta/gamma on the monocytes, NK cells and T cells.

Adult↗
PubMed2000

Coil occlusion of the parent artery for treatment of symptomatic peripheral intracranial aneurysms.

BACKGROUND AND PURPOSE: Peripheral intracranial aneurysms can be difficult to treat with traditional surgical or embolization techniques that spare the parent vessel. We report the results of our use of coil occlusion of the parent vessel for the treatment of nine peripheral intracranial aneurysms. METHODS: During approximately a 4-year period, nine patients (six men and three women, 27 to 68 years old; average age, 42 years) presented to our institution with peripheral intracranial aneurysms. The aneurysms were located on branches of the right posterior inferior cerebellar artery (n = 2), the right superior cerebellar artery (n = 1), the right anterior inferior cerebellar artery (n = 1), the right posterior cerebral artery (n = 3), the left middle cerebral artery (n = 1), and the left anterior cerebral artery (n = 1). Parent vessel occlusion was performed using microcoils after test injection with amobarbital (Amytal) in eight of the nine cases (one patient was comatose and could not be tested before occlusion). RESULTS: Angiography immediately after the procedure showed aneurysmal occlusion in every patient. Follow-up arteriography, performed in six patients 2 to 12 months after treatment, documented continued aneurysmal occlusion in every case. Three patients exhibited mild, nondisabling neurologic deficits after coil placement; the rest had no new deficits, although one patient was severely disabled from the initial hemorrhage and one patient died of an unrelated cause. CONCLUSION: Our results lend support to the use of parent vessel occlusion for peripheral aneurysms that are difficult to treat surgically or that are not amenable to intra-aneurysmal coil placement.

Adult↗
PubMed2000

[3D finite element analysis of stress distributions in supporting tissues of clasp-type partial dentures of transferring occlusion force].

OBJECTIVE: To provide a theoretical basis for clinical application of the clasp-type partial denture of transferring occlusion force, the stress distributions in its supporting tissue were analyzed. METHODS: The 3D finite element model was made, and the features of stress distributions in the supporting tissues of the clasp-type partial dentures which had different rest recess angles were discussed. RESULTS: The medial stress component was significantly larger than the distal one in the supporting tissue around main abutment root. When the rest recess angles were equal or greater to 60 degrees, the stress values were greatly reduced in the supporting tissue around the roots of the three abutments. When the rest recess angle reached 75 degrees, the stress values in the supporting tissue around the main abutment root wore decreased by 30% than the angle 0 degree and by 52% than the angle 45 degrees, the stress values in the supporting tissue around the root of second abutment (adjacent to the main abutment) were wholly increased by 44% than the angle 0 degree and reduced by 23% than the angle 45 degrees, the stress values in supporting tissue around the root of third abutment (outside of the second abutment) were wholly increased by 53% than the angle 0 degree and decreased by 31% than the angle 45 degrees, the stress values in the medial root of the main abutment were reduced by 73%. CONCLUSION: The clasp-type partial denture can resolve part of the occlusion force into two components the medial and distal components, and the occlusion force is distributed to several abutments, at the same time the conversed force components can be canceled by each other. So the whole stress values are greatly reduced and the stress around abutment root is greatly decreased. As a result the damage to the abutment will be eliminated. A design with centralized multi abutments and multi fixed bodies are used in this denture, which are more fixed and stable and have no obvious sinkage in the plate base. Therefore, the mastication function can be satisfactorily recovered, and the reasonable rest recess angle for the clasp-type partial denture of transferring occlusion force is about 75 degrees.

Bite Force↗
PubMed1999

The calcium/calmodulin-dependent phosphodiesterase PDE1C down-regulates glucose-induced insulin secretion.

To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic beta-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in betaTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Optimal storage conditions for preserving granulocyte viability as monitored by Annexin V binding in whole blood.

It is important in the laboratory to develop techniques to preserve leucocyte viability in blood specimens for subsequent flow cytometric analysis. This article describes a new simple whole blood lysis method using Annexin V FITC staining which can be used to define both early and late apoptosis of granulocytes in heterogeneous cell populations, without the need for additional stains or to purify the cells (which may result in loss of the cells of interest). The differential Annexin V binding assay was in good agreement with the light microscopy reference method and showed excellent correlation with 7-aminoactinomycin D (7-AAD) staining. It was not affected by problems of morphological interpretation and artifactal changes of granulocyte deformability noted using light microscopy, or the technical difficulties encountered due to red cell contamination using the 7-AAD method. Using this new differential Annexin V staining method, we determined the optimum conditions that maintain granulocyte viability for subsequent flow cytometric analysis and are now employed in our laboratory. These conditions were lithium heparin (Hep) anticoagulated whole blood specimens kept at 4 degrees C with the addition of nutrient medium. Specimens that are anticoagulated with acid citrate dextrose (ACD) or ethyl-diacetyl-tetraacetic acid (EDTA) should also be treated similarly to preserve granulocyte viability and to overcome problems associated with identification of cell populations by flow cytometry.

Adult↗