Trigger-happy resident memory CD4+ T cells inhabit the human lungs.

Resident memory T cells (TRM) reside in the lung epithelium and mediate protective immunity against respiratory pathogens. Although lung CD8+ TRM have been extensively characterized, the properties of CD4+ TRM remain unclear. Here we determined the transcriptional signature of CD4+ TRM, identified by the expression of CD103, retrieved from human lung resection material. Various tissue homing molecules were specifically upregulated on CD4+ TRM, whereas expression of tissue egress and lymph node homing molecules were low. CD103+ TRM expressed low levels of T-bet, only a small portion expressed Eomesodermin (Eomes), and although the mRNA levels for Hobit were increased, protein expression was absent. On the other hand, the CD103+ TRM showed a Notch signature. CD4+CD103+ TRM constitutively expressed high transcript levels of numerous cytotoxic mediators that was functionally reflected by a fast recall response, magnitude of cytokine production, and a high degree of polyfunctionality. Interestingly, the superior cytokine production appears to be because of an accessible interferon-γ (IFNγ) locus and was partially because of rapid translation of preformed mRNA. Our studies provide a molecular understanding of the maintenance and potential function of CD4+ TRM in the human lung. Understanding the specific properties of CD4+ TRM is required to rationally improve vaccine design.

Modulation of the activated protein C pathway in severe haemophilia A patients: The effects of thrombomodulin and a factor V-stabilizing fab.

INTRODUCTION: The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APC's inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next-generation therapeutic targets for haemophilia treatment. AIMS: To determine ex vivo the respective concentration-dependent effects of TM and a FV-stabilizing Fab on the APC regulatory pathway in severe FVIII-deficient blood and plasma. METHODS: Ten severe haemophilia A subjects and one healthy control were enrolled. Blood was spiked with TM (0, 1, 2.5, 5, 10, 20.0 nmol/L) and FV-stabilizing Fab (0, 3, 15, 65, 300 nmol/L). The respective effects were compared to FVIII concentrations of 3- and 10% using rotational thromboelastometry clotting time (CT) and thrombin generation analysis (TGA). RESULTS: With 1 and 2.5 nmol/L TM, 5% FVIII resulted in CT similar to the absence of TM, suggesting it completely reversed the effect of APC. Increasing TM concentrations also reduced peak thrombin generation and ETP. The addition of 300 nmol/L FV-stabilizing Fab returned CT to nearly baseline, but for most subjects was less than the effects of 3- or 10% FVIII. The FV-stabilizing Fab produced similar or greater thrombin generation compared to samples with 3- or 10% FVIII. CONCLUSIONS: The FV-stabilizing Fab resulted in enhanced CT and TGA parameters consistent with FVIII levels of 3- and 10%. Additional studies need to further characterize how modulating the APC pathway may prove beneficial in developing new haemophilia drug targets.

Enhanced uptake of blood coagulation factor VIII containing immune complexes by antigen presenting cells.

Essentials Anti-factor (F) VIII antibody formation is a major complication in the treatment of hemophilia A. We investigated uptake of FVIII and FVIII immune complex by bone marrow derived dendritic cells. Immune complex formation increased uptake of FVIII 3-4 fold in a Fcγ receptor dependent manner. FVIII immune complex binding to Fcγ receptors may modulate immune tolerance induction. SUMMARY: Background A major complication in the treatment of hemophilia A is the development of inhibitory antibodies targeting coagulation factor VIII (FVIII). Eradication of these inhibitors can be established by immune tolerance induction (ITI), which consists of daily administration of high dosages of FVIII. FVIII immune complexes (FVIII-IC) could be formed following FVIII infusion in patients with pre-existing anti-FVIII antibodies. Objectives Here we studied endocytosis of FVIII-IC by bone marrow-derived dendritic cells (BMDCs). Methods BMDCs were pulsed with FVIII/FVIII-IC and uptake was assessed by flow cytometry and confocal imaging. Results BMDCs were able to efficiently internalize FVIII-IC in a dose-dependent manner, 3-4-fold more efficiently when compared with equimolar concentrations of non-complexed FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with anti-Fcγ receptor (FcγR) antibody 2.4G2, indicating functional involvement of FcγR. No internalization of FVIII-IC was observed in BMDCs lacking FcγRI, FcγRIIb, FcγRIII and FcγRIV. Genetic ablation of FcγRIIb, FcγRIII or FcγRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FcγRI showed lower FVIII-IC uptake levels when compared with other single FcγR null BMDCs. Expression of the inhibitory FcγRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions FcγR are critical in the internalization of FVIII-IC by BMDCs and multiple FcγR can contribute independently to this process. Our findings provide a basis for future studies to address whether the outcome of ITI is dependent on the interplay between FVIII-IC and inhibitory and activating FcγR.

Coated platelets and severe haemophilia A bleeding phenotype: Is there a connection?

INTRODUCTION: Coated platelets are a subpopulation of platelets that possess highly prothrombotic properties. Previous observational data suggest that bleeding phenotype in severe haemophilia A is associated with coated platelet levels. Haemophilia A patients with higher coated platelet levels may have a mild bleeding phenotype; those with lower levels may have a more severe bleeding phenotype. AIM: The aim of the study was to test the hypothesis that coated platelet levels are correlated with clinical bleeding phenotype. METHODS: This cross-sectional, observational study enrolled 20 severe haemophilia A patients, including 15 with severe and five with a mild bleeding phenotype, and a control group of 12 healthy volunteers. The haemophilia bleeding phenotype was determined by the patient's medical history and haemophilia treatment centre records. Blood was obtained from each patient by venipuncture and platelets were analysed by flow cytometry. RESULTS: Patients categorized as having a severe bleeding phenotype experienced a median eight bleeds per year compared to one bleed annually in the mild bleeding phenotype group. Both groups had similar total platelet counts and fibrinogen levels. There was no difference in coated platelet percentage between severe and mild bleeding phenotype (17 and 16% respectively), however, both groups had significantly lower % coated platelets compared to controls (44%, P < 0.0001). CONCLUSION: Coated platelet levels were not associated with bleeding phenotype in this study; however, these data may suggest coated platelet levels are lower in haemophilia patients relative to healthy volunteers.

Monitoring rFVIII prophylaxis dosing using global haemostasis assays.

Secondary factor VIII (FVIII) prophylaxis converts severe haemophiliacs (FVIII:C < 1 IU dL(-1)) to a moderate phenotype (FVIII:C ≥ 1 IU dL(-1)), however, plasma FVIII:C is a poor predictor of bleeding risk. This study used thromboelastography (TEG) and thrombin generation assay (TGA) to quantify coagulation across a 48 h rFVIII prophylaxis period. 10 severe haemophiliacs with varying clinical bleeding phenotypes received their standard rFVIII prophylaxis dose and blood samples were obtained over 48 h. Measured parameters included FVIII:C, TEG and TGA at each time point. FVIII:C pharmacokinetics (PK) and correlation between global assay parameters was performed. The FVIII:C PK parameters were consistent with previous literature. There was significant correlation between FVIII:C and TEG R-time and aPTT (both P < 0.001). Significant correlations existed between FVIII:C and TGA peak, ETP and velocity parameters (all P < 0.001). At 24 h the TEG parameters were sub-therapeutic despite median FVIII:C of 13.0 IU dL(-1). TGA was sensitive to FVIII:C below 1 IU dL(-1). Those with the severest bleeding phenotype had the lowest TGA parameters. There was significant correlation between FVIII:C and TEG and TGA. TEG lost sensitivity at 48 h, but not TGA. Prospective studies are needed to determine whether these data can be used to design individualized rFVIII prophylaxis regimens.

Enhanced costimulation by CD70+ B cells aggravates experimental autoimmune encephalomyelitis in autoimmune mice.

OBJECTIVE: Assess whether CD70+ B cells contribute to EAE. MATERIALS AND METHODS: MOG-specific TCR transgenic mice (2D2) were crossed with mice with constitutive CD70 expression on B cells. The development of EAE and the phenotype of B-T lymphocytes were studied in 2D2xCD70 animals. RESULTS: Spontaneous EAE developed in 20% of 2D2xCD70 and 3% of 2D2 mice. EAE was also more severe in 2D2xCD70 versus 2D2 animals upon MOG immunization. The susceptibility of 2D2xCD70 to EAE was associated with fewer FoxP3+ T cells. CONCLUSIONS: Expression of CD70 by B cells aggravates EAE possibly by reducing the number of regulatory T cells.

Improved kinetics of rIX-FP, a recombinant fusion protein linking factor IX with albumin, in cynomolgus monkeys and hemophilia B dogs.

BACKGROUND: Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half-life has the potential to greatly improve the convenience of, and adherence to, prophylaxis. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profile of a recombinant fusion protein linking coagulation FIX with albumin (rIX-FP). METHODS: Cynomolgus monkeys and hemophilia B dogs received single intravenous doses of rIX-FP (50-500 IU kg(-1)). rIX-FP plasma levels were determined by an activity-based assay (dogs only) and anti-FIX ELISA methods. Additionally, activated partial thromboplastin time (APTT) was determined in hemophilia B dogs. Data were compared with a direct study comparator (recombinant FIX [rFIX]) or previously published data. RESULTS: The terminal half-life of rIX-FP was prolonged in both species compared with FIX reference data. In hemophilia B dogs, human FIX antigen levels remained above 0.05 IU mL(-1) more than three times longer after rIX-FP (7.3 days) compared with rFIX (2.3 days), whereas respective calculations based on activity levels confirmed the observed superior profile. Prolonged PDs of rIX-FP were demonstrated with APTT<60 s sustained around four times longer with rIX-FP (5.9 days) than rFIX (1.5 days). CONCLUSIONS: These studies indicate that the recombinant albumin fusion technology successfully improves the PK profile of FIX. Clinical studies will test whether the improved kinetics result in a significant half-life extension in patients with hemophilia B.

Monitoring rFVIIa 90 µg kg⁻¹ dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h.

Recombinant FVIIa is a haemostatic agent administered to patients with severe FVIII or FIX deficiency with inhibitors. Although rFVIIa is effective at stopping bleeding, a reliable assay to monitor its effect is lacking. To characterize the pharmacokinetics and global coagulation effects of rFVIIa for 6 h following a IV dose of 90 µg kg⁻¹. Ten non-bleeding subjects with severe FVIII or FIX deficiency were infused with a single-dose of rFVIIa 90 µg k⁻¹ body weight and blood was collected before and at 0.5, 1, 2, 4 and 6 h postdose. Global haemostasis was characterized throughout the study utilizing whole blood analyses (Hemodyne HAS, TEG, ROTEM). The clearance and half-life of factor FVII:C was estimated as 39.0 ± 8.8 mL h⁻¹ kg⁻¹ and 2.1 ± 0.2 h respectively. There was good inter-assay agreement with respect to clot initiation parameters (R, CT and FOT) and these parameters all fell to a mean of approximately 9 min following rFVIIa dosing. The platelet contractile force (PCF) and clot elastic modulus (CEM) were positively correlated to FVII:C (P < 0.0001), and these parameters were dynamic throughout the 6-h period. The MA and MCF did not correlate to FVII:C nor did they significantly change during the study. Prothrombin F1 + 2 significantly increased following rFVIIa dosing (P < 0.001), but remained steady throughout the study. There was no change in D-dimer concentrations over time. The FOT, R and CT characterized clot initiation following rFVIIa dosing. The PCF and CEM were correlated to FVII:C and characterized the dynamics of platelet function and clot strength over the rFVIIa dosing interval. The clinical significance of these findings needs additional study.

DNA microarrays for identifying fishes.

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a "Fish Chip" for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

Effect of recombinant factor VIIa variant (NN1731) on platelet function, clot structure and force onset time in whole blood from healthy volunteers and haemophilia patients.

NN1731 is a novel variant of recombinant factor VIIa (rFVIIa) that binds to activated platelets, but has greater enzymatic activity than rFVIIa in generating FXa and thrombin. The effect of NN1731 on clot structure and platelet function was characterized ex vivo in whole blood from healthy volunteers and haemophilic patients. Blood samples from six healthy volunteers, nine haemophilia A patients with and without inhibitors and one acquired haemophilia A patient, were spiked with increasing concentrations (0.32, 0.64 and 1.28 microg mL(-1)) of rFVIIa and NN1731. Platelet contractile force (PCF) or platelet function, clot elastic modulus (CEM) or clot structure, and force onset time (FOT) or the thrombin generation time (TGT) were determined using the Hemodyne Hemostasis Analysis System (HAS). Baseline PCF, CEM and FOT values in patients were abnormal compared to healthy volunteers' baseline values. Overall, haemophilia blood samples with or without inhibitors spiked with NN1731 had significantly greater PCF, CEM and shorter FOT values relative to samples spiked with corresponding doses of rFVIIa. The variability in response to treatment between patients was greater with rFVIIa compared to NN1731. At 1.28 microg mL(-1) (90 microg kg(-1)), NN1731 normalized PCF, CEM and FOT in nine of 10 patients, while rFVIIa normalized these parameters in four of 10 patients. Increasing in vitro concentrations of NN1731 normalized platelet function, clot structure and thrombin generation consistently in haemophilia blood with or without inhibitors. NN1731 may be a promising haemostatic agent for patients with bleeding disorders. These results should be confirmed in an in vivo study.

Bacterial protein patterning by micro-contact printing of PLL-g-PEG.

Biomimetic micro-patterned surfaces of three S-layer (fusion) proteins, wild type (SbpA), enhanced green fluorescence protein (SbpA-EGFP) and streptavidin (SbpA-STV), were built by microcontact printing of poly-L-lysine grafted polyethylene glycol (PLL-g-PEG). The functionality of the adsorbed proteins was studied with atomic force microscopy and fluorescence microscopy. Atomic force microscopy (AFM) measurements showed that wild-type SbpA recrystallized on PLL-g-PEG free areas, while fluorescent properties of SbpA-EGFP and the interaction of SbpA-streptavidin heterotetramers with biotin were not affected due to the adsorption on the micro patterned substrates.

Percutaneous liver biopsy in adult haemophiliacs with hepatitis C virus: safety of outpatient procedure and impact of human immunodeficiency virus coinfection on the spectrum of liver disease.

Both HCV and HIV are common in haemophiliacs previously treated with non-viral-inactivated clotting factor concentrates. Because of increased bleeding risks, little data are available on the safety of percutaneous outpatient liver biopsy (LBx) and impact of HIV coinfection in this population. This study aims at reporting our experience with percutaneous LBx in a cohort of haemophiliacs infected with HCV and describe the spectrum of disease and impact of HIV coinfection. A retrospective review of consecutive patients with haemophilia and HCV who underwent percutaneous LBx was performed. All patients were positive for HCV RNA by commercial assay and received factor concentrate prior to biopsy. A total of 29 male patients (mean age 36, 24 haemophilia A, five haemophilia B, and 44% coinfected with HIV) underwent successful outpatient percutaneous LBx without bleeding complication. Histologic activity index was 6.44 with advanced fibrosis (bridging fibrosis/cirrhosis) in 31%. When patients were stratified by HIV positive (n = 13) vs. HIV negative (n = 16), coinfected patients had higher fibrosis scores and higher proportion advanced fibrosis (54% vs. 12%; P = 0.0167) with no differences in age, demographic or other laboratory parameters. Multivariate logistic regression found that HIV positivity was independently associated with advanced fibrosis (OR = 3.7; 95% CI: 1.17-11.8; P = 0.026). Outpatient percutaneous LBx can be safely performed in patients with haemophilia. Despite similar age, HIV coinfection was an independent predictor of advanced fibrosis. These data support the hypothesis that HIV accelerates fibrosis progression in those coinfected with HCV and highlights the importance of liver histology in this population.

Freestanding polyelectrolyte multilayers as functional and construction elements.

This article reviews the progress in the field of polyelectrolyte multilayer membranes with special attention to freestanding membranes. These can be prepared both in the form of hollow capsules and as flat membrane sheets. While (bio) functionality, or bioactivity as it is known, from solid supported multilayers is maintained, additional applications arise for the freestanding membranes in the fields of encapsulation, separation and micromechanics. The production processes and functionalities achieved for capsules and flat sheets. The integration of membranes into larger scale structures is essential for their use and an overview of existing strategies is given. In particular, the way in which arrays of micro-compartments can be built up is shown, and their potential for sensing and combinatorial chemistry discussed. Recent results on the applications of such systems as membrane sensors in the case of flat membrane sheets are also discussed.

[Rapidly lethal progression of a therapy-resistant status epilepticus]. / Refraktärer generalisiert konvulsiver Status epilepticus - Fallbericht eines rasch letalen Verlaufs.

We present a case of death after first manifestation of generalised convulsive status epilepticus in a young man. A previously healthy 23-year-old man was admitted to our emergency department by ambulance service with approximately 20 min of generalised convulsive seizures. First line treatment in the emergency ward with benzodiazepines failed. The patient was cardiopulmonary stable until, after more than 30 min of status epilepticus, he developed tachycardia and became bradypnoeic. Intubation and ventilation was performed and anticonvulsive treatment was escalated with thiopental. Fifteen minutes later he developed ventricular fibrillation. CPR was started. The patient became asystolic after 90 min CPR following the ILCOR (International Liaison Committee on Resuscitation) Instructions. CPR was continued for another 30 min without success. The patient died after 120 min of maximal efforts. Autopsy and toxicology were performed, neuropathologic examination showed general brain edema and neuronal cell loss in purkinje cell layers of the cerebellum and olive knots which may be the consequence of generalised convulsive status epilepticus. We conclude: status epilepticus becomes refractory in approximately 30 % of cases. Until now, there are no randomised trials on the optimal treatment of refractory status epilepticus. Better treatment algorithms are urgently needed.

Outcome analysis of 189 consecutive cancer patients referred to the intensive care unit as emergencies during a 2-year period.

The referral of critically ill cancer patients to an intensive care unit (ICU) is a matter of controversial debate. This study was conducted by an interdisciplinary clinical group to evaluate the outcome of ICU treatment in cancer patients according to their characteristics at the time of referral. A retrospective analysis was used to identify relevant subgroups among 189 consecutive cancer patients referred as emergencies to one of four ICUs during a 2-year period. Reasons for ICU referral were pneumonia (29.6%), sepsis (27.0%), fungal infection (11.1%), another infection (9.5%), gastrointestinal emergency (16.9%), treatment-related organ toxicity (6.9%), or other, non-infectious complications (43.9%). Vasopressor support was required in 50.3%, mechanical ventilation in 49.7%, and haemodialysis/-filtration in 26.5% of the patients. Overall, 41.3% died during ICU treatment, 12.2% died after transfer from ICU to a non-ICU ward, and 35.4% were discharged alive. Sepsis, mechanical ventilation, vasopressor support, renal replacement therapy and neutropenia were independent risk factors for fatal outcome, but no single risk factor unequivocally predicted death. All patients with fungal infection who required vasopressor support and either had sepsis (n=13) or needed mechanical ventilation (n=14) died during ICU treatment, while all non-septic patients. who did not require mechanical ventilation, were younger than 74 years of age and had a non-infectious underlying complication (n=29), survived. This analysis may help to early identify relevant subgroups of cancer patients with different prognoses under ICU treatment. A prospective study to confirm the predictive usefulness of this approach is needed. Cancer patients should not be excluded from referral to the intensive care unit in an emergency solely due to their underlying malignant disease or a single unfavourable prognostic factor.

[Incidence and differential diagnosis of chronic eosinophilic leukemia]. / Häufigkeit und Differenzialdiagnose der chronischen Eosinophilenleukämie.

The diagnosis of chronic eosinophilic leukemia (CEL) is based on the evidence of an autonomous, clonal proliferation of eosinophilic precursors and the exclusion of other myeloid neoplasms with eosinophilia. Histopathological evaluations of bone marrow are rare, and reliable data on the frequency of CEL do not yet exist. A total of 100 cases characterized by eosinophilia >/=1.5x10(9)/l blood for more than 6 months were evaluated. In 87 cases, the eosinophilia turned out to be secondary and a reactive genesis was likely, but not proven in 3 further cases. Idiopathic hypereosinophilic syndrome was diagnosed in three cases. The diagnosis CEL was considered in four out of a total of seven cases with a myeloid neoplasia and all four disorders showed an abnormal karyotype. However, only one of them could be classified as CEL. We conclude that CEL is a rare disease concerning only a minority of cases with chronic eosinophilia.

[Endovascular gamma-irradiation for prevention or restenosis after angioplasty of femoropopliteal de-novo-stenoses-long-term results of a feasibility study]. / Die endovaskuläre gamma-Bestrahlung zur Prävention der Restenose nach Perkutaner Transluminaler Angioplastie von de-novo-Stenosen femoropoplitealer Arterien-Langzeitergebnisse einer Pilotstudie.

OBJECTIVES: To evaluate the performance and efficacy of endovascular irradiation after percutaneous transluminal angioplasty (PTA) of de-novo femoropopliteal stenoses in a pilot study. METHODS: 6 patients received non-centered endovascular irradiation (12 Gray at surface of the vessel wall) immediately after angioplasty of de-novo femoropopliteal stenosis, 1 patient was given centered endovascular irradiation using 192-iridium (12 Gray at surface of the vessel wall) Centered irradiation was considered for two other patients. Duplex sonographies and interviews were performed the day before and after PTA and after 1, 3, 6, 9, 12, 18, 24 months up to 4 years. Intraarterial angiography was performed in symptomatic patients. RESULTS: Non-centered endovascular irradiation was possible in all patients without problems or complications. Centered irradiation was not possible in two patients with the cross-over approach. One thromboembolic complication occurred during centered irradiation. Both restenosis and new stenosis at the edge of irradiated distance occurred in 1/7 patients. No other side effects were observed during follow-up. CONCLUSIONS: In our pilot study endovascular irradiation after angioplasty of de-novo femoropopliteal stenosis was possible with low rates of complications and restenosis and taking vessel anatomy into account.

Defining community integration of persons with brain injuries as acculturation: a Canadian perspective.

In this study, the AIMS, a measure of community integration developed from acculturation theory, was used to explore the various ways that people with brain injuries, a smaller cultural or minority group, are involved with the larger cultural group and to evaluate rehabilitation outcome (i.e., reintegration into the community). Specifically, the AIMS assesses whether the needs of individuals with brain injuries in a number of areas are identified and supported in a way that supports community participation. In addition, the opinions of persons with a brain injury regarding their level of community integration were compared to the opinions of individuals who knew them well. The results of this study indicate that while a large proportion of the participants with brain injuries were integrated in traditional areas of service delivery such as medical services, housing and social activity, their disability-related needs were not being identified and supported adequately in the areas of spirituality and productive activity.