In vitro biological characteristics of human cord blood-derived megakaryocytes.

INTRODUCTION: Umbilical cord blood (CB) has been used as an alternative source for haematopoietic stem cell transplantation (HSCT) in recent years. However, delayed platelet recovery is frequently associated with CB HSCT. Megakaryocytes (Mk) are the specialised precursors of platelets and they are among the rarest haemopoietic cell types. Despite the rapid expansion of our knowledge of megakaryopoiesis in recent years, many questions, such as the molecular regulatory mechanisms in Mk differentiation and maturation, platelet formation and release, remain unanswered in CB-derived megakaryopoiesis. Variations can be seen from the literature by individual investigators using different approaches for Mk-specific differentiation and maturation induction. The development of in vitro culture methods to obtain sufficient numbers of Mks from readily available haematopoietic stem cells is of value for both basic research and clinical applications. MATERIALS AND METHODS: The CD34+ cells from cord blood samples were cultured in serum-free medium with haematopoietic growth factors (GFs), such as IL-3, stem cell factor (SCF), and thrombopoietin (Tpo). The differentiation of Mk was monitored using Mk- and platelet-specific monoclonal antibodies and flow cytometric analysis. The morphology of the cultured cells was studied by both light and electronic microscopy (LM and EM). The involvement of the human Notch gene family members was studied by real time-polymerase chain reaction (RT-PCR). Maturation of the cultured Mks was studied using flow cytometric analysis for both platelet-specific surface markers and enodomitosis. Platelet activation was assessed in the cytoplasmic fragments harvested from the cultures. RESULTS: Specific Mk differentiation of >70% resulted from a 2-step culture approach using IL-3, SCF and Tpo for 7 days followed by Tpo only for another 14 days. RT-PCR showed high-level expression of both Notch-1 and its ligand, Jagged-1, in the cultured Mks. Limited levels of polyploidy (>4N, endomitosis, EnM) were observed in the cultured Mks. The results also showed that the cytoplasmic fragments from the cultures responded to platelet activation reagents, including ADP and collagen, marked by upregulation of platelet-specific activation markers, such as CD62P (P-selectin) and PAC-1 (gpalphaIIbbeta3). CONCLUSION: The methods used in this study are specific for differentiation of Mk from CB CD34+ cell, which can partially mature and produce functional platelets in vitro. This approach for human Mk differentiation could be further optimised and may be adapted on larger scales for clinical purposes.

Demonstration of mixed properties of RU486 in progesterone receptor (PR)-transfected MDA-MB-231 cells: a model for studying the functions of progesterone analogues.

Progesterone antagonist RU486 (mifepristone) has been implicated for many anti-neoplastic and obstetrical applications. But the compound has demonstrated undesired agonist-like effect depending on cell, tissue and species studied. Using PR-transfected breast cancer cells MDA-MB-231, this report describes the similarities and differences between progesterone- and RU486-mediated effects on cell growth, cell differentiation and, at the molecular level, on the activation of p44/p42 MAP kinases (MAPK). Like progesterone, RU486 inhibited cells growth by arresting the cells in G0/G1 phase of the cell cycle. In contrast to progesterone that induced cell spreading, RU486 induced a multipolar, stellate morphology. RU486-treated cells showed no increase of stress fibers, nor was there any increase of focal adhesions as progesterone-treated cells did. Furthermore, despite of the fact that both compounds inhibited cell growth, RU486 significantly stimulated the activation of p44/p42 MAP kinases whereas progesterone markedly inhibited the activation. Nonetheless, the effects of RU486 were PR-mediated and RU486 was able to antagonize the effect of progesterone on cell growth and focal adhesion. In conclusion, RU486 can act not only as a progesterone antagonist, a progesterone agonist but also induced morphological and molecular changes that were distinct from progesterone-mediated effects in PR-transfected MDA-MB-231 cells. The non-progesterone-like effect of RU486 may be mediated through a pathway that is different from the progesterone-mediated pathway, or it is the result of a blockade of certain critical step(s) in the progesterone-mediated pathway. In any case, undesired side effects of antiprogestin may create clinical complications. PR-transfected MDA-MB-231 breast cancer cells provide a model for studying the functions of progesterone analogues.

Changes in prostaglandin and nitric oxide levels in the hyperdynamic circulation following liver resection.

BACKGROUND AND AIMS: Significant changes in hepatic haemodynamics occur after major hepatectomy, but the pathogenesis of this phenomenon is unclear. This study investigates the serum profile of prostaglandin and nitric oxide in the hepatic and systemic circulation before and after hepatectomy and the temporal relationship of these to changes in the hepatic blood flow. METHODS: Blood samples were collected from the internal jugular, portal and hepatic veins of six Yorkshire pigs (17-25 kg) before, immediately after and 48 h after partial hepatectomy. RESULTS: Serum levels of prostacyclin I2 (PGI2) and prostaglandin E2 (PGE2) from the systemic circulation, the portal circulation and from the hepatic veins were found to differ considerably even before hepatectomy. After the hepatectomy was performed, there was a significant rise in PGI2 levels in the systemic circulation (P=0.027). Hepatic blood flow and cardiac output were measured before hepatectomy, 24 and 48 h after hepatectomy in another six pigs. A significant increase in hepatic blood flow (P=0.029) occurred after the hepatectomy and this was accompanied by a concomitant increase in the cardiac output (P=0.042). The increase in PGI2 concentration in the systemic circulation after hepatectomy appears to accompany the development of hyperdynamic hepatic and systemic circulations. No significant change was found in circulating PGE2 levels in the systemic, portal and hepatic veins at the three collection intervals (i.e. pre, post and 48 h after hepatectomy). The PGE2 levels in the hepatic vein, however, were significantly higher from than in the portal vein (P=0.028). No significant changes were observed in the level of nitric oxide. CONCLUSION: This study demonstrates an increase in the total hepatic blood flow after hepatectomy together with a threefold increase in prostacyclin in the systemic circulation. The liver was identified as the main source of circulatory prostaglandin.

A non-invasive isotope dilution technique for quantifying hepatic blood flow using radiolabelled red blood cells.

Clinically significant changes in hepatic haemodynamics accompany the development of portal hypertension, hepatocellular carcinoma, liver metastases and liver cirrhoses, and after major liver resection. Hepatic blood flow parameters, such as hepatic arterial flow (HAF), hepatic portal flow (HPF), total hepatic blood flow (THBF) and hepatic perfusion index (HPI), are useful adjuncts to the diagnosis of liver pathology, the evaluation of disease progress and prognostication. Here, we describe a non-invasive method that combines the measurement of these parameters in a single study in real time. Red blood cells from eight pigs were labelled with 99Tc(m) using an in-vitro method and re-injected into the pigs. Data acquisition over the heart, lungs, liver and kidneys was started immediately and a blood sample was obtained 15 min post-injection. Hepatic arterial flow was determined from the ratio of the maximum gradients between the integrated time-activity curve of the left ventricle and the first-pass time-activity curve of the liver before the peak of the kidneys time-activity curve. The hepatic perfusion index was determined by comparing the slope of the liver time-activity curve before and after the kidney peak. Hepatic portal flow was determined from the hepatic arterial flow and the hepatic perfusion index, and total hepatic blood flow was determined as the sum of arterial and portal flow. The results were compared against those obtained from a clearance method using 99Tc(m)-DISIDA. The average hepatic perfusion index was 0.38, and the average hepatic arterial flow and hepatic portal flow were 168.3 +/- 52.9 and 274.6 +/- 60.1 ml x min(-1) respectively. The average total hepatic blood flow was 442.8 +/- 53.5 ml x min(-1), while the total hepatic flow determined by 99Tc(m)-DISIDA clearance was 419.7 +/- 62.6 ml x min(-1). No significant difference in total hepatic blood flow was found between the two methods. The results of this study show that it is possible to obtain all hepatic haemodynamics data in a single study using a non-invasive method.

Influence of respiration and portal pressure on transabdominal duplex Doppler ultrasound measurement of portal blood flow: a porcine model for experimental studies.

Transabdominal duplex Doppler ultrasonography (TDDU) is commonly used for measuring hepatic blood flow (HBF) in clinical practice. Flow velocity and the cross-sectional area (CSA) of vessels are obtained separately and used to compute blood flow. Respiration and changes in portal pressure are known to cause variations in the CSA of the portal vein, but the impact of these parameters on TDDU measurement of portal blood flow is unclear. Eight Yorkshire pigs (20.7-25.1 kg) were used for the study. TDDU determination of portal blood flow was carried out using CSA of the portal vein obtained at inspiration (maximal) and at expiration (minimal) for computation, and the differences obtained were compared. Determination of HBF was carried out simultaneously on the same animals using diisopropyliminodiacetic acid (DISIDA) clearance. A physiological increase in portal pressure was then created by 50% hepatectomy and TDDU measurement similarly carried out on the second postoperative day. Computing portal blood flow in the intact liver using maximal and minimal CSA gave rise to a mean difference of 7.0 ml kg(-1) min(-1) (P < 0.001). A significant correlation was obtained between HBF and portal flow computed from maximal CSA (Pearson's correlation = 0.85, P < 0.033). The respiratory index of the portal vein (maximal CSA/minimal CSA) decreased from 1.5 to 1.2 after hepatectomy, which also caused a 90% increase in portal pressure. Respiration and portal pressure thus significantly impact on TDDU determination of HBF, and in this porcine model, computation using maximal CS more accurately reflects HBF.

Progesterone induces focal adhesion in breast cancer cells MDA-MB-231 transfected with progesterone receptor complementary DNA.

Since the effects of progesterone are mediated mainly via estrogen-dependent progesterone receptor (PR), the expression of the effects of progesterone may be masked or overridden by the influence of estrogen under conditions in which priming with estrogens is required. We have established a PR-positive but estrogen receptor-alpha (ER-alpha) negative breast cancer cell model by transfecting PR cDNA into ER-alpha- and PR-negative MDA-MB-231 cells in order that the functions of progesterone can be studied independently of estrogens. We have demonstrated using this model that progesterone markedly inhibited cell growth. We have also discovered that progesterone induced remarkable changes in cell morphology and specific adhesion structures. Progesterone-treated cells became considerably more flattened and well spread than vehicle-treated control cells. This was associated with a striking increase of stress fibers, both in number and diameter, and increased focal contacts as shown by the staining of focal adhesion proteins paxillin and talin. There were also distinct increases in tyrosine phosphorylation of focal adhesion protein paxillin and focal adhesion kinase in association with increased focal adhesion. The staining of tyrosine-phosphorylated proteins was concentrated at focal adhesions in progesterone-treated cells. More interestingly, monoclonal antibody (Ab) to beta1 integrin was able to inhibit progesterone-induced cell spreading and formation of actin cytoskeleton. To our knowledge, this is the first report describing a direct effect of progesterone in inducing spreading and adhesion of breast cancer cells, and beta1-integrin appeared to play an essential role in the effect. It is known that the initial step of tumor metastasis is the breakaway of tumor cells from primary tumor mass when they lose the ability to attach. Hence, progesterone-induced cell spreading and adhesion may have significant implications in tumor metastasis.

Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels.

The aims of this study were to (a) determine the prevalence of patients without elevated thyroid hormone levels in Graves' ophthalmopathy (GO) using current generation free thyroid hormone assays, (b) measure the prevalence of thyrotropin receptor antibodies (TRAb) in these cases, and (c) identify possible predictors of hyperthyroidism. Over a 30-month period, 1020 cases of thyroid eye disease were evaluated, of which only 19 (1.9%) met the diagnostic criteria. Ten (1%) had subclinical thyrotoxicosis, 7 (0.7%) were euthyroid, and 2 (0.2%) were hypothyroid as determined by a third-generation thyrotropin (TSH) assay. TRAb levels were measured in 16 of these 19 patients. The prevalence of TRAb varied according to the assay used. Polyethylene glycol-extracted thyroid-stimulating immunoglobulin (PEG-TSI), unfractionated thyroid-stimulating immunoglobulin (uTSI), first-generation porcine TSH-binding inhibitory immunoglobulin (pTBII), and second-generation human TSH-binding inhibitory immunoglobulin (hTBII) assays were positive in 93.8%, 50%, 18.8%, and 81.3% of patients, respectively. TRAb was detected by at least one method in all patients. Patients were followed up for 15 to 45 months. Hyperthyroidism developed in 4 patients (25%). Suppressed TSH levels and elevated TBII were predictors of hyperthyroidism. When sensitive assays are used, the prevalence of GO patients without elevated thyroid hormone levels is extremely low. The sensitivities of assays for TRAb detection differ substantially in these cases. PEG extraction improves the detection rate of TSI (p = 0.02), and hTBII assays improve the detection of TBII in these patients (p = 0.002). The high prevalence of TRAb in such cases supports a role for these antibodies in the pathogenesis of thyroid-associated eye disease.

Liver regeneration after partial hepatectomy is non-uniform: flow cytometric bromodeoxyuridine incorporation and cell cycle studies in a porcine model.

The rate of hepatocyte regeneration at different anatomical locations of the remnant liver after partial hepatectomy was assessed in porcine hepatocytes by bromodeoxyuridine (BrdUr) incorporation and cell cycle kinetics using flow cytometric analysis. Partial hepatectomy was performed in five Yorkshire pigs. A single intravenous injection of BrdUr at 50 mg/kg was administered on the 2nd post-operative day and the animals were sacrificed 1 h later. The remnant liver tissue was harvested and divided into four equal zones, from the liver periphery towards the surgical cut-edge. Biopsy samples were obtained from the centre of each of these zones and similarly from identical anatomical locations in two control pigs that had undergone sham surgery. Hepatocyte nucleus suspension was prepared, double labelled with anti-BrdUr and propidium iodide and analysed by a flow cytometer. The cells in S-phase was used as the parameter to measure the regeneration status. A gradient increase in S-phase from the periphery to the cut edge was observed in all five pigs that had undergone partial hepatectomy. The percentage of S-phase cells in all four zones from the hepatectomy group was significantly higher when compared with that in the controls. Liver regeneration after partial hepatectomy was not uniform but was greatest adjacent to the surgical cut edge and decreased towards the periphery of the liver.

Five micro-curie urea breath test for the diagnosis of Helicobacter pylori infection: evaluation in a South-East Asian population.

BACKGROUND: Helicobacter pylori is an important pathogen responsible for significant morbidity and mortality. Its prevalence varies widely in different geographical locations and is especially high in parts of Asia. METHODS: A double-blind study was carried out to evaluate the use of the 5 microCi (185 KBq) [14C]-urea breath test ([14C]-UBT) in a South-East Asian population by validating its diagnostic accuracy against histology and the CLO test. RESULTS: The sensitivity and specificity of the [14C]-UBT was 100% when compared against the CLO test. When histology was used as the 'gold standard', the sensitivity and specificity were 100% and 97.2%, respectively. There was no overlap or indeterminate values between positive and negative results on the [14C]-UBT. CONCLUSIONS: Among South-East Asian populations where the prevalence of H. pylori infection is high, the high sensitivity of the 5 microCi [14C]-UBT makes it a very important test in the detection of H. pylori.

Progestins inhibit the growth of MDA-MB-231 cells transfected with progesterone receptor complementary DNA.

Because progesterone exerts its effects mainly via estrogen-dependent progesterone receptor (PgR), the expression of progesterone's effects may be overshadowed by the priming effect of estrogen. PgR expression vectors were transfected into estrogen receptor (ER)-alpha and PgR-negative breast cancer cells MDA-MB-231; thus the functions of progesterone could be studied independent of estrogens and ERs. Eight stable transfectant clones expressing both PgR isoform A and B were studied for their growth response to progesterone and its analogues. Although progesterone had no effect on growth in the control transfectant, the hormone markedly inhibited DNA synthesis and cell growth in all of the PgR-transfectants dose-dependently from 10(-12)-10(-6) M. This growth inhibition was associated with an arrest of cells in the G0/G1 phase of the cell cycle. Progestins medroxyprogesterone acetate, Org2058, and R5020 also strongly inhibited DNA synthesis, and their doses required for maximal inhibition of 60-70% were 10(-17) M, 10(-13) M, and 10(-7) M, respectively. Antiprogestin ZK98299 alone had no effect, but the compound was capable of counteracting the inhibitory effect of progesterone. In contrast, RU486 inhibited DNA synthesis, and it showed no further effects when it was used concurrently with progesterone. These results indicate that progestins are per se antiproliferative via a PgR-mediated mechanism in breast cancer cells. More importantly, we have shown that progestins may exert effective inhibitory control over the cell growth if the PgR expression is reactivated in ER- and PgR-negative breast cancer cells.

The combination of absent thyroid peroxidase antibodies and high thyroid-stimulating immunoglobulin levels in Graves' disease identifies a group at markedly increased risk of ophthalmopathy.

Among Graves' Disease (GD) patients, we have observed an unexpectedly high prevalence of antithyroperoxidase antibody (TPOAb) and antithyroglobulin antibody (TgAb) negativity in those with severe ophthalmopathy. To study the possible role of thyroid autoantibodies in the pathogenesis of Graves' ophthalmopathy (GO), TPOAb, TgAb, thyroid-stimulating immunoglobulin (TSI), and thyrotropin-binding inhibitory immunoglobulin (TBII) levels were measured, and the presence or absence of GO was assessed by a single observer in 100 consecutive patients with newly diagnosed, untreated GD who were nonsmokers. Ophthalmopathy was present in 43 patients. TSI levels (p = 0.001), and the prevalence of TPOAb-negativity (p = 0.002) were significantly higher in patients with ophthalmopathy compared to those without. Logistic regression analysis showed that TSI levels (p = 0.005) and the absence of TPOAb (p = 0.0025) were independent predictors of GO. No correlation between TBII or TgAb and eye disease was found. The prevalence of GO increased with each quartile of TSI levels. The prevalence was 20%, 36%, 52%, and 64% in the first, second, third and fourth quartiles of TSI, respectively. The odds ratio of GO (with 95% confidence intervals) when TSI levels were above the median level (1640%) was 3.6 (1.5-8.0), when TPOAb was negative it was 5.0 (1.7-14.4), and with both risk factors it was 36.6 (4.3-313.5). The prevalence of ophthalmopathy in this last group was 92.9%. The combination of negative TPOAb and high TSI levels appears to be associated with a markedly increased risk of clinically evident ophthalmopathy.

Vinorelbine induces apoptosis and caspase-3 (CPP32) expression in leukemia and lymphoma cells: a comparison with vincristine.

Vinorelbine (NVB) is a novel vinca alkaloid FDA approved for use in some advanced carcinomas. However, its role in non-Hodgkin's lymphoma (NHL) is still not well defined. NVB is an antimicrotubule agent, but as yet, it is not known whether it induces apoptosis. By flow cytometry using nuclear staining (propidium iodide) and annexin V, we demonstrated that NVB and vincristine (VCR) induced both mitotic arrest and apoptosis in leukemia and lymphoma cells, in a drug exposure time dependent manner. Cell cycle kinetics in 3 different cell lines varied during vinca alkaloid treatment. The annexin V method showed that apoptosis, as opposed to necrosis, was the dominant mode of cell kill of chemosensitive leukemia and lymphoma cells. Phosphatidylserine expression on the cell surface was detectable as a hallmark of apoptosis at earlier drug exposure when compared to conventional flow cytometry with PI staining. By Western blot analysis, we demonstrated that CPP32 or caspase-3, a critical apoptosis inducer, and its active subunits p20 and p11 were upregulated in chemo- and apoptosis-sensitive lymphoma and leukemia cells treated with NVB. Our data contributes to the emerging hypothesis suggesting that widely divergent exogenous stimuli and chemotherapeutic agents can effect apoptosis in cancer cells via different pathways involving the caspases. We believe that vinorelbine may be a potentially important drug in the treatment of NHL in the future.

99Tcm-polyclonal IgG and 99Tcm nanocolloid scans in orthopaedics: a comparison with conventional bone scan.

The bone scan is sensitive in detection of active bone/joint lesions. A normal bone scan virtually excludes the presence of an inflammatory process with high precision, but the poor specificity of bone scans is well known. In recent years, various new agents including 99Tcm-hexamethylpropylene amine oxime (HMPAO)-labelled white blood cells, nanocolloid, polyclonal IgG, anti-granulocyte antibody, 111In-labelled IgG, leucocytes, chemotactic peptides etc. have been widely evaluated in inflammatory imaging, especially in the orthopaedic context. This study was undertaken to compare the usefulness of 99Tcm-nanocolloid and 99Tcm-polyclonal IgG in the detection of focal bone/joint inflammation. Twenty-seven patients with a common presentation of bone/joint pain resulting from various pathologies were included in the study. A total of 47 lesions were imaged. The overall sensitivity and specificity of both nanocolloid scan and IgG scan were identical with 95% sensitivity and 100% specificity, in detecting inflammatory foci. However, specificity dropped to 18% with nanocolloid scans and 16% with IgG scans when an attempt was made to distinguish noninfective from infective inflammatory processes; thus neither type of scan permits differentiation between septic and nonseptic inflammatory processes with sufficient accuracy. As both nanocolloid and IgG scans are equally sensitive and specific in detecting inflammation, the choice of type of scan will depend on cost, imaging time and availability of the radiopharmaceutical.

Differential oxygen sensitivities in G6PDH activities of cultured keloid and normal skin dermis single cells.

Cell cultures of excised keloids and biopsied normal skin were established from patients of the Plastic Surgery Department of Singapore General Hospital and their single cells quantitated for glucose-6-phosphate dehydrogenase (G6PDH) activity using microspectrophometry. G6PDH has been cited as a transformation-linked discriminant. Analysis of variance shows no difference in overall activity between skin and keloid cells, but under oxygen saturation conditions, keloid G6PDH activity was significantly higher than skin G6PDH activity, although they were almost identical under nitrogen saturation conditions. Differential oxygen sensitivity in G6PDH activities between malignant and non-malignant cells have been reported, but its occurrence between keloid and normal skin cells is novel, especially as the keloid is regarded as a benign tumor with a zero carcinogenicity rate.

Hyperinsulinaemia in thyrotoxic hypokalaemic periodic paralysis.

To test the hypothesis that there is an abnormal serum insulin response to a carbohydrate load in thyrotoxic hypokalaemic periodic paralysis (THPP), 18 men with THPP and 15 with uncomplicated thyrotoxicosis were studied during an oral glucose tolerance test. The THPP group had significantly higher fasting insulin concentrations (27.6 [3.6] vs 13.4 [1.8] mU/l; p less than 0.005) and a higher overall insulin response to oral glucose (p less than 0.001 by ANOVA) than the thyrotoxicosis group. There were no significant differences in fasting or stimulated glucose. Hyperinsulinaemia may be an important factor in the precipitation of acute paralysis in THPP.