Preparation and in vitro evaluation of 111In-CHX-A"-DTPA-labeled anti-VEGF monoclonal antibody bevacizumab

Bevacizumab is a humanized monoclonal antibody that binds to vascular endothelial growth factor (VEGF) and prevents tumor angiogenesis. Radionuclide imaging using radiolabeled bevacizumab might be useful for selection of patients for anti-VEGF therapy. This study describes preparation of a potential imaging agent, 111In-CHX-A"-DTPA-bevacizumab, and evaluation of specificity of its binding to three tumor cell lines, SKOV3, LS174T and DU 145. Bevacizumab was conjugated with CHX-A"-DTPA and radiolabeled with 111In with high yield and excellent stability. Specificity of cellular binding was examined by a saturation assay using 100-fold excess of non-radiolabeled antibody. SKOV3 and LS174T tumor cell lines showed significantly specific binding, while DU 145 cells did not showed any specific binding. The specific binding is dependent to type of cell lines, which it is important for selection of tumor model for scintigraphic imaging of the VEGF expression.     

Factor XI antigen and activity in human platelets

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.     

Quantifying the risks and benefits of efavirenz use in HIV‐infected women of childbearing age in the USA

Objectives

The aim of the study was to quantify the benefits (life expectancy gains) and risks (efavirenz‐related teratogenicity) associated with using efavirenz in HIV‐infected women of childbearing age in the USA.

Methods

We used data from the Women's Interagency HIV Study in an HIV disease simulation model to estimate life expectancy in women who receive an efavirenz‐based initial antiretroviral regimen compared with those who delay efavirenz use and receive a boosted protease inhibitor‐based initial regimen. To estimate excess risk of teratogenic events with and without efavirenz exposure per 100 000 women, we incorporated literature‐based rates of pregnancy, live births, and teratogenic events into a decision analytic model. We assumed a teratogenicity risk of 2.90 events/100 live births in women exposed to efavirenz during pregnancy and 2.68/100 live births in unexposed women.

Results

Survival for HIV‐infected women who received an efavirenz‐based initial antiretroviral therapy (ART) regimen was 0.89 years greater than for women receiving non‐efavirenz‐based initial therapy (28.91 vs. 28.02 years). The rate of teratogenic events was 77.26/100 000 exposed women, compared with 72.46/100 000 unexposed women. Survival estimates were sensitive to variations in treatment efficacy and AIDS‐related mortality. Estimates of excess teratogenic events were most sensitive to pregnancy rates and number of teratogenic events/100 live births in efavirenz‐exposed women.

Conclusions

Use of non‐efavirenz‐based initial ART in HIV‐infected women of childbearing age may reduce life expectancy gains from antiretroviral treatment, but may also prevent teratogenic events. Decision‐making regarding efavirenz use presents a trade‐off between these two risks; this study can inform discussions between patients and health care providers.     

Cellular infiltration and cytokine mRNA expression in perennial allergic rhinitis

BACKGROUND: Allergen challenge in allergic rhinitis patients leads to local eosinophilia and Th2-type cytokine expression. Natural exposure to grass pollen is additionally characterized by epithelial mast-cell infiltration. We hypothesized that perennial allergic rhinitis is also associated with T-cell and eosinophil infiltration of the nasal mucosa, local Th2-type cytokine expression, and increased numbers of nasal epithelial mast cells. METHODS: Nasal biopsies from perennial allergic rhinitis patients and controls were analysed by immunocytochemistry for different cell populations and in situ hybridization for cytokine mRNA-expressing cells. RESULTS: Perennial allergic rhinitis was associated with increased numbers of submucosal CD3+ T cells (P=0.05), EG2+ activated eosinophils (P=0.01), and CD68+ macrophages (P=0.01) compared to controls. Epithelial, but not submucosal, tryptase-positive mast cells were also elevated in rhinitics compared to controls (P=0.01). The numbers of cells expressing interleukin (IL)-5 were higher (P=0.01) and the numbers of cells expressing IL-2 were lower (P=0.04) in rhinitic patients than controls. There were no significant differences for either IL-4 or interferon-gamma between the groups. CONCLUSIONS: Perennial allergic rhinitis is characterized by mast-cell migration into the epithelium; submucosal infiltration by T cells, eosinophils, and macrophages; and an imbalance in local T-cell cytokine production in favour of enhanced IL-5 and reduced IL-2 expression.     

Platelet kinetics in patients with bone marrow hypoplasia: evidence for a fixed platelet requirement

We have studied 16 normal subjects and 27 patients with stable, untreated thrombocytopenia secondary to bone marrow failure and platelet counts ranging from 12,000 to 70,000/microL. Autologous platelets were labeled with 51Cr for measurement of mean platelet life span in the normal subjects and in 20 patients. Labeled donor cells were used in the remaining subjects. Platelet survival, as determined with both autologous and homologous platelets, correlated directly with platelet count in the thrombocytopenic patients. Platelet life span was only modestly reduced in patients having counts in the range of 50,000 to 100,000/microL (7.0 +/- 1.5 days v 9.6 +/- 0.6; P less than .01) but was markedly reduced when the count fell below 50,000/microL (5.1 +/- 1.9 days, P less than .001). The recovery of donor platelets in severely thrombocytopenic recipients (60% +/- 15%) was equivalent to control values (66% +/- 8%; P greater than .2). The recovery of autologous platelets was normal when the platelet count exceeded 50,000/microL (74% +/- 15%) but was reduced in patients with lower counts (50% +/- 22%; P less than .01). All patient and normal data were well correlated by a model predicting a maximum platelet life span of 10 1/2 days and a fixed requirement for 7,100 platelets per microliter of blood per day, or about 18% of the normal rate of platelet turnover, which averaged 41,200 platelets per microliter per day. We conclude that although relatively few platelets are used to support vascular integrity, this requirement is reflected by a reduced platelet life span in marrow hypoplasia and may contribute to the shortening of platelet survival observed in other thrombocytopenias.     

脑死亡状态巴马小型猪肺水含量及肺超微结构变化

目的:观察脑死亡状态下巴马小型猪肺水含量及肺组织常规及超微结构改变。方法:实验于2003-08/2004-12在河南省实验动物中心及河南省病理学重点实验室完成。选用健康巴马小型猪11只,采用随机数字表法随机分为2组,对照组5只,脑死亡组6只。对照组持续麻醉维持24h,不建立脑死亡模型,24h后撤除麻醉,动物苏醒后处死;脑死亡组6只,应用改进的缓慢间断颅内加压法建立脑死亡模型,判定脑死亡后通过呼吸、循环支持维持动物脑死亡状态24h,24h后撤除呼吸、循环支持,动物心肺死亡。维持脑死亡状态下巴马小型猪的生命体征达到脑死亡供体要求,即平均动脉压≥60mmHg(1mmHg=0.133kPa),血氧饱和度≥95%,血氧分压≥100mmHg,体温≥35℃,中心静脉压≤10~12mmHg,血流动力学稳定。观察脑死亡组动物脑死亡24h后肺组织肺水含量、肺脏常规组织形态(光镜,苏木精-伊红染色)及超微结构变化(电镜),并与对照组比较。结果:脑死亡组1只动物在脑死亡造模过程中因心肺死亡脱失。①两组动物肺水含量比较:脑死亡组肺水含量显著高于对照组[(0.83±0.00,0.76±0.02)g/g(P<0.05)]。②两组动物肺脏常规组织形态及超微结构变化:脑死亡组动物肺组织光镜下见肺间质弥漫性水肿,肺泡间隔增宽,肺泡腔有渗出液,局灶性肺不张,肺毛细血管充血;电镜下可见肺泡Ⅱ型上皮细胞线粒体肿胀,线粒体嵴存在排列紊乱、消失,微绒毛缺失,板层小体数量减少、脱颗粒,结构有破坏。对照组光镜及电镜下未见明显的损伤性变化。结论:脑死亡状态可导致肺水含量增加,肺组织出现损伤性改变,可能对移植后肺组织功能造成一定影响。     

Point mutations in the conserved box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel

The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL- 3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy- terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF- mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG- CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR.     

Exploring quantitative methods for evaluation of lip function

Summary The objective was to explore quantitative methods for the measurement of lip mobility and lip force and to relate these to qualitative assessments of lip function. Fifty healthy adults (mean age 45 years) and 23 adults with diagnoses affecting the facial muscles (mean age 37 years) participated in the study. Diagnoses were Möbius syndrome (n = 5), Facioscapulohumeral muscular dystrophy (n = 6) and Myotonic dystrophy type 1 (n = 12). A system for computerised 3D analysis of lip mobility and a lip force meter were tested, and the results were related to results from qualitative assessments of lip mobility, speech (articulation), eating ability and saliva control. Facial expressions studied were open mouth smile and lip pucker. Normative data and cut‐off values for adults on lip mobility and lip force were proposed, and the diagnostic value of these thresholds was tested. The proposed cut‐off values could identify all inviduals with moderate or severe impairment of lip mobility but not always the milder cases. There were significant correlations between the results from quantitative measurements and qualitative assessments. The examined instruments for measuring lip function were found to be reliable with an acceptable measuring error. The combination of quantitative and qualitative ways to evaluate lip function made it possible to show the strong relation between lip contraction, lip force, eating ability and saliva control. The same combination of assessments can be used in the future to study if oral motor exercises aimed at improving lip mobility and strength could have a positive effect on lip function.     

In vitro and in silico evaluation of P‐glycoprotein inhibition through 99mTc‐methoxyisobutylisonitrile uptake

P‐glycoprotein (P‐gp) is a multidrug resistance (MDR) transporter with unknown structural details. This macromolecule is normally responsible for extruding xenobiotics from normal cells. Overexpression of P‐gp in tumor cells is a major obstacle in cancer chemotherapy. In this study, human 3D model of P‐gp was built by homology modeling based on mouse P‐gp crystallographic structure and stabilized through 1 ns molecular dynamics (MD) simulation. Stabilized human P‐gp structure was used for flexible docking of 80 drugs into the putative active site of P‐gp. Accordingly, digoxin, itraconazole, risperidone, ketoconazole, prazosin, verapamil, cyclosporine A, and ranitidine were selected for further in vitro assay. Subsequently, cell‐based P‐gp inhibition assay was performed on Caco‐2 cells while ^99mTc‐methoxyisobutylisonitrile (MIBI) was used as a P‐gp efflux substrate for calculating IC50 values. Results of the ^99mTc‐MIBI uptake in drug‐treated Caco‐2 cells were in agreement with the previously reported activities. This study for the first time described the relation between molecular dynamics and flexible docking with cellular experiments using ^99mTc‐MIBI radiotracer for evaluation of potencies of P‐gp inhibitors. Finally, results showed that our radiotracer–cell‐based assay is an accurate and fast screening tool for detecting P‐gp inhibitors and non‐inhibitors in drug development process.