Corrigendum to "Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices" (J. Dairy Sci. 105:9995-10006).

Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with ferritin iron supplementation. OnlyStaphylococcus hominis strains originating from quarter milk were able to significantly utilize ferritin as an iron source to reverse the growth inhibition caused by chelating agent 2,2'-bipyridyl in varying degrees. Both S. chromogenes strains (IM and TA) and all S. hominis strains were unable to significantly use lactoferrin as an iron source for growth recovery.

Comparison of non-aureus staphylococcal and mammaliicoccal species found in both composite milk and bulk-tank milk samples of dairy cows collected in tandem.

Non-aureus staphylococci and the closely related mammaliicoccal species (NASM) are the most common causes of bovine subclinical mastitis on modern dairy farms and are highly prevalent in bulk-tank milk. The purpose of this study was to determine the distribution of NASM in both composite cow milk (CCM) and bulk-tank milk (BTM) samples collected in tandem in commercial Flemish dairy herds and to estimate the origin of the different (subgroups of) NASM species present in BTM by applying strain typing (random amplification of polymorphic DNA or random amplified DNA [RAPD]). A single cross-sectional sampling was performed over 5 herds that volunteered to participate in the study. Composite cow milk samples (n = 356) were collected from all lactating cows (except those with clinical mastitis) during a milking in tandem with 6 BTM samples per herd sequentially collected immediately post that milking (n = 30). In total, 421 and 80 NASM isolates were recovered and identified by MALDI-TOF mass spectrometry from the CCM and BTM samples, respectively and a total of 21 and 12 different NASM species were identified from CCM and BTM samples, respectively. Staphylococcus cohnii was the most prevalent NASM species found in BTM followed by Staphylococcus haemolyticus, Staphylococcus epidermidis, Mammaliicoccus lentus, and Staphylococcus equorum, whereas from CCM samples the most common species were S. hemolyticus, S. cohnii, S. equorum, S. epidermidis, and Staphylococcus chromogenes. The prevalent NASM species in both CCM and BTM samples was distinct for each herd, corroborating other studies observing a herd-specific NASM microbiota. Random amplified DNA analysis was performed on 9 NASM species (S. chromogenes, S. epidermidis, S. haemolyticus, S. equorum, Mammaliicoccus sciuri, Staphylococcus xylosus, S. cohnii, Staphylococcus debuckii, and M. lentus) because these species were isolated from both sample types in a herd. The same RAPD types were found in both sample types for all NASM species selected for strain typing in varying degrees. When assessing the distribution of NASM species, differences within NASM species should be examined meaning a closer look should be taken at the strain level rather than at the species level only.

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

Short communication: Barrier characteristics of 3 external teat sealants to prevent bacterial penetration under in vitro conditions using rubber calf-feeding nipples.

The main objective of this study was to evaluate the barrier characteristics of 3 external teat sealants for dry cows in preventing bacterial penetration by 3 common major mastitis pathogens (Escherichia coli, Staphylococcus aureus, and Streptococcus uberis) via a novel in vitro simulation model using rubber calf-feeding nipples. All feeding nipples were filled with a sterile cotton plug soaked in sterile broth heart infusion medium and were treated as follows: rubber teats 1 and 5 were sealed with Ubera Dry (Inovet, Arendonk, Belgium); rubber teats 2 and 6 were sealed with T-Hexx Dry (Huvepharma Livestock, St. Louis, MO); rubber teats 3 and 7 were sealed with Uddergold Dry (Ecolab Food and Beverage Division, St. Paul, MN); and rubber teats 4 and 8 remained unsealed and served as positive and negative controls, respectively. After the dips had dried, rubber teats 1 to 4 were immersed in a suspension of E. coli (experiment 1), Staph. aureus (experiment 2), or Strep. uberis (experiment 3; ≥1.5 × 108 cfu/mL) for 24 h, whereas rubber teats 5 to 8 were not exposed to the bacterial suspensions. All external teat sealants adhered well to the rubber teats. All cotton plugs collected from the teats that were not exposed to E. coli, Staph. aureus, or Strep. uberis (rubber teats 5 to 8) remained culture-negative, except for 1 (due to contamination). Of the rubber teats that were exposed to the major mastitis pathogens, all cotton plugs collected from the teats dipped with Ubera Dry and T-Hexx Dry remained culture-negative for the mastitis pathogen they were exposed to. The cotton plugs of the rubber teats that were sealed with Uddergold Dry and exposed to E. coli and Strep. uberis showed positive cultures for the respective bacteria, as demonstrated using strain-typing. The cotton plugs collected from the rubber teats that were not sealed with an external teat sealant and that served as positive controls became culture-positive for the mastitis pathogens they were exposed to. We conclude that Ubera Dry showed comparable and superior barrier performance against penetration of E. coli, Staph. aureus, and Strep. uberis compared with T-Hexx Dry and Uddergold Dry, respectively, under in vitro conditions using a novel in vitro simulation model. Although one should be aware that the method has not yet been validated to predict risk of intramammary infections, the proposed technique can be a meaningful starting point to evaluate and compare the barrier characteristics of external teat sealants in preventing bacterial penetration. A large-scale clinical trial is needed before any definite conclusions can be drawn as to the adherence, duration of adherence, barrier performance, and efficacy in protection against intramammary infections of the 3 external teat sealants under field conditions.

Coxiella burnetii immunogenic proteins as a basis for new Q fever diagnostic and vaccine development.

Coxiella burnetii is the etiological agent of the zoonosis Q fever, which can cause an acute or a chronic, life-threatening disease in humans. It presents a highly stable cell form, which persists in the environment and is transmitted via contaminated aerosols. Ruminants are considered as the main reservoir for human infections but are usually asymptomatic. Subclinical infection in these animals and the occurrence of serologically negative shedders hamper the identification of infected animals with the currently used diagnostic techniques. This suboptimal sensitivity limits reliable identification of infected animals as well as the well-timed implementation of countermeasures. This review summarizes compounds, focusing on C. burnetii seroreactive proteins, which were discovered in recent immunoproteomic studies. We analyzed these proteins regarding their localization, function, frequency of citation, differences seen in various host species as well as sensitivity and specificity. Finally, proteins useful for the development of new diagnostic test systems as well as subunit vaccines were discussed.

Two-Particle Interference of Electron Pairs on a Molecular Level.

We investigate the photodouble ionization of H_{2} molecules with 400 eV photons. We find that the emitted electrons do not show any sign of two-center interference fringes in their angular emission distributions if considered separately. In contrast, the quasiparticle consisting of both electrons (i.e., the "dielectron") does. The work highlights the fact that nonlocal effects are embedded everywhere in nature where many-particle processes are involved.

Low zinc and selenium concentrations in sepsis are associated with oxidative damage and inflammation.

BACKGROUND: Oxidative stress with dysregulated inflammation are hallmarks of sepsis. Zinc and selenium have important antioxidant functions, such that they could be important in patients with sepsis. We used an in vitro approach to assess the effect of zinc and selenium on oxidative stress, mitochondrial function, and inflammatory responses in conditions mimicking sepsis and related the findings to plasma concentrations and biomarkers in patients with and without sepsis. METHODS: Human endothelial cells were exposed to a range of zinc and selenium concentrations in conditions mimicking sepsis. Zinc, selenium, and a series of biomarkers of oxidative stress and inflammation were measured in plasma from critically ill patients with and without sepsis. RESULTS: Culturing cells with different concentrations of zinc caused altered zinc transporter protein expression and cellular zinc content, and selenium affected glutathione peroxidase 3 activity. Although zinc or selenium at physiological concentrations had no effect on interleukin-6 release in vitro, higher concentrations of the trace elements were associated with improved mitochondrial function. Plasma zinc and selenium concentrations were low in patients [zinc: median (range) 4.6 (2.1-6.5) µM in control patients without sepsis and 3.1 (1.5-5.4) µM in patients with sepsis, P=0.002; and selenium: 0.78 (0.19-1.32) µM in control patients and 0.42 (0.22-0.91) µM in sepsis patients, P=0.0009]. Plasma concentrations of interleukin-6, other biomarkers of inflammation, and markers of oxidative damage to proteins and lipids were elevated, particularly in patients with sepsis, and were inversely related to plasma zinc and selenium concentrations. CONCLUSIONS: Zinc and selenium concentrations were reduced in critically ill patients, with increased oxidative stress and inflammatory biomarkers, particularly in patients with sepsis. Oxidative stress as a result of suboptimal selenium and zinc concentrations might contribute to damage of key proteins. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: registration number NCT01328509.

Monitoring of modified factor VIII and IX products.

The dawning era of novel recombinant factor VIII and factor IX concentrates, many of which have been bioengineered to achieve prolonged activity, brings with it the need to consider the most appropriate clinical laboratory approaches for potency assignment, as well as the measurement of postinfusion levels. This session will highlight the known limitations and inconsistencies between existing assay methodologies with respect to currently available products, and discuss some of the early data with respect to the novel agents.

Early warnings from automatic milk yield monitoring with online synergistic control.

Sensors play a crucial role in the future of dairy farming. Modern dairy farms today are equipped with many different sensors for milk yield, body weight, activity, and even milk composition. The challenge, however, is to translate signals from these sensors into relevant information for the farmer. Because the measured values for an individual cow show nonstationary behavior, the concepts of statistical process control, which are commonly used in industry, cannot be used directly. The synergistic control concept overcomes this problem by on-line (real-time) modeling of the process and application of statistical process control to the residuals between the measured and modeled values. In this study, the synergistic control concept was developed and tested for early detection of anomalies in dairy cows based on detection of shifts in milk yield. Compared with the combination of visual observation and milk conductivity measurements, the developed strategy had a sensitivity of 63% for detecting clinical mastitis. Consequently, this technique could have added value on many farms, as it extracts practical information out of inexpensive data that are already available. As it can be easily extended to other measured parameters, the technique shows potential for early detection of other nutrition and health problems.

Compliance with international prevention guidelines for central-line-associated bloodstream infections in neonatal intensive care units in Belgium: a national survey.

BACKGROUND: Central-line-associated bloodstream infections (CLABSIs) are a preventable cause of morbidity among patients in neonatal intensive care units (NICUs). AIMS: To assess compliance with international guidelines for prevention of CLABSIs in Belgian NICUs, and to study unit characteristics contributing to CLABSIs. METHODS: A survey was undertaken to measure the adherence of various NICUs to the CLABSI prevention guidelines related to catheter insertion, catheter maintenance and quality control measurements. A Poisson regression model was used to estimate the CLABSI adjusted relative risk for each prevention guideline item implemented. Multi-variable linear regression was used to estimate associations between guideline compliance rate and facility characteristics and the incidence of CLABSIs for 2015-2016. FINDINGS: In Belgium, the overall CLABSI incidence density was 8.48/1000 central-line-days, and was higher in larger NICUs: 10.87‰ vs 6.69‰ (P<0.05). Adherence was highest for prevention items at catheter insertion (64%), and low for catheter maintenance and quality control items (47% and 50%, respectively). Superior adherence to insertion items (P=0.051) and quality performance items (P=0.004) was associated with decreased risk of CLABSIs, but this was not found for maintenance prevention items (P=0.279). After adjustment for guideline adherence, the size of the NICU was found to be an independent determinant for CLABSIs (P=0.002). CONCLUSIONS: In Belgium, the adherence of NICUs to international CLABSI prevention guidelines is moderate to poor. Compliance of NICUs with the guidelines is significantly associated with decreased CLABSI rates. The reasons for the gap between current practice in Belgian NICUs and international prevention guidelines need further investigation.

Metabolic tumour volume. Prognostic value in locally advanced squamous cell carcinoma of the head and neck.

PURPOSE: Evaluate the predictive and prognostic value of semi-quantitative FDG-PET variables derived from pretreatment FDG-PET images in patients suffering from locally advanced squamous cell carcinoma of the head and neck (SCCHN), treated by means of concomitant radiochemotherapy. PATIENTS, METHODS: 40 patients with newly diagnosed SCCHN that were treated with concomitant radiochemotherapy underwent FDG-PET/CT for treatment planning; 18 patients had neck dissection prior to their baseline scan and to receiving radiochemotherapy. FDG-PET images were used to calculate metabolic tumour volumes using region growing and a threshold of 50% (MTV50) of primary lesions and involved lymph nodes as well as the mean and maximum standard uptake value (SUVmean and SUVmax) of the primary tumours. RESULTS: Neither SUVmean nor SUVmax values of the primary tumour were significantly different between responders and non-responders whereas MTV50 values of the primary tumour proved significantly higher in non-responders. SUVmean, SUVmax and MTV50 of the primary tumour were not predictive for overall or disease free survival. Contrariwise, dichotomized summed MTV50 values (cut-off≥31 cm3) of the primary tumour and involved lymph nodes in patients that didn't have neck dissection prior to radiochemotherapy were predictive for disease free and overall survival in both univariate and multivariate analysis (p≤0.05). CONCLUSION: Summed MTV50 values of both the primary tumour and involved lymph nodes provided independent prognostic information on disease free and overall survival.

The assessment of viscosity measurements on the albumen of consumption eggs as an indicator for freshness.

Viscosity values of albumen reported in the literature are difficult to compare because different shear rates and different methods of sample preparation have been adopted. Therefore, we first investigated the effect of shear rate on the viscosity measurements of thick albumen. For intact albumen, a large intersample fluctuation in viscosity with increasing shear rate was observed. Furthermore, a large hysteresis effect was observed, indicating that the structural properties were substantially altered by the rotational behavior of the rotor. From this, we concluded that to obtain reliable measurements, the albumen needed to be mixed. After mixing, a smooth evolution in viscosity with increasing shear rate was observed. Compared with intact albumen, the hysteresis effect was smaller but still present. We next investigated the correlation between albumen viscosity and Haugh units. For this, we compared the viscosity of fresh eggs with the viscosity of eggs stored for 24 d at a temperature of 18°C. The Haugh units were first determined, and the viscosity was next determined on mixed albumen at a shear rate of 200 rad/s. Mean viscosity equaled 0.0304 and 0.0181 Pa/s for fresh eggs and eggs stored for 24 d, respectively. The decrease in viscosity during storage was significant (α = 0.05). Furthermore, we observed that the correlation between Haugh units and viscosity measured on the same egg was low. Fresh eggs having comparable Haugh units differed substantially in their measured viscosities, whereas for stored eggs, the Haugh units differed substantially but the viscosities were comparable. It is unlikely that the very large variation in rheological properties observed among fresh eggs reflects the natural variation in albumen freshness present after lay. Results suggest that these differences were partly due to the structural changes caused by albumen sampling and by the turning motion of the rotor. We conclude that determination of the rheological properties of albumen is practically infeasible and that albumen viscosity cannot be used as an index for albumen freshness.

The transmission color value: a novel egg quality measure for recording shell color used for monitoring the stress and health status of a brown layer flock.

Stress and diseases have the potential to influence the deposition of eggshell pigmentation during egg formation. Therefore, defining the shell color of eggs on a daily basis could be a representative method for monitoring stress or health status of a flock and maintaining good performance. A novel way of measuring eggshell color based on visible-near infrared transmission spectroscopy transmission spectra was defined: the transmission color value (TCV). The TCV was calculated as the ratio between the transmission at 643 nm (maximum absorbance of the pigmentation molecule protoporphyrin IX) and the transmission at 610 nm (a reference wavelength). Experiments were carried out to investigate the relevance of TCV for monitoring flock stress and health or even anticipating any factors unfavorable to performance. In 2 small experimental flocks, deliberate heat stress challenges were applied. A medium-scale experimental flock in an aviary was monitored on a daily basis during the whole productive period. From the deliberate heat stress challenges, it was seen that stress had a significant effect on eggshell pigmentation. This observation was confirmed in a daily monitored flock, in which, for example, an infectious bronchitis infection occurred. These stress situations were quickly reflected by an increased TCV value: more transmission due to less pigmentation and hence less absorbance at the pigmentation wavelength. Furthermore, for the observed problems in the daily monitoring, the TCV value signaled the problem earlier (4 d) than the average egg weight or even signaled when the other parameters did not signal anything. Measuring the TCV of all eggs produced on a daily basis provides relevant information on the stress or health status of a flock of brown layers. This could be used as an early detection of stress situations or emerging diseases, even before important quality and health damage can occur.

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix.

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.

Nation-wide prospective surveillance of Clostridium difficile infections in hospitals in Belgium, July 2007-June 2008.

We report here baseline data from the first year of compulsory surveillance of Clostridium difficile infections (CDI) in hospitals in Belgium. Between 1 July 2007 and 30 June 2008, 2,704 CDI were reported: 12% were recurrent and 66% were hospital-associated (half of which occurred 15 days or more after admission). CDI was considered the cause of death (direct or indirect) for 10% of the episodes. The median incidence of CDI was 1.5 per 1,000 admissions and 1.9 per 10,000 hospital-days for all cases, and 0.9 per 1,000 admissions, and 1.1 per 10,000 hospital-days for hospital-associated cases. Further investigation of risk stratification by average length of stay in the reporting hospitals is warranted as a way to improve the comparability of indicators across hospitals and surveillance systems. In spite of methodological issues, the surveillance of CDI in Belgian hospitals has been able to produce robust baseline data that should allow monitoring of trends at hospital and national level, and provide a basis for international comparisons. Remaining challenges are to define and monitor targets for the control of CDI, and to improve the individual feed-back of data at hospital level.

Postpartum C-Reactive Protein: A limited value to detect infection or inflammation.

BACKGROUND: During pregnancy the maternal immune system adjusts to preserve the foetoplacental unit. These adjustments lead to an increase in CRP, continuing into the postpartum. The objective of this study was to determineantepartal, peripartal and postpartal factors associated with an elevated CRP on the second postpartum day. METHODS: A retrospective quantitative, monocentric file analysis in which antepartal, peripartal and postpartal factors were collected from a convenience sample was performed. On the second day postpartum CRP was taken according to local protocol. Uni- and multi-variate analysis was performed to determine factors that are related to postpartum level of CRP. The total sample size consisted of 1400 patients. RESULTS: Multiple regression analysis indicated 11 factors related to increased CRP on the second day postpartum: gestational age (p=0.002), maternal blood leukocyte count on day 2 postpartum (p<0.001), artificial rupture of the membranes (p<0.001), fever during labor (p<0.001), indwelling urinary catheter (p=0.008), epidural anesthesia (p<0.001), fetal scalp electrode (p<0.001), primary planned caesarean (p=0.019), secondary caesarean h (p<0.001), formula feeding (p=0.030) and fever during postpartum (p=0.001). CONCLUSION: This research indicates that many antepartal, peripartal and postpartal factors are related to high postpartum CRP. CRP can not be used as a screening test test in the postpartum to discriminate between normal and pathologic inflammatory/infectious changes.

Note: Soft X-ray transmission polarizer based on ferromagnetic thin films.

A transmission polarizer for producing elliptically polarized soft X-ray radiation from linearly polarized light is presented. The setup is intended for use at synchrotron and free-electron laser beamlines that do not directly offer circularly polarized light for, e.g., X-ray magnetic circular dichroism (XMCD) measurements or holographic imaging. Here, we investigate the degree of ellipticity upon transmission of linearly polarized radiation through a cobalt thin film. The experiment was performed at a photon energy resonant to the Co L3-edge, i.e., 778 eV, and the polarization of the transmitted radiation was determined using a polarization analyzer that measures the directional dependence of photo electrons emitted from a gas target. Elliptically polarized radiation can be created at any absorption edge showing the XMCD effect by using the respective magnetic element.

Publisher Correction: Imaging the square of the correlated two-electron wave function of a hydrogen molecule.

The original version of this Article contained an error in the fifth sentence of the first paragraph of the 'Application on H2' section of the Results, which incorrectly read 'The role of electron correlation is quite apparent in this presentation: Fig. 1a is empty for the uncorrelated Hartree-Fock wave function, since projection of the latter wave function onto the 2pσu orbital is exactly zero, while this is not the case for the fully correlated wave function (Fig. 1d); also, Fig. 1b, c for the uncorrelated description are identical, while Fig. 1e, f for the correlated case are significantly different.' The correct version replaces 'Fig. 1e, f' with 'Fig. 2e and f'.

Requirements for cellular co-trafficking of factor VIII and von Willebrand factor to Weibel-Palade bodies.

BACKGROUND: von Willebrand factor (VWF) serves a critical role as a carrier of factor (F)VIII in circulation. While it is generally believed that FVIII and VWF assemble in circulation after secretion from different cells, an alternative view is that cells should exist that co-express FVIII and VWF. OBJECTIVES: In this study, intracellular co-expression of FVIII and VWF was studied, with particular reference to complex assembly and high-affinity interaction. METHODS: Using yellow fluorescent protein-tagged FVIII (FVIII-YFP) and cyan fluorescent protein-tagged VWF (VWF-CFP), we studied intracellular trafficking in human embryonic kidney (HEK293) cells and human umbilical vein endothelial cells (HUVEC). The role of the high-affinity interaction between FVIII and VWF was assessed using a FVIII-YFP variant carrying a Tyr1680Phe substitution, which abolishes high-affinity binding to VWF. Cellular trafficking studies were complemented by binding studies employing purified proteins. RESULTS: Solid phase binding assays employing FVIII-YFP demonstrated that the presence of the fluorescent moiety did not compromise high-affinity binding (K(d) = 0.065 +/- 0.008 nm) whereas the binding of the Tyr1680Phe FVIII-YFP variant was significantly reduced. Co-expression studies in HEK293 cells revealed intracellular co-storage of both FVIII-YFP and Tyr1680Phe FVIII-YFP within VWF-containing storage organelles. In addition, expression of FVIII-YFP and Tyr1680Phe FVIII-YFP in HUVEC demonstrated co-trafficking with endogenous VWF to authentic Weibel-Palade bodies (WPBs). CONCLUSIONS: Our findings demonstrate that FVIII trafficking to WPBs is independent of Tyr1680 and high-affinity binding to VWF. We therefore conclude that the structural requirements that determine intracellular co-trafficking differ from those that determine complex assembly in circulation.

Efficiency of von Willebrand factor-mediated targeting of interleukin-8 into Weibel-Palade bodies.

BACKGROUND: After de novo synthesis in endothelial cells, the chemokine interleukin-8 (IL-8) is targeted to endothelial cell-specific storage vesicles, the Weibel-Palade bodies (WPBs), where it colocalizes with von Willebrand factor (VWF). OBJECTIVE: In this study we investigated a putative regulator function for VWF in the recruitment of IL-8 to WPBs. METHODS: We performed a quantitative analysis of the entry of IL-8 into the storage system of the endothelium using pulse-chase analysis and subcellular fractionation studies. RESULTS: Using pulse-chase analysis of IL-1beta-stimulated human umbilical vein endothelial cells, we found that a small part of de novo synthesized IL-8 was retained in endothelial cells after 4 h. In density gradients of endothelial cell homogenates nearly equimolar amounts of VWF and IL-8 were present in subcellular fractions that contained WPBs. Furthermore, we found that IL-8 binds to immobilized VWF under the slightly acidic conditions thought to prevail in the lumen of the late secretory pathway. CONCLUSIONS: These observations indicate that the sorting efficiency of IL-8 into the regulated secretory pathway of the endothelium is tightly controlled by the entry of VWF into WPBs.