Real-world experience with the all-oral, interferon-free regimen of ombitasvir/paritaprevir/ritonavir and dasabuvir for the treatment of chronic hepatitis C virus infection in the German Hepatitis C Registry.

Real-world studies are relevant to complement clinical trials on novel antiviral therapies against chronic hepatitis C; however, clinical practice data are currently limited. This study investigated effectiveness and safety of ombitasvir/paritaprevir/ritonavir (OBV/PTV/r)±dasabuvir (DSV)±ribavirin (RBV) for treatment of HCV genotype (GT) 1 and GT4 infection in a large real-world cohort. The German Hepatitis C Registry is an observational cohort study prospectively collecting clinical practice data on direct-acting antiviral therapies. Patients with GT1/4 infection treated with OBV/PTV/r±DSV±RBV were analysed. Effectiveness was assessed by sustained virologic response in 558 patients who reached post-treatment week 12 (SVR12). Safety is reported in 1017 patients who initiated treatment. Of the patients, 892 (88%) had GT1 and 125 (12%) had GT4 infection. Prior treatment experience and cirrhosis were reported in 598 (59%) and 228 (22%) patients, respectively. Overall, SVR12 (mITT) was 96% (486/505) in GT1- and 100% (53/53) in GT4 patients. SVR12 rates were high across subgroups including patients with cirrhosis (95%, 123/129), patients with moderate to severe renal impairment (100%, 34/34), and subgroups excluded from registrational trials like patients ≥70 years (96%, 64/67) and failures to prior protease inhibitor treatment (96%, 46/48). Adverse events (AEs) and serious AEs were reported in 52% (525/1017) and 2% (21/1017) of patients, respectively, and led to treatment discontinuation in 1.5% (15/1017) of patients. OBV/PTV/r±DSV±RBV was effective and generally well tolerated for treatment of HCV infection in clinical practice.

Chronic hepatitis C patients prior to broad access to interferon-free treatments in Germany.

In 2014, the first interferon-free treatment options for chronic Hepatitis C (CHC) became available in Europe introducing a new era of highly effective and well tolerated oral treatment options for CHC. The data from the cross-sectional study CURRENT-C highlights the epidemiological characteristics of patients with CHC in Germany. During the period that the study was conducted, the approval of the combination drugs for the treatment of CHC was imminent.Between June and November 2014, 1471 CHC-patients not receiving anti-HCV treatment were included nationwide in 40 German centers specializing in viral hepatitis. The mean age was 52.4 years with 41.2 % of the patients being female. Presumed route of infection in male patients was most frequently drug use (46.2 %) and blood products in females (22.8 %). The route of infection was unknown in 28.2 % of male and 43.1 % of female patients. Compared to male patients, female patients were older (55.6 vs. 50.1 years) and longer diagnosed with HCV (18 vs. 15 years). First language of the patients was most frequently German (72.2 %), followed by Russian (14.2 %), and Polish (2.9 %). HCV genotype (GT) 1 was found in 73.8 % (1a 29.0 %, 1b 38.4 %), GT2 in 3.5 %, GT3 in 18.3 %, GT4 in 4.2 %, GT5 in 0.2 %, and GT6 in 0.3 %. Liver cirrhosis was diagnosed in 15.7 % of the patients (17.1 % male, 13.7 % female). 43.2 % of the patients had already received HCV treatment, most frequently dual therapy with pegIFN + RBV (75.8 %) or triple therapy with telaprevir or boceprevir (20.3 %). Compared to treatment-naïve patients, pretreated HCV patients were older (55.1 vs. 50.3 years) and more frequently had liver cirrhosis as clinical diagnosis (22.2 % vs. 10.8 %). Patients scheduled for HCV treatment within the next 3 months had higher rates of pre-treatment (49.4 % vs. 37.0 %), and liver cirrhosis (21.4 % vs. 10.0 %).Compared to epidemiological data of Hüppe et al. 1 from 2003 to 2006, Klass et al. 2 stated in 2012 in a comparable setting that the German CHC population were older and had more advanced liver disease. The current data seem to support this ongoing trend towards more difficult to treat patients with an urgent need for new treatment options.

Pressure measurements in laboratory-scale blast wave flow fields.

The present study examines the effects that temporal and spatial averagings due to finite size and finite response time of pressure transducers have on the pressure measurements in blast wave flow fields generated by milligram charges of silver azide. In such applications, the characteristic time and length scales of the physical process are of the same order of magnitude as the temporal and spatial characteristics of the transducer. The measured pressure values will then be spatially and temporally averaged, and important parameters for the assessment of blast effects may not be properly represented in the measured trace. In this study, face-on and side-on pressure transducer setups are considered. In the experiments, face-on and side-on readings at the same distance from the charge as well as time-resolved optical visualization of the whole flow field are obtained simultaneously for the same explosive event. The procedure of data extraction from the experimental pressure traces is revisited and discussed in detail. In the numerical modeling part of the study, numerical blast flow fields are generated using an Euler flow solver. A numerical pressure transducer model is developed to qualitatively simulate the averaging effects. The experimental and numerical data show that the results of pressure measurements in experiments with small charges must be used with great caution. The effective averaging of the pressure signal may lead to a significant underestimation of blast wave intensities. The side-on setup is especially prone to this effect. The face-on setup provides results close to those obtained from optical records only if the pressure transducer is sufficiently remote from the charge.

Discovery of N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methylpyrimidine-4-carboxamide. A Highly Selective and Orally Bioavailable Matrix Metalloproteinase-13 Inhibitor for the Potential Treatment of Osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.

Cellular uptake and localization of liposomal-methylphosphonate oligodeoxynucleotides.

Nuclease digestion and intracellular delivery are major factors limiting the potential use of oligodeoxynucleotides as antisense molecules. Structural analogues of phosphodiester oligodeoxynucleotides, such as phosphorothioates and methylphosphonates, are resistant to nuclease degradation and can still bind to their mRNA targets. However, their limited ability to escape from the endosomal/lysosomal compartments and reach the intracellular sites of action have dampened their potential clinical application. To circumvent this problem we have incorporated methylphosphonate oligodeoxynucleotides into liposomes. We found that the level of uptake of liposome-incorporated methylphosphonate oligodeoxynucleotides is time and concentration dependent. Maximal up take occurred at 8 h when 4-8 microM liposome-incorporated methylphosphonate oligodeoxynucleotides was added. Approximately 50% of liposome-incorporated methylphosphonate oligodeoxynucleotides were retained in cells after 24 h of incubation. Using fluorescent microscopy, intracellular fluorescence could be seen within 2.5 h of incubation. Diffused fluorescence was found throughout the cytoplasm, suggesting that the liposome-incorporated methylphosphonate oligodeoxynucleotides were not confined within the endosomal/lysosomal structures. We conclude that liposomes can effectively deliver methylphosphonate oligodeoxynucleotides to the cytoplasm, which is the major intracellular site of action for translational arrest.

Multivariate analyses of prognostic factors in acute myeloid leukemia: relevance of cytogenetic abnormalities and CD34 expression.

Identification of additional prognostic factors besides karyotype is important for the improvement of the risk adapted treatment strategies in acute myeloid leukemia (AML). The aim of this study was to investigate whether other factors besides karyotype could be used as a prognostic tool in newly diagnosed AML. Biological and disease related established and potential prognostic factors were retrospectively analysed in 124 consecutive AML patients treated between 1993 and 2002 at the University hospital Rostock (Germany). One hundred patients received a potential curative intensive chemotherapy (81%), of whom 28 received an allogeneic HSCT at some point of their treatment course, 17 patients (14%) received palliative therapies and 7 patients (5%) received supportive care only. In patients that received potential curative therapies LDH >or=2000 U/l, WBC >50 GPT/l, CD34 surface expression on the AML blasts, secondary AML, unfavorable karyotype and no allogeneic HSCT at some point of treatment course were associated with unfavorable prognosis. However, in the multivariate risk factor analyses only unfavorable karyotype (p=0.012), CD34 positivity of AML blasts (p=0.046), no allogeneic HSCT (p=0.008) and first diagnosis after 1997 (p=0.025) were independent unfavourable prognostic factors. In conclusion, karyotype and CD34 expression are independent prognostic markers in newly diagnosed AML. Furthermore, receiving an allogeneic HSCT at some point of the treatment course seems to be of benefit for AML patients.

Optimizing follicular dendritic cell isolation by discontinuous gradient centrifugation and use of the magnetic cell sorter (MACS).

Follicular dendritic cells (FDC) contribute minimally to the total cell population of lymphatic tissue. In order to obtain higher numbers of viable FDC with only a small fraction of contaminating cells the following procedure was developed. Subsequent to the usual mechanical and enzymatical digestion of human tonsils, single cells were layered on top of a discontinuous bovine albumin gradient and centrifuged at 8500 x g. The suspension collected from the 1.052-1.030 interphase contained an average of 10.5% FDC. Next, the preparation was subjected to a new step involving separation of FDC previously treated with biotin-labelled KiM4 monoclonal antibody, raised against FDC, and attached via biotin-streptavidin bonding to streptavidin-conjugated paramagnetic beads. Purification on a magnetic cell sorter (MACS) yielded 3.3-10.1 x 10(6) cells with an average FDC content of 78.4%. The viability and morphology of the resulting FDC population was examined using trypan blue staining or electron microscopy. This technique will permit in vitro studies and long term cultures with FDC isolated from human lymphatic tissue.

Large-scale preparation of highly purified, frozen/thawed CD34+, HLA-DR- hematopoietic progenitor cells by sequential immunoadsorption (CEPRATE SC) and fluorescence-activated cell sorting: implications for gene transduction and/or transplantation.

The purification of early hematopoietic progenitor cells for autologous transplantation is based on two rationales: (1) elimination of clonogenic tumor cells, and/or (2) gene transfer into indefinitely self-replicating hematopoietic stem cells. Primitive CD34+ stem cells can be separated from more mature stem cells, or probably from clonogenic tumor cells, by differences in HLA-DR surface antigen expression. The objective of this study was to establish a large-scale technique for purification of CD34+, DR- progenitor cells from a large volume marrow harvest. In five different experiments, CD34+ cells were purified to between 76% and 91% by avidin-biotin immunoadsorption (CEPRATE SC) as a first step. This was followed by fluorescence-activated cell sorting to separate DR+ and DR- cells, which resulted in the generation of between 1.75 and 11.3 x 10(5) CD34+, DR- cells. The purity of DR- cells increased from between 0.5% and 4.3% in the immuno-adsorbed fraction up to 99% in the DR- sorted fraction. As shown in a single experiment, the purity of CD34+, DR- cells immediately after thawing increased from 0.01% to 94.3% while losing 99% of those early progenitor cells during the multistep purification procedure. We were able to physically separate one CD34+, DR- cell from up to 8000 nucleated cells in the prepurified cell suspension. One million highly purified CD34+, DR- progenitor cells is potentially an adequate cell dose for autologous transplantation equivalent to what is contained in an unselected and functioning marrow autograft.

Effects of tumour necrosis factor alpha on bone marrow aspirates of patients with acute myelogenous leukemia determined by flow-cytometric cell-cycle analysis.

Tumour necrosis factor alpha (TNF alpha) exerts cytotoxic and antiproliferative effects on neoplastic cells. It has been used as a therapeutic agent for solid tumours and haematological malignancies. We report on the ex vivo determination of the effect of recombinant human rhuTNF alpha on bone marrow aspirates by a bromodeoxyuridine/propidium iodide method. Cell samples were drawn after 0.5, 2, 4, 6, 8, 10, 22, and 25 h from short-term suspension bone marrow cultures from patients with acute myelogenous leukemia (AML). Flow-cytometric cell-cycle analysis was performed after double DNA staining with propidium iodide and anti-BrdU antibodies. By this method the effect of rhuTNF alpha on cell proliferation can be evaluated after only 35 h. In about two-thirds of the bone marrow aspirates of AML an inhibiting effect on rhuTNF alpha can be demonstrated, developing to its full extent after 10 h.

Granulocyte colony-stimulating factor enhances the expression of CD62 on platelets in vivo.

The expression of CD41, CD42 and CD62 on platelets was determined in ten patients with and without G-CSF treatment. CD41 and CD42 is expressed in nearly 100% of the platelets without change after G-CSF treatment. The expression of CD62 on the platelets' surface is significantly enhanced by G-CSF indicating a depletion of the alpha-granules. No platelet aggregation was observed. The enhanced secretion of thrombocyte-specific proteins does not induce aggregation but may promote ADP-induced aggregation. Furthermore, the surface-bound and soluble CD62 binds specifically to macrophages and endothelial cells and is involved in the regulation of inflammatory processes. Thus indirect mechanisms may supplement direct effects of G-CSF after chemotherapy.

Absence of intrinsic electric conductivity in single dsDNA molecules.

The intrinsic dc conductivity of long, individual lambda phage dsDNA molecules has been investigated by ultrasensitive low current-voltage-spectroscopy (IV) under ambient conditions and controlled low humidity inert gas atmosphere on microfabricated metal-insulator-metal gap structures. We found a strong dependence of the measured conductivity on the apparent humidity, which we attribute to capillary condensation of water to the immobilized DNA molecules, giving rise to additional ionic currents. Additional IV-spectroscopy experiments under controlled argon atmosphere always revealed a significant drop in electrical conductivity to 4 x 10(-15)AV(-1)microm(-1), indicating almost no considerable contribution of electrical long range charge transport.

Paroxysmal nocturnal hemoglobinuria in a 63-year-old patient.

Intermittently occuring hemolytic anemia can be the expression of paroxysmal nocturnal hemoglobinuria (PNH). Diagnosis is made via the detection of decreased resistance of the erythrocytes to acidified serum or osmotic hemolysis. Furthermore, diagnostic proof is provided by the cytometric detection of several erythrocyte populations caused by the altered expression of an anchor protein (PIG-A protein) of the cell membrane. This report is of a case where "black morning urine" has been in existence for more than 10 years and in which the additional occurrence of icterus led to the diagnosis. The patient's spleen had been removed in 1964 because of idiopathic thrombopenia, in retrospect, however, thrombopenia within the framework of PNH should be considered.

Cytokine expression of T cells in chronic myeloid leukemia.

OBJECTIVE: To evaluate the function of T cells in chronic myeloid leukemia (CML). METHODS: Interleukin-1 beta (IL-1 beta), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF alpha), and granulocyte macrophage-colony stimulating factor (GM-CSF) gene expressions were investigated by reverse transcription polymerase chain reaction (RT-PCR) assay in fluorescence active cell sorter (FACS) sorted peripheral blood CD2+/CD56-T cells from 12 CML patients, 10 meylodysplastic syndrome (MDS) patients and 7 normal individuals. RESULTS: TNF alpha mRNA was transcribed in T cells from all of the CML, MDS and normal individuals. IL-1 beta mRNA was transcribed in T cells from 10 CML, 9 MDS and 6 normal individuals. Low levels of IL-2 and IL-4 mRNA were detected in 5 CML patients. IL-3, IL-6 and GM-CSF mRNA were undetectable in all samples. CONCLUSION: IL-4 and IL-2 were expressed abnormally in T cells of CML.

[Therapy of chronic myeloid leukemia with interferon-alpha. A decade of experiences]. / Therapie der chronischen myeloischen Leukämie mit Interferon alpha. Erfahrungen aus einem Jahrzehnt.

PATIENTS AND RESULTS: One hundred and fifty-nine patients with chronic myelogenous leukemia have been treated in six studies during 10 years at Hannover Medical School University Center. The prognosis of 111 patients without pretreatment has been improved compared to conventional therapy with a median survival of 5.7 years. Cytogenetic remissions have been induced in all studies followed for a longer time. The most pronounced improvement of prognosis has been observed in these patients. CONCLUSIONS: Several conclusions can be drawn on the basis of the results on the different treatment concepts: 1. Patients with pretreatment have an unfavourable response to interferon. 2. There is a likely effect of the dose of Interferon alpha on the frequency of cytogenetic remissions. 3. The combination of Interferon alpha and interferon gamma has been toxic and ineffective in a pilot study. 4. The combination of interferon and cytosine arabinoside has a positive impact on the frequency of cytogenetic remissions. A continuous parallel application of both drugs seems to be most effective in this respect. An ongoing trial has been initiated to compare a fixed combination of Interferon alpha and cytosine arabinoside and with hydroxyurea respectively. Additionally the feasibility of autologous peripheral blood stem cell transplantation will be studied in patients with insufficient response to the interferon treatment.

[Emission of air pollutants in removal of bone cement with ultrasound instruments in revision endoprosthetics]. / Schadstoffemissionen bei der Entfernung von Knochenzement mit Ultraschallgeräten in der Revisionsendoprothetik.

The use of ultrasonic instruments to remove PMMA bone cement during revision hip arthroplasty results in the production of fumes. These emissions were analysed under standardized laboratory conditions, when it was found that the MMA concentration measured was only 1/10th of the MAC value. In terms of occupational safety, the MMA fumes emitted may therefore be considered non-hazardous for the medical personnel.