Patrick Man Pan Yuen

Effect of Hemodialysis on Protein C, Protein S, and Antithrombin III Levels

We studied the effect of hemodialysis on natural coagulation inhibitors including protein C (PC), protein S (PS), and antithrombin III (AT III), as well as the correlations between the antigen level (immunological activity) and functional activity of individual coagulation inhibitor. Plasma AT III, PS, and PC were measured in 20 uremic patients on maintenance hemodialysis immediately before, during, and after dialysis treatment. These values were compared with those obtained from 20 matched healthy controls. Plasma PC and total PS antigen levels were measured by enzyme immunoassay. The plasma AT III antigen level was determined by Laurell rocket immunoelectrophoresis. Functional activities of PC and AT III were determined by the amidolytic method. Free PS antigen level was quantitated by measuring the free PS-related antigen after the sample was treated with polyethylene glycol to remove the C4b-binding protein. Uremic patients on maintenance hemodialysis had a higher total PS antigen level, but a lower free PS antigen level compared with the controls. Both the antigen level and functional activity of AT III in uremic patients were significantly lower than those of controls. Their predialysis plasma PC antigen level and functional activity were not different from those of normal controls. A significant correlation between the antigen level and functional activity of PC, PS, and AT III was demonstrated in healthy controls, but not in hemodialysis patients. No significant change in the level of AT III or PS was observed with hemodialysis, but a progressive increase of functional activity of PC was documented with hemodialysis. Furthermore, the coefficient of correlation between the antigen level and functional activity of PC improved significantly with dialysis treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice.

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This studys purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum‐free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.

Promoting effects of serotonin on hematopoiesis : Ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5‐hydroxytryptamine (5‐HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony‐forming unit‐fibroblast (CFU‐F) formation, and antiapoptosis of megakaryoblastic M‐07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony‐forming unit‐mixed [CFU‐GEMM]) and multilineage committed progenitors (burst‐forming unit/colony‐forming unit‐erythroid [BFU/CFU‐E], colony‐forming unit‐granulocyte macrophage, colony‐forming unit‐megakaryocyte, CD61+CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU‐E, and CFU‐GEMM engraftment in BM of animals 6 weeks post‐transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet‐derived growth factor, or vascular endothelial growth factor stimulated BM CFU‐F formation. In M‐07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase‐3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5‐HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation.

Mutational analysis of 65 Wilson disease patients in Hong Kong Chinese: Identification of 17 novel mutations and its genetic heterogeneity

AbstractWilson disease (WD), an autosomal recessive disorder of copper transport, is the most common inherited liver disorder in Hong Kong Chinese. This was the first local study to elucidate the molecular basis and establish an effective DNA-based diagnostic protocol. The ATP7B genes of 65 patients were amplified by polymerase chain reaction (PCR) and sequenced. Haplotype analysis was performed using D13S301, D13S314, and D13S316. The p.L770L/p.R778L status in 660 subjects was determined to estimate WD prevalence. Allele age of p.R778L was determined by the smallest homozygosity region between D13S301 and D13S270. We identified 42 different mutations with 17 being novel. p.R778L (17.3%) was the most prevalent. Exons 2, 8, 12, 13, and 16 harbored 70% mutations. Thirty-two haplotypes were associated with WD chromosomes. The estimated prevalence rate was 1 in 5,400. Three out of 660 normal subjects had p.L770L/p.R778L. In the remaining 657 individuals, neither p.L770L nor p.R778L was found. We characterized a Hong Kong Chinese-specific ATP7B mutation spectrum with great genetic diversity. Exons 2, 8, 12, 13, and 16 should be screened first. The perfect linkage disequilibrium suggested that p.R778L and its private polymorphism p.L770L originated from a single ancestor. This East-Asian-specific mutation p.R778L/p.L770L is aged at least 5,500 years.

Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long-term culture-initiating cells, and nonobese diabetic-SCID repopulating cells.

BACKGROUND : The cryopreservation of HPCs in DMSO has been practiced by cord blood (CB) banks worldwide. Inevitably, some detriment to biologic function occurs as the result of freezing injuries and DMSO toxicity. Trehalose, a disaccharide, is a natural cryoprotectant in organisms capable of surviving extreme dehydration and cold. The objective of this study was to establish the cryopreservation of CB under preclinical conditions using trehalose as a supplement to DMSO.

Liver disease in transfusion dependent thalassaemia major

Aims: To study the prevalence and severity of liver diseases of transfusion dependent thalassaemia major patients, and correlate the histological and biochemical changes of iron overload in liver with the peripheral blood markers. Method: Liver biopsy was performed to assess the histological changes and liver iron content (LIC). Results: One hundred patients were evaluated (median age 11.7 years, range 1.5–27). A total of 81 liver biopsies were performed in 73 patients; 43 samples were analysed for LIC. Grade 3–4 haemosiderosis and hepatic fibrosis was found in 44% and 30% of patients respectively; both were significantly associated with higher serum ferritin, liver enzymes, and LIC. Very high LIC (>15 mg/g dry weight) was present in 16.3% of patients. Conclusion: Severe haemosiderosis and hepatic fibrosis were common in patients with thalassaemia major despite the use of chelation therapy. Liver biopsy provided information on fibrosis and LIC which could not be accurately predicted from peripheral blood markers.

Platelet-derived growth factor enhances ex vivo expansion of megakaryocytic progenitors from human cord blood.

Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is a strategy for shortening the duration of thrombocytopenia after haematopoietic stem cell transplantation. The cell dose after expansion has emerged as a critical factor for achieving the desired clinical outcomes. This study aimed to establish efficient conditions for the expansion of the MK lineage from enriched CD34+ cells of umbilical cord blood and to investigate the effect of platelet-derived growth factor (PDGF) in this system. Our results demonstrated that thrombopoietin (TPO) alone produced a high proportion of CD61+CD41+ cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of interleukin-1β (IL-1β), Flt-3 ligand (Flt-3L) and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1β, IL-3, IL-6 and Flt-3L. CD34+ cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61+CD41+ cells, CFU-MK and CD34+ cells increased by 101%, 134% and 70%, respectively. On day 14, the CD61+ population consisted of diploid (86.5%), tetraploid (11.8%) and polyploid (8N–32N; 1.69%) cells. Their levels were not affected by PDGF. TPO, IL-1β, IL-3, IL-6, Flt-3L and PDGF represented an effective cytokine combination for expanding MK progenitors while maintaining a moderate increase of CD34+ cells. This study showed, for the first time, that PDGF enhanced the ex vivo expansion of the MK lineage, without promoting their in vitro maturation. PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. Bone Marrow Transplantation (2001) 27, 1075–1080.

Platelet‐derived growth factor promotes ex vivo expansion of CD34+ cells from human cord blood and enhances long‐term culture‐initiating cells, non‐obese diabetic/severe combined immunodeficient repopulating cells and formation of adherent cells

Summary. Platelet‐derived growth factor (PDGF) is a major mitogen for connective tissue cells. In this study, we investigated the effects and mechanism of PDGF on the ex vivo expansion of cord blood CD34+ cells. Our data demonstrated that among various cytokine combinations of thrombopoietin (TPO), interleukin 1 beta (IL‐1β), IL‐3, IL‐6 and Flt‐3 ligand (Flt‐3L), TPO + IL‐6 + Flt‐3L was most efficient in promoting the expansion of CD34+ cells, CD34+CD38– cells, mixed‐lineage colony‐forming units (CFU‐GEMM) and long‐term culture‐initiating cells (LTC‐IC) by 21·7 ± 5·00‐, 103 ± 27·9‐, 10·7 ± 7·94‐ and 6·52 ± 1·51‐fold, respectively, after 12–14 d of culture. The addition of PDGF increased the yield of these early progenitors by 45·0%, 66·5%, 45·1% and 79·8% respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in non‐obese diabetic (NOD)/severe‐combined immunodeficient (SCID) mice. The expression of PDGF receptor (PDGFR)‐β was not detectable in fresh CD34+ cells but was upregulated after culture for 3 d. PDGF also enhanced the development of adherent cells/clusters that expressed the endothelial markers VE‐cadherin and CD31. These findings suggest that PDGF is an effective cytokine for the ex vivo expansion of early stem and progenitor cells. The mechanism could be mediated by PDGFR‐β on committed CD34+ progenitor cells and/or secondary to the stimulation of autologous, stromal feeder cells.

Detection of micrometastasis of neuroblastoma to bone marrow and tumor dissemination to hematopoietic autografts using flow cytometry and reverse transcriptase-polymerase chain reaction.

The identification of neuroblastoma metastases to bone marrow (BM) is requisite in staging disease for risk‐adopted therapy. However, micrometastases were not elucidated fully.

Elevated Serum Interleukin-15 Level in Acute Graft-versus-host Disease After Hematopoietic Cell Transplantation

Objective To correlate serum cytokine levels and the development of acute graft-versus-host disease (GVHD), the authors conducted a prospective study on serial measurements of interferon (IFN)-&ggr; and interleukin (IL)-10, IL-12 and IL-15. Methods The cytokines were measured in 27 subjects by enzyme-linked immunosorbent assay serially for the first 2 months after hematopoietic cell transplantation. Results Nineteen subjects with acute GVHD had significantly higher mean peak serum levels of IFN-&ggr; and IL-15 than the baseline levels at the start of conditioning. The peak level occurred soon after stem cell infusion and returned to the pretransplantation state in the second month. In contrast, there was lesser difference between the mean peak serum levels of IFN-&ggr;, IL-10, and IL-15 and the baseline level in the eight subjects without GVHD. The peak serum level for IL-15 was, in addition, significantly higher among GVHD subjects than those without GVHD in the first month posttransplantation. However, the level of IL-15 showed no correlation with the severity of GVHD. Conclusions These changes point to a possible role of systemic cytokine secretion in the development of acute GVHD. Elevated levels of IL-15 early in the posttransplant period could be a helpful laboratory predictor of acute GVHD.