Biomedical subjects
Publications and source records attributed to C H Toh.
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Although specific assays of coagulation factors are essential for diagnostic purposes they only give partial information about an individual's haemostatic state. This can be better assessed by various global tests, and recent developments and evaluations of five such tests are described in this symposium: the PFA-100; waveform analysis; thrombin generation; overall haemostasis potential; thrombelastography. Each test has advantages in various applications, but the thrombin generation test and waveform analysis have been found most useful in haemophilia, whilst the PFA-100 is helpful in von Willebrand's disease.
CD154 is an important regulator of chronic lymphocytic leukaemia (CLL)-cell survival. In CLL, high serum levels of VEGF are a feature of advanced disease, and we and others have previously shown that CLL cells produce and secrete this growth factor. Since CD154 stimulates VEGF production in other cell types, and VEGF is known to promote cell survival, we examined whether the cytoprotection of CLL cells by CD154 involves VEGF. We report for the first time that treatment of CLL cells with CD154 results in increased VEGF production and demonstrate involvement of NF-kappaB in this process. Moreover, we show that CD154-induced CLL-cell survival is reduced by anti-VEGF-neutralising antibody and by inhibiting VEGF receptor (VEGFR) signalling with SU5416. However, addition of exogenous VEGF alone or blocking secreted autocrine VEGF had little or no effect on CLL-cell survival. We therefore conclude that CLL-cell cytoprotection in the presence of CD154 requires combined signalling by both CD40 and VEGFR. This combined signalling and resulting cytoprotection were shown to involve NF-kappaB activation and increased survivin production. In conclusion, our findings identify autocrine VEGF as an important mediator of the antiapoptotic effect of CD40 ligation, and thus provide new insights into CLL-cell rescue by CD154 in lymphoreticular tissues.
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Summary. The biphasic waveform is an early marker of disseminated intravascular coagulation (DIC). Neutrophil elastase (NE) cleaves coagulation factors; thus, elevated elastase levels or its dysregulation by alpha-1-protease inhibitor (Alpha1PI) may be linked to DIC. Time courses over a period were determined for factors associated with NE and coagulation in 14 Intensive Care Unit patients with a biphasic waveform who developed DIC. The data were analyzed using a random coefficient linear regression model to predict the variables' mean values on day 0 and their mean rates of change over the period in which the biphasic waveform appeared. The biphasic waveform was normal on day 0, maximized on day 1, and approached normal again by day 4. Alpha1PI/NE complex levels were 2.5-fold greater than normal for the entire period. The A1PI activity, antigen, and specific activity levels were normal on day 0 and increased thereafter by 21.0, 10.5, and 8.9% of normal per day, respectively. Factor II, V, VII, IX, and X activity levels were, respectively, 57, 46, 46, 77, and 46% of normal on day 0, whereas factor VIII and fibrinogen levels were normal. All coagulation factor levels trended upward with time but not significantly. The prothrombin time, but not the activated partial thromboplastin time, was prolonged, and the platelet counts and hematocrits were below normal on day 0 and remained so thereafter. We conclude that events associated with neutrophil activation, elastase release, and perturbations of coagulation precede both the appearance of the biphasic waveform and the diagnosis of DIC in these patients.
Combined hepatocellular-cholangiocarcinoma (HCC-CC) is a rare primary liver tumour. We report a carrier of both HBV and HCV presenting with intermittent abdominal pain, fever, chillness and elevated á-fetoprotein (AFP) of 1197 ng/ml. Computed tomography showed an irregular hypodense mass in the left lateral segment of the liver with vague contrast enhancement and multiple regional lymphadenopathy. Hepatic angiogram showed that the mass was hypovascular and the left portal vein was occluded with a tapered end. Percutaneous ultrasound-guided core needle biopsy of the liver yielded HCC-CC. We suggest that HCC-CC should be considered in hypovascular liver tumours with striking elevation of serum AFP and multiple regional lymphadenopathy.
OBJECTIVE: To assess the maternal response to low molecular weight heparin during pregnancy, by estimation of plasma anti-Xa activity, at three specified gestation points and in the nonpregnant state. METHODS: A longitudinal, prospective, observational study was set in a tertiary referral recurrent miscarriage clinic. Twenty-four women, attending consecutively, were invited to participate and gave informed consent. Each woman had a history of recurring pregnancy loss and positive preconception screening for antiphospholipid syndrome. After confirmation of a viable pregnancy all subjects began taking 5000 IU of dalteparin once daily subcutaneously. Serial measurement of plasma anti-Xa activity after administration of dalteparin was performed at three standard gestation points (12, 24, and 36 weeks) and in the nonpregnant state (6 weeks postpartum). RESULTS: Peak anti-Xa levels occurred at 4 hours postbolus in pregnancy, as compared with 2 hours in the nonpregnant state. The mean anti-Xa levels at 12, 24, and 36 weeks' gestation were significantly reduced, at 2 hours postinjection, as compared with the nonpregnant state (P <.001, P <.01, P <.001, respectively). The lowest dose-response curve was at 36 weeks' gestation. A repeated-measures analysis of variance found a significant difference (P <.05) between the 36-week group and the postterm group but not between any of the other groups. CONCLUSION: During pregnancy, differences in the pharmacokinetics of low molecular weight heparin were observed, with an overall reduction in anti-Xa activity. On the basis of this study it is questionable to extrapolate dosing and lack of dose monitoring, in pregnant women, using data derived from a nonpregnant population.
Transmittance waveform charts the changes in light transmittance on standard coagulation assays, such as the prothrombin time (PT) and activated partial thromboplastin time (APTT). Analysis and characterization of these data on photo-optical coagulation analysers provides additional qualitative and quantitative information to that obtained using the clotting time alone. The most thoroughly evaluated clinical application is that of the biphasic APTT waveform with disseminated intravascular coagulation (DIC). The degree of waveform abnormality correlates directly with the severity of haemostatic dysfunction and allows for both the prediction and monitoring from non-overt to overt DIC. As its performance is simple and rapid, this provides the means for targeting therapeutic intervention to an earlier stage of DIC. The recent identification that the mechanism underlying the biphasic waveform is a complex that exists in vivo between C reactive protein with very low density lipoprotein, provides potentially important insights into the molecular pathogenesis of DIC. Thus, in addition to the immediate clinical utility in diagnostic practice, it has important applications as a research tool. Preliminary experience in the application of this technology to the diagnosis and management of the haemophilias and the lupus anticoagulant syndrome has also provided evidence of the power and utility of waveform analysis in essentially simple clotting assays.
The 3' end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF. (Blood. 2001;98:674-680)
OBJECTIVE: To examine the prevalence of maternal thrombophilia in women with severe preeclampsia/eclampsia, placental abruption, fetal growth restriction, and unexplained stillbirth. METHODS: We studied 102 women who had pregnancy complications and 44 healthy women with uncomplicated pregnancies. All women were tested 10 weeks postpartum for mutations of factor V Leiden, methylenetetrahydrofolate reductase (MTHFR) C677T, and G20210A prothrombin gene; deficiencies of protein C, protein S, and antithrombin III; and the presence of lupus anticoagulant and anticardiolipin antibodies. We aimed to recruit 100 cases and 300 controls to detect a 10% difference in thrombophilia between the groups. However, we were able to recruit only 44 controls. RESULTS: Abnormal thrombophilia screen was found in 54 women with pregnancy complications (53%) and in 17 women (39%) with normal pregnancies (odds ratio [OR] 1.8; 95% confidence interval [CI] 0.87, 3.67). Mutations encoding for factor V Leiden, G20210A prothrombin gene, and MTHFR C677T (homozygous) were identified in 18% of women with complications compared with 16% of controls (OR 1.1; 95% CI 0.44, 2.94). Activated protein C resistance, not due to factor V Leiden mutation, was the most common thrombophilic defect, found in 26% of women with pregnancy complications compared with 18% of controls (OR 1.5; 95% CI 0.63, 3.73). Twenty women with complications (20%) had multiple thrombophilic defects compared with four controls (9%) (OR 2.4; 95% CI 0.78, 7.61). CONCLUSION: In our cohort of women with pregnancy complications, maternal thrombophilia was less common than previously thought, and multiple thrombophilias were not a major additional risk factor.
The lack of improvement in the clinical prognosis of disseminated intravascular coagulation (DIC) poses searching questions on the value of current laboratory testing in this area. There is little immediate chance of advancement unless new tests or new modes of analyzing currently available tests place an emphasis on identifying the early phase of DIC. An equal emphasis is also required on practicality to ensure that recommendations are applicable to the routine clinical setting. These two priorities need to be fulfilled to facilitate earlier therapeutic intervention in the clinical trials required for formal assessment of promising and emerging new molecules from successful animal studies. This article therefore discusses the advantages and disadvantages of available assays in DIC with these perspectives in mind. It also proposes a format to bring together the necessary elements required to make an impact on ultimately delivering clinical improvement in this critical area.
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Expansion of primary solid tumors and their malignant dissemination are angiogenesis-dependent. Vascular endothelial growth factor (VEGF) is the key factor playing a pivotal role in solid tumor-induced angiogenesis. Recent studies indicate that angiogenesis may also be involved in the pathogenesis of certain hemic malignancies, including B-cell chronic lymphocytic leukemia (B-CLL). Mechanisms underlying angiogenesis in B-CLL and the role of VEGF in this process are incompletely understood. In this study, it was examined whether angiogenically functional VEGF is produced by B-CLL cells. Immunohistochemical staining with antibodies against VEGF and CD34, an endothelial cell marker, demonstrated the presence of VEGF protein and abundant blood vessels in infiltrated lymphoreticular tissues. Low levels of VEGF were detected by ELISA in the culture media of unstimulated cells; this was enhanced up to 7-fold by hypoxic stimulation. SDS-PAGE and Western blot analysis of the concentrated culture media showed 2 isoforms of VEGF protein with molecular weights of 28 and 42 kd, respectively. RNA hybridization showed that these cells expressed VEGF mRNA. Reverse transcription-polymerase chain reaction, combined with nucleotide sequence analysis, revealed that the predominantly expressed isoforms were VEGF121 and VEGF165. Moreover, (3)H-thymidine incorporation and an in vivo angiogenic assay demonstrated that the VEGF produced by CLL cells can induce angiogenesis by stimulating endothelial cell proliferation. In conclusion, this study shows that B-CLL cells produce VEGF and demonstrates the angiogenic effects of this growth factor, which may be relevant for the tissue phase of the disease.
The finding of low factor VIII levels in pregnancy immediately raises concern of haemophilia A carriage, especially with a history of bleeding in the maternal grandfather. However, the diagnosis of type 2N von Willebrand disease (2N vWD) should also be considered as illustrated here. This is also the first reported case of the management of 2N vWD in pregnancy.
Vascular endothelial growth factor (VEGF) is one of the most potent factors for stimulating angiogenesis, an essential process required for expansion of primary tumour and dissemination of malignant cells. To investigate the possible role of VEGF in facilitating metastasis of prostate cancer via stimulating angiogenesis, we have used Northern and slot blotting, reverse transcription polymerase chain reaction, nucleotide sequence analysis and enzyme-linked immunosorbent assay to compare the VEGF expression in series of human and rat cell lines with either benign or malignant characteristics. We have also employed the chick chorioallantoic membrane (CAM) assay to measure the angiogenic activity of the VEGF derived from both benign and malignant cells. The level of VEGF mRNA expressed in the seven malignant human and rat cell lines is 3.5- to 10-fold higher than that expressed in the benign cell lines. The three metastatic variants, generated by transfection of a benign cell line with DNA extracted from prostate carcinoma cells, expressed 2.5 to 5 times more VEGF mRNA than their parental benign cells. While VEGF 121 and 165 were predominantly expressed by both the benign and malignant cells, the transcript representing VEGF 189 isoform was only detected in the malignant cells. At protein level, three human malignant cell lines produced more VEGF (2.7-7.9 ng ml(-1)) than the benign cell line (1.3 ng ml(-1)). CAM assay detected a VEGF-dependent angiogenic activity in the medium from malignant cells, but only a relatively weak VEGF-independent activity in the medium from benign cells. These results demonstrated that malignant cells did over-express VEGF and only the VEGF derived from malignant cells was angiogenically active. Thus, we suggest that the VEGF produced by malignant cells might play an important role in facilitating metastasis of prostatic cancer.
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