Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz-1 domain.

Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

Rapid molecular diagnosis of the Gilbert's syndrome-associated exon 1 mutation within the UGT1A1 gene.

Gilbert's syndrome is suspected in patients with unconjugated hyperbilirubinemia caused by decreased activity of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene in the absence of abnormal liver function and hemolysis. The major genetic variants underlying Gilbert's syndrome are TATA-box repeats of the promoter region and exon 1 G211A of the coding region, particularly in Asians. The efficacy of DNA melting curve analysis, however, has not been established for the G211A mutation. For rapid and accurate molecular diagnosis of Gilbert's syndrome, DNA melting curve analysis was evaluated for its genotyping capability not only for TATA-box repeats of the UGT1A1 promoter, but also for G211A of UGT1A1 exon 1. TA repeats within the TATA-box sequence and the exon 1 G211A mutation of the UGT1A1 gene were analyzed by DNA melting curve analysis. To evaluate the assay reliability, direct sequencing or polyacrylamide gel electrophoresis was used as a comparative method. All homozygous and heterozygous polymorphisms of A(TA)7TAA within the TATA-box allele and of exon 1 G211A mutants of the UGT1A1 gene were successfully identified with DNA melting curve analysis. DNA melting curve analysis is, therefore, an effective molecular method for the rapid diagnosis of Gilbert's syndrome, as it detects not only TATA-box polymorphisms but also the exon 1 G211A mutation located within the UGT1A1 gene.

A phase II study of erlotinib in combination with bevacizumab versus chemotherapy plus bevacizumab in the first-line treatment of advanced non-squamous non-small cell lung cancer.

BACKGROUND: Molecularly targeted agents for non-small cell lung cancer (NSCLC) can provide similar efficacy to chemotherapy without chemotherapy-associated toxicities. Combining two agents with different modes of action could further increase the efficacy of these therapies. The TASK study evaluated the efficacy and safety of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in combination with the anti-angiogenic agent bevacizumab as first-line therapy in unselected, advanced non-squamous NSCLC patients. METHODS: Patients were recruited from December 2007 to September 2008. Planned sample size was 200 patients, a total of 124 patients were randomized. Patients were randomized using a minimization algorithm 1:1 to receive bevacizumab (iv 15 mg/kg day 1 of each 21-day cycle) plus chemotherapy (gemcitabine/cisplatin or carboplatin/paclitaxel standard doses, 4-6 cycles) (BC arm) or bevacizumab plus erlotinib (p.o. 150 mg/day; BE arm) until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS). If the hazard ratio (HR) of PFS for BE relative to BC was above 1.25 at the pre-planned interim analysis in favor of BC, the study would be re-evaluated. Secondary endpoints included overall survival, response rate and safety. RESULTS: All randomized patients (n = 63 BE; n = 61 BC) were evaluated for the efficacy analyses. At the updated interim analysis, median PFS was 18.4 weeks (95% confidence interval [CI] 17.0-25.1) versus 25.0 weeks (95% CI 20.6-[not reached]) for BE versus BC, respectively (HR for death or disease progression, BE relative to BC, 2.05, p = 0.0183). The incidence of death was 19% for BE treatment compared with 11.5% for BC treatment. The HR for PFS at the updated interim analysis was above 1.25, therefore patients on the BE arm were permitted to change arms or switch to another drug and the study was terminated. Adverse events reported were as expected. CONCLUSIONS: The TASK study did not show a benefit in terms of PFS for the combination of erlotinib with bevacizumab in unselected first-line advanced non-squamous NSCLC compared with chemotherapy plus bevacizumab.

Ensembled artificial neural networks to predict the fitness score for body composition analysis.

OBJECTIVES: To predict the nutrition and health status of staff and students in Yuan Ze University and select the influential variables from the total body composition variables, which should have similar predictive ability with the whole factors. DESIGN: Spontaneous and voluntary physical examination. SETTING: Sanitary and Health Care Section of Yuan Ze University in Taiwan. PARTICIPANTS: 1227 staff and students. MEASUREMENTS: With the help of Inbody720TM, 139 body composition variables were measured and 60 variables were retained after data pre-processing. An ensembled artificial neural networks (EANN) prediction model was established and seven different methods for assessing variables importance were applied. Besides, classical linear and logistic regression models were developed for comparison with EANN prediction results. RESULTS: The prediction performance of EANN model was satisfactory (RMSE (train) = 0.2686, RMSE (validation) = 0.2648, RMSE (test) = 0.3492). Since both the actual and simulation fitness score were at the range of 0 to 100, according to rounding off rule, the simulated value was almost the same with actual value. Besides, 12 important variables were obtained by seven methods for quantifying variable importance in EANN, which had similar predictive capability with 60 variables (RMSE (train) = 0.3263, RMSE (validation) = 0.322, RMSE (test) = 0.3226). The linear and logistic regression models results were both evidently worse than EANN results. CONCLUSION: The results confirm that EANN is appropriate to approximate such a complicated, non-invasive and highly non-linear problem as body composition analysis. It can be helpful for nutritionists to manage and improve the nutrition and health condition of staff and students, by adjusting the 12 most important variables.

Genetic analysis of haemophilia A in Taiwan.

Haemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene. Identification of these mutations is becoming increasingly important in a variety of clinical settings. The purpose of this report is to describe our experience of FVIII gene mutation analysis in the largest cohort of patients in Taiwan including the discovery of 21 novel mutations. We tested 115 HA patients from 91 unrelated families, including 79 severe, 15 moderate and 21 mild types starting with an assay for the intron 22 inversion by long range-PCR followed if necessary by additional genetic studies. Intron 22 inversion accounted for 27.8% of the total and 36.7% of severe HA patients respectively while intron 1 inversion comprised 7.6% of severe patients. These were clearly different from the known data in caucasian populations. Of 75 patients without intron 22 or 1 inversion, 70 from 62 unrelated families revealed 56 different mutations by denaturing high-performance liquid chromatography (DHPLC), of which 21 were novel. Also, the only female patient with severe HA was found to have heterozygous non-sense mutation (c.6683G>A) of exon 24. Seven patients, including five without amplified PCR product and two without encoded DNA defect turned out to have exon(s) deletion or insertion by reverse transcript PCR (RT-PCR). In our study, the combination of various molecular techniques including LR-PCR, multiplex PCR, DHPLC and RT-PCR analysis enabled definitive detection of the causative FVIII gene defects in 112 of 113 (99%) HA patients.

Efficacy and tolerability of bevacizumab plus capecitabine as first-line therapy in patients with advanced hepatocellular carcinoma.

BACKGROUND: Molecularly targeted agents with anti-angiogenic activity, including bevacizumab, have demonstrated clinical activity in patients with advanced/metastatic hepatocellular carcinoma (HCC). This multicentre phase II study involving patients from several Asian countries sought to evaluate the safety and efficacy of bevacizumab plus capecitabine in this population. METHODS: Histologically proven/clinically diagnosed advanced HCC patients received bevacizumab 7.5 mg kg(-1) on day 1 and capecitabine 800 mg m(-2) twice daily on days 1-14 every 3 weeks as first-line therapy. RESULTS: A total of 45 patients were enrolled; 44 (96%) had extrahepatic metastasis and/or major vessel invasion and 30 (67%) had hepatitis B. No grade 3/4 haematological toxicity occurred. Treatment-related grade 3/4 non-haematological toxicities included diarrhoea (n=2, 4%), nausea/vomiting (n=1, 2%), gastrointestinal bleeding (n=4, 9%) and hand-foot syndrome (n=4, 9%). The overall response rate (RECIST) was 9% and the disease control rate was 52%. Overall, median progression-free survival (PFS) and overall survival (OS) were 2.7 and 5.9 months, respectively. Median PFS and OS were 3.6 and 8.2 months, respectively, for Cancer of the Liver Italian Programme (CLIP) score < or =3 patients, and 1.4 and 3.3 months, respectively, for CLIP score 4 patients. CONCLUSION: The bevacizumab-capecitabine combination shows good tolerability and modest anti-tumour activity in patients with advanced HCC.

Prevalence of von Willebrand disease in women with iron deficiency anaemia and menorrhagia in Taiwan.

Iron deficiency anaemia (IDA) is a frequently encountered disease, which can be attributed to menorrhagia. Most female patients with von Willebrand disease (VWD) have menorrhagia. The aim of this study was to investigate the prevalence of VWD in women with both IDA and menorrhagia in Taiwan. From January to December 2005 and November 2006 to January 2007, 56 consecutive patients with both IDA and menorrhagia were enrolled in this study. Their median age was 41 years (range 18-53). IDA was diagnosed by anaemia plus either low ferritin or transferrin saturation. Menorrhagia was evaluated by patient's menses history. Both von Willebrand factor antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) were measured for each patient. Bleeding time (BT) and platelet function analyser (PFA)-100 assay were determined as ancillary tests. The VWD diagnosis was established if: (i) both VWF:Ag (<50%) and VWF:RCo (<50%) were low; (ii) either VWF:Ag or VWF:RCo was low plus prolonged BT or prolonged PFA closure times. VWF multimer analysis was performed for subtype confirmation of VWD. Nine of the 56 (16.1%) patients were identified to have VWD. VWD patients with menorrhagia might develop IDA at younger age (34.3 vs. 39.7, P = 0.09) and had more IDA recurrence (75% vs. 16%, P = 0.03) than those patients without VWD. Of the eight VWD patients with VWF multimer analyses, all were revealed to have type I VWD. Our study demonstrates that VWD was not uncommon in women with both IDA and menorrhagia in Taiwan.

Influence of temperature on the ontogenetic expression of neural development-related genes from developing tilapia brain expressed sequence tags.

The developing central neural circuits in teleosts are genetically controlled and temperature-initiated. We compiled a list of transcripts expressed in the developing tilapia (Oreochromis mossambicus) brain using expressed sequence tags derived from the developing brain, and investigated genes with thermosensitive ontogenetic expression. Of 1084 clones, 893 were unique genes, 445 of which were known. Fourteen of the latter were neural development-related, and the ontogenetic expression of nine was temperature-influenced. Discs large homolog 5, myelin expression factor 2, plasticity-related protein-2, tsc2 gene product-related genes, and an inhibitor of differentiation protein 2 (Id2) were differentially temperature-influenced according to their developmental stages. Endothelial differentiation-related factor 1, midkine-related growth factor b, and mitogen-activated protein kinase 14b were specifically influenced by elevated temperature, and beta-catenin-like isoform 1 by lower temperature. Neural development-related genes, particularly those with thermosensitive ontogenetic expression, might be important for developing central neural circuits in teleosts.

An open, multi-centre, phase II clinical trial to evaluate the efficacy and safety of paclitaxel, UFT, and leucovorin in patients with advanced gastric cancer.

The aim of the study was to evaluate the response rate and safety of weekly paclitaxel (Taxol((R))) combination chemotherapy with UFT (tegafur, an oral 5-fluorouracil prodrug, and uracil at a 1 : 4 molar ratio) and leucovorin (LV) in patients with advanced gastric cancer. Patients with histologically confirmed, locally advanced or recurrent/metastatic gastric cancer were studied. Paclitaxel 1-h infusion at a dose of 100 mg m(-2) on days 1 and 8 and oral UFT 300 mg m(-2) day(-1) plus LV 90 mg day(-1) were given starting from day 1 for 14 days, followed by a 7-day period without treatment. Treatment was repeated every 21 days. From February 2003 to October 2004, 55 patients were enrolled. The median age was 62 years (range: 32-82). Among the 48 patients evaluated for tumour response, two achieved a complete response and 22 a partial response, with an overall response rate of 50% (95% confidence interval: 35-65%). All 55 patients were evaluated for survival and toxicities. Median time to progression and overall survival were 4.4 and 9.8 months, respectively. Major grade 3-4 toxicities were neutropenia in 25 patients (45%) and diarrhoea in eight patients (15%). Although treatment was discontinued owing to treatment-related toxicities in nine patients (16%), there was no treatment-related mortality. Weekly paclitaxel plus oral UFT/LV is effective, convenient, and well tolerated in treating patients with advanced gastric cancer.

Hepatitis B virus reactivation in breast cancer patients undergoing cytotoxic chemotherapy and the role of preemptive lamivudine administration.

BACKGROUND: Recent data suggest that hepatitis B virus (HBV) reactivation develops in 41% of breast cancer (BC) patients carrying HBV after chemotherapy. Our study aimed to determine the role of preemptive use of lamivudine in BC patients undergoing chemotherapy. PATIENTS AND METHODS: The test group consisted of 11 female patients with BC who were seropositive for hepatitis B surface antigen (HBsAg). Of these, 10 patients were treated in an adjuvant setting and one for metastatic disease. Lamivudine was given from the start of chemotherapy and was maintained until 1 month after the last infusion of chemotherapy. The control group consisted of nine historical BC patients carrying HBV and received similar systemic chemotherapy without preemptive lamivudine. Variables including HBsAg, HBV envelope antigen, anti-HBV envelope antibody, serial serum alanine transaminase (ALT), quantitative HBV viral DNA analysis, and HBV-DNA precore promoter and precore sequence were monitored. Test for emergence of mutant strains, notably nucleotide 550, was performed 6 months after the completion of chemotherapy. RESULTS: All patients tolerated lamivudine well without development of evident HBV reactivation or overt hepatitis. Serum ALT remained unchanged without rebound hepatitis after cessation of chemotherapy and withdrawal of lamivudine. No emergence of lamivudine-selective resistant strain (so-called tyrosine-methionine-aspartate-aspartate mutations) was observed. CONCLUSIONS: Our results encourage preemptive use of lamivudine for prevention of HBV reactivation in patients who need short-term chemotherapy.

Phase II study of weekly oxaliplatin and 24-h infusion of high-dose 5-fluorouracil and folinic acid in the treatment of advanced gastric cancer.

To investigate the efficacy and safety of combining weekly oxaliplatin with weekly 24-h infusion of high-dose 5-fluorouracil (5-FU) and folinic acid (FA) in treatment of patients with advanced gastric cancer. Patients with histologically confirmed, locally advanced or recurrent/metastatic gastric cancer were studied. Oxaliplatin 65 mg m(-2) 2-h intravenous infusion, and 5-FU 2600 mg m(-2) plus FA 300 mg m(-2) 24-h intravenous infusion, were given on days 1 and 8, repeated every 3 weeks. Between January 2001 through January 2002, 55 patients were enrolled. The median age was 64 years (range: 22-75). In all, 52 patients (94.5%) had recurrent or metastatic disease and three patients had locally advanced disease. Among 50 patients evaluable for tumour response, 28 patients achieved partial response, with an overall response rate of 56% (95% confidence interval (CI): 41.8-70.3%). All 55 patients were evaluated for survival and toxicities. Median time to progression and overall survival were 5.2 and 10.0 months, respectively, during median follow-up time of 24.0 months. Major grades 3-4 toxicities were neutropenia in 23 cycles (7.1%) and thrombocytopenia in 16 cycles (5.0%). Treatment was discontinued for treatment-related toxicities in nine patients (16.4%), of whom eight were due to oxaliplatin-related neurotoxicity. One patient (1.8%) died of neutropenic sepsis. This oxaliplatin-containing regimen is effective in the treatment of advanced gastric cancer. Except for neurotoxicity that often develops after prolonged use of oxaliplatin, the regimen is well tolerated.

Restoration of immunity and reactivation of hepatitis B virus after immunosuppressive therapy in a patient with severe aplastic anaemia.

We recently treated a patient with severe aplastic anaemia (SAA) who also had chronic hepatitis B virus (HBV) infection. The HBV serological status at the time of diagnosis of SAA was HBsAg(+) and HBeAg(+). Subsequent analysis of the precore region of HBV DNA showed wild-type. He received anti-thymocyte globulin (ATG) and cyclosporin A (CsA) therapy twice. After each course of ATG infusion and during CsA therapy he developed lymphopenia for 1 and 2.5 months, respectively. His serum alanine aminotransferase (ALT) became normalized during the period of lymphopenia, but the serum HBV viral load increased. When his peripheral lymphocytes count recovered, his ALT became elevated again. Lamivudine was effective to normalize his elevated ALT and suppress viral replication. The phenomenon observed in this case supports the prevailing notion that hepatitis B flare-up in HBV carriers after chemotherapy is caused by an immune-mediated mechanism. Meanwhile, this is the first documented case of SAA who developed HBV reactivation upon recovery of lymphopenia after immunosuppressive therapy. This also highlights the necessity of pre-emptive therapy with lamivudine in SAA/HBsAg(+) patients to receive immunosuppressive therapy with ATG/CsA.

Suppurative salmonella thyroiditis in a patient with chronic lymphocytic leukemia.

We describe an 82-year-old man with undiagnosed chronic lymphocytic leukemia (CLL) who presented with acute swelling of the thyroid goiter. Subsequent thyroid aspirate and blood culture yielded group B Salmonella thyroid abscess with septicemia. Infectious complications are the major cause of morbidity and mortality in patients with CLL since most of them can be timely detected and few can arise from innocent-looking lesions.

The high value of high-resolution computed tomography in predicting the activity of pulmonary tuberculosis.

SETTING: A 2500-bed medical centre in southern Taiwan. OBJECTIVE: To study the clinical value of high-resolution computed tomography (HRCT) in predicting the activity of pulmonary tuberculosis (TB). DESIGN: HRCTs were performed prospectively in 148 patients whose chest radiographs (CXRs) showed highly suspicious signs of pulmonary TB, predominantly upper lung field infiltration. The HRCT findings, interpreted independently by a pulmonologist and a radiologist, were used to predict the activity of pulmonary TB. RESULTS: Pulmonologist-interpreted and radiologist-interpreted HRCTs showed high sensitivity (both 93%), specificity (83 vs. 88%), accuracy (86 vs. 90%), positive predictive values (76 vs. 83%) and negative predictive values (both 95%). Kappa statistic indicates good inter-reader agreement. CONCLUSION: HRCT has a high value in predicting the activity of pulmonary TB. It is a useful tool in this regard when a patient with suspected pulmonary TB lacks microbiologic proof, when clinical condition makes invasive diagnosis impossible or when a patient has completed anti-tuberculosis treatment with no compatible unequivocal CXR.