Search

Cell-based approaches in medicine, biotechnology and in pharmaceutical research offer unique prospects to cope with future challenges in the field of public health. Stem cell research, autologous cell therapies and tissue engineering are only a few possible key applications. Progress in these fields will depend on the successful implementation of versatile and flexible tools for the gentle manipulation and characterization of cells. In recent years, we and others have introduced microfluidic lab-on-chip systems that include dielectrophoretic elements for the contact-less handling and the analysis of cells. Here, we present results that were obtained by combining our labon-on-chip devices with a low-cost centrifugation stage for the efficient and gentle separation of microparticles and live human cells. Our approach is supposed to overcome limitations that arise from the use of bulky and expensive external pumping stages.

Objective: Repinotan hydrochloride (repinotan) is a selective, high-affinity, full serotonin receptor agonist at the 5-HT1a subtype that is undergoing clinical development in acute stroke. To investigate whether gender is an important covariable for repinotan pharmacokinetics, two studies were performed in subjects of different age and gender who had been phenotyped as extensive metabolisers for cytochrome P450 (CYP)2D6 using dextromethorphan as model substrate. Subjects and methods: Both studies were placebo-controlled, double-blind, randomised, parallel-group studies. Study I was conducted in six healthy young males, five healthy elderly males, and four healthy elderly females receiving a continuous intravenous (IV) infusion of repinotan 0.45 μg/kg/h or placebo for a duration of 12 hours. Study II was performed in healthy elderly male and female volunteers aged ≥65 years with 67 subjects receiving repinotan and 34 receiving placebo. Subjects received a 12-hour infusion of repinotan at doses of 0.1, 0.3, 0.5, 1.0, 2.0 or 3.0 μg/kg/h. Results: Following IV infusion, the steady-state plasma concentration (Css) for repinotan was reached after 4–6 hours consistent with its half-life of 0.8–1.8 hours. In both male and female subjects, the volume of distribution at steady-state and plasma clearance (CL) were independent of dose, indicating linear pharmacokinetics and dose-proportionality for area under the concentration-time curve (AUC) and peak plasma concentration (Cmax) over the dose range of 0.1–3.0 μg/kg/h. Compared with elderly subjects, repinotan CL was unchanged in a subgroup of young subjects. The pooled evaluation of elderly subjects (n = 67) showed that gender had no influence on the pharmacokinetics of repinotan. The ratios male : female and their 90% CIs were: 0.91 (0.789, 1.052) for dose/ bodyweight-normalised Cmax (Cmax, norm), 0.95 (0.808, 1.110) for half-life, 0.97 (0.900, 1.044) for Vss, and 1.11 (0.923, 1.336) for CL. Conclusion: These results indicate that gender does not affect the pharmacokinetics of repinotan in healthy subjects whose age (≥65 years) was representative of the target patient population for repinotan in acute stroke.

Objective To examine the effects of DNase1 treatment on testicular damage after testicular torsion (TT). It has been demonstrated that TT induces thrombus formation and that anticoagulation significantly reduces testicular damage after TT. It was hypothesized that these thrombi are dependent on neutrophil extracellular traps (NETs) and thus NETs disintegration would reduce testicular cell damage. Methods A sham operation was performed in 10 rats. Thirty-four rats underwent induction of iatrogenic TT for 3 hours. After de-torsion and randomization, 24 rats received DNase1 or inactivated DNase1. The following parameters were assessed: testicular damage via Cosentino grading; spermatogenesis via Johnsen score; stem cell factor and c-Kit, apoptosis via Bax, Bcl2, Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling assay, and cleaved caspase3 staining; oxidative stress via superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde; neutrophil recruitment via myeloperoxidase and neutrophil elastase staining; and NET formation via cell-free DNA. Results Forty-three rats were included in the study. Subjects treated with DNase1 showed significantly less cellular damage, oxidative stress, and apoptosis. Further, DNase1-treated rats demonstrated a significant improvement of spermatogenesis, compared with the controls. Conclusion The results of the study indicate that thrombus formation during TT is quite likely NET associated, and that dissolution of cell-free DNA (including NETs) significantly improves testicular damage in rats. As treatment with DNase1 reduced apoptosis, oxidative stress, and inflammation, without adversely affecting coagulation, it might be a suitable treatment for (neonatal) TT and ought to be evaluated in humans.

Background Severe congenital diaphragmatic hernia (CDH) is prenatally managed by fetoscopic tracheal occlusion (FETO) to improve lung growth and maturation. As FETO is not able to reduce the pressure onto the developing lungs originating from the intestine growing into the thoracic cavity, fetal abdominal decompression may alleviate this pressure effect by directing the growing intestine into the amniotic cavity away from the lungs. Therefore, aim of this study was to establish an animal model for fetoscopic abdominal decompression in fetal sheep with CDH. Methods CDH was created surgically on day 75 of 145 day gestation in eight fetuses. 2-3 weeks later, an opening was created in the fetal abdomen by fetoscopic surgery. The fetuses were retrieved by cesarean section at the end of pregnancy and evaluated. Results Five fetuses with CDH were treated with fetoscopic abdominal decompression. Three fetuses with CDH were taken as controls. One fetus was lost after creation of the CDH and two other after creation of the abdominal defect. Preliminary sterological results showed that the septal thickness of the experimental group was smaller than in the CDH group. Conclusion This study demonstrates the general feasibility of fetoscopic abdominal decompression for diaphragmatic hernia in our new animal model. Although not statistically significant, the lungs of treated fetuses were larger and heavier than those of untreated controls. Our findings support the hypothesis of palliative fetal surgery for severe CDH compared to tracheal occlusion. More controlled animal trials are needed.

Prognosis of midgut volvulus in neonates and infants younger than 1 year remains poor, as diagnostic findings may not be apparent until gut infarction had occurred. To characterize factors that help to predict complex midgut volvulus early was aim of this study.Institutionally approved retrospective analysis of all children younger than 1 year treated for midgut volvulus at the author's center from January 2002 to December 2011. Medical history, symptoms, laboratory and radiologic findings as well as sequelae of midgut volvulus were evaluated.In 10 years, 37 children fulfilled the inclusion criteria. Of these, 43% developed complications, and mortality rate was 16%. In 30% of the patients, the only clinical sign was a sudden worsening of the general condition and abdominal distension (complex 19% vs. simple 38%). In one child with simple midgut volvulus, all clinical, laboratory and radiologic signs were negative. CART analysis identified a base excess below -1.70 and preterm birth (36 weeks) as the best discriminators of complex and simple midgut volvulus. A score1pt (comprised of these two factors) was found in all children with complex and in 14% of simple midgut volvulus (p0.001). A positive score (1pt) offers a sensitivity of 100% (81.7-100%), specificity of 85.7% (71.8-85.7%), a PPV of 84.2% (68.8-84.2%) and NPV 100% (83.8-100%).The study shows that midgut volvulus has a substantial morbidity and mortality. Unfortunately, not all affected children get picked up by history, laboratory and imaging. However, the proposed score helps to identify subject with increased risk of complications. It has the potential to facilitate and accelerate diagnosis of complex midgut volvulus; ultimately, it might help to reduce morbidity and mortality.

Background Prospective data of transumbilically laparoscopic-assisted appendectomies (TULAA) is absent in the pediatric population. We therefore compared the midterm outcome of TULAA with open (OA) and laparoscopic (LA) appendectomies in children with appendicitis in a matched prospective study. Methods A total of 20 patients operated with TULAA were matched to 20 cases operated by LA and OA, respectively, according to sex, age, and histology of the resected appendix. All 60 children were evaluated during a 3-month follow-up visit. Results The subjective pain level after discharge, the rate of complications, and persistent painful wound as well as the duration of days refraining from school or kindergarten were similar in all three groups. The wound satisfaction was significantly higher in TULAA and LA. Children operated with TULAA had a faster return to full physical activity compared with OA. Conclusion Our data suggest that TULAA, LA, and OA have a similar outcome 3 months after surgery apart from cosmetic appearance of the wound and return to full physical activity in pediatric patients. Whether parental bias or the increased wound satisfaction act as confounders for early return to full physical activity should be evaluated in larger prospective randomized trials.

Background: Synthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well. Results: Here, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism. Conclusions: The identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients.

Patients with spina bifida are particularly vulnerable to developing immunoglobulin E (IgE)-mediated latex sensitization. Even though many risk factors leading to latex allergy in these patients have been described, it is still unclear whether the increased prevalence of latex sensitization is disease associated or due to the procedures used to treat spina bifida. The aim of this study was to assess prenatal latex sensitization in patients with spina bifida by examining IgE levels in umbilical cord blood.Patients with spina bifida and matched healthy infants were recruited from the University Medical Center Hamburg-Eppendorf and Children's Hospital Altona. Latex-specific and total IgE were assessed in umbilical cord blood using ImmunoCAP testing to evaluate the degree of prenatal latex sensitization.Twenty-two subjects, 10 with spina bifida and 12 healthy individuals, were included. Subjects were selected after matching for sex, gestational age, weight, parental allergy profile, number of prenatal examinations, and utilization of latex tools during pregnancy (propensity score estimates, p = 0.36). In patients with spina bifida, latex-specific and total IgE levels were significantly higher than those in healthy individuals (p = 0.001). After normalization to total IgE, latex-specific IgE levels were higher, yet not significantly increased (p = 0.085).Perinatally, there is a significant augmentation of total and latex-specific IgE in patients with spina bifida. After correcting for total IgE, latex-specific IgE was increased, yet not significantly higher than in matched, healthy controls. This pilot study gives novel insights in the immunological reactions related to spina bifida. The increased latex-specific IgE levels could possibly be associated with the occurrence of a latex allergy in the future.

Biogenic physical weathering and leaching of the quartzite dominated table mountain systems of South America is a slow but strongly biologically influenced process. Observations and analyses on the basis of sample materials collected during an expedition to the protected areas of the most conspicuous tepuis of Venezuela are reported. The rock material consists of more than 98% silica, and the waters collected reflect rainwater quality further deprived of some essential elements. Wear-down of these rocks is recognized as a biogeomorphogenetic process ruled by the microbiota surviving under harsh and nutrient-poor conditions. Poikilotrophic subaerial biofilms of cyanobacteria, fungi, and some lichens perforate quartz grains and idiomorphic quartz crystals, as well as the subcrystalline cement. The typical pattern of biopitting is regarded as proof of biogenic quartz destruction. Within the subaerial biofilms, which form a massive cover of the slowly biocorroded rock surface, lichens were found that minerali...

The pathogenesis of intestinal dysmotility in gastroschisis is not completely understood. Peel formation and disorganization of interstitial Cajal cells (ICC) have been proposed in humans. The aim of this study was to evaluate the impact of prenatal coverage of gastroschisis on gut inflammation and expression of ICC in a fetal lamb model. Twenty-one German blackhead sheep with an abdominal wall defect that was created fetoscopically on day 77 of 145 days gestation were used in this study. Intrauterine surgery with the aim to cover the defect was performed 3 weeks later; two fetuses were covered completely, 5 partially and 11 remained uncovered. Three fetuses without gastroschisis were used as controls. All fetuses were retrieved by cesarean section at day 135. Samples of the small intestine were stained with hematoxylin and eosin for histologic analysis of peel formation and serosal and muscular thickness. For ICC detection, immunohistochemistry using anti-CD117 (c-Kit) antibody was used. In all samples with exposure to amniotic fluid, peel formation and significantly decreased ICC were found. Complete coverage reduced peel formation and disorganization of ICC compared to uncovered animals almost to the level of controls. Peel formation and ICC derangement were significantly reduced by prenatal coverage of gastroschisis. Moreover, this animal model mimics the histopathological bowel changes as seen in human gastroschisis and may, therefore, be used for further research on the pathophysiology and fetal therapy of this malformation.

To test the clinical and sonographic predictors of testicular torsion (TT) with the aim of reducing negative exploration rates.We performed a prospective study of all boys treated for 'acute scrotum' at our institute between January 2001 and April 2012 and clinical findings were documented. If available, ultrasonography (US) was added to the diagnostic evaluation. A prediction of the diagnosis was based on clinical and sonographic features, and was followed by surgical exploration in all patients.A total of 104 patients were included in the 16-month period of the study. No single finding excluded TT. The clinical features (pain24 h, nausea/vomiting, abnormal cremasteric reflex, high position of the testis) appeared predictive (100% sensitivity) and the clinical scoring system was proven to be reliable, reducing the negative exploration rate by55%. Ultrasound predictors alone were not able to identify all boys with TT.It is safe to refrain from routine surgical exploration in every child with acute scrotum if the clinical score is applied, which results in a marked reduction of negative explorations. A reliable diagnosis could not be obtained based on US alone. As scrotal US is unpleasant for the child, we propose to refrain from this if the clinical score is positive. Patients with a negative clinical score are suitable candidates for US to establish and secure diagnosis.

Impalement injuries are uncommon, especially in the pediatric population. Because of the rarity of these injuries, physicians may have difficulty recognizing and treating impalement injuries appropriately. Clinical findings are sometimes innocuous, but can be life threatening. Therefore, evaluation of suspected impalement injury should involve careful history and thorough physical examination, even if there is no evidence of trauma to the perineum. We report a very rare case of rectum perforation after transanal introduction of a broomstick with almost no clinical findings. Impalement injuries are difficult to recognize, and severity may not be reflected by their external appearance. To diagnose these injuries in time, it is important to use a well-organized workup. Language: en

Introduction: Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties. Methods: We describe a sub-population expressing the stem-like marker CD44+/CD24-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44+/CD24low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments. Results: Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo. Conclusion: In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A.

Chemotherapy of advanced pancreatic cancer has mainly been gemcitabine-based for the past fifteen years, with only limited effect. Recently, combination therapy that also targets checkpoint kinase 1 (CHK1) has become an attractive option. The central role of CHK1 in many DNA damage response pathways, however, may result in undesired cytotoxicity in normal cells causing side effects. We were searching for other target molecules of similar function that may be more specific and thus better suited for combination therapy. To this end a negative selection RNAi screen was performed in cell lines with small hairpin RNA molecules targeting over 10,000 genes. Genes that were found to be synthetically lethal with gemcitabine and whose proteins are acting upstream of CHK1 were characterised in more detail. In particular, the inhibition of RAD17 potentiated gemcitabine cytotoxicity in the pancreatic cancer cell lines BxPC-3, MiaPaca-2 and the primary cell line JoPaca-1 that closely resembles primary tumour tissue. Further analysis showed that the synergistic effect of RAD17 knockdown and gemcitabine leads to forced mitotic entry of cells arrested in S-phase by gemcitabine treatment, resulting in asymmetric DNA distribution during anaphase followed by DNA fragmentation and finally cell death by mitotic catastrophe. Our data suggest RAD17 as a novel target for gemcitabine combination therapy supplementing or complementing inhibition of checkpoint kinase 1. As opposed to CHK1, RAD17 knockdown by itself does not lead to abnormal DNA segregation, suggesting a more specific action.

Particulate matter (PM) or soot sensing technologies are needed for feedback and control of exhaust after-treatment systems to meet increasingly stringent environmental regulations. A novel low-cost electrostatic PM sensing approach has recently been developed based on a concentric electrode design with a central cylindrical high-voltage electrode surrounded by an inner baffle that serves as the ground/negative terminal. The sensing principle is not completely understood, but seems to rely on capturing naturally combustion charged particles on electrode surfaces where a delay in sensor startup time occurs during initial testing. The influence of three-dimensional dendritic Ni-Co coatings on PM sensor performance was examined. Nickel and cobalt were co-electrodeposited on cylindrical stainless steel substrates from an electrolytic bath using a square waveform cathodic current. The effect of altering the applied current density and the number of current cycles on the height and spacing of dendritic features was monitored in attempts to correlate surface morphology to the performance of the materials as PM sensors. Coatings were characterized using scanning electron microscopy and confocal laser microscopy, and Ni-Co coated cylinders were used as the central high-voltage electrodes of electrostatic PM sensors to determine the effect of different microstructures on sensor performance. The coated electrodes demonstrated an improvement in startup time for an optimized coating morphology when compared to bare control electrodes. The surface features of the dendritic Ni-Co coatings are believed to alter the interaction of PM with the high-voltage electrode and can induce a faster response of the sensor when optimal electrodeposition conditions are employed.

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.

To distinguish the prognostic factors that decrease the probability of a negative exploration for "acute scrotum." In some institutes, patients with "acute scrotum" undergo immediate exploration after clinical evaluation. Because testicular torsion (TT) accounts only for a fraction of these cases, most infants can be treated conservatively.We performed a retrospective study of all patients treated at our institute from January 2008 to December 2009 for the diagnosis of "acute scrotum." Differences between groups were calculated using the chi-square test or analysis of variance and Mann-Whitney-Wilcoxon test for univariate or multivariate analysis, expressed as odds ratios (ORs) and 95% confidence intervals (CIs).The data from 138 patients were analyzed. The mean age was 9 years, 8 months. Of the 138 patients, 19 (13.8%) had TT. This group was compared with the boys without TT at exploration. The patients with TT were older on average (11 years, 1 month vs 9 years, 1 month, p = .035). Pain for24 hours (OR 4.2, 95% CI 1.3-13.4), nausea and/or vomiting (OR 21.6, 95% CI 4.9-93.4), abnormal cremasteric reflex (OR 4.8 95% CI 0.7-35.2), and a high position of the testis (OR 18.0 95% CI 1.8-177.1) were associated with an increased likelihood of torsion. In the group of boys with ≥ 2 of these findings present, 100% had TT at exploration, with 0% false-positive results.TT is uncommon among the group of boys treated for "acute scrotum." In particular, a pain duration24 hours, nausea or vomiting, a high position of the testis, and an abnormal cremasteric reflex had a positive prognostic value for TT. A clinical score might help to avoid unnecessary explorations. In the future, we intend to test the diagnostic set described combined with ultrasonography.

Purpose To quantify intermuscular adipose tissue (IMAT) of the lower leg as well as to investigate associations with other adipose tissue (AT) compartments. The relationship between IMAT and insulin sensitivity was also examined. Materials and Methods Standardized quantification of IMAT was performed in a large cohort (N = 249) at increased risk for type 2 diabetes in the right calf by T1-weighted fast spin-echo imaging at 1.5T (Magnetom Sonata; Siemens Healthcare). Additionally, whole-body AT distribution was assessed. Insulin sensitivity was determined by glucose clamp. Results Males showed significantly more IMAT than females (2.1 ± 1.1 cm2 vs. 1.5 ± 0.9 cm2; P < 0.001). IMAT correlated well with other AT depots, especially with visceral AT (VAT; rfemales = 0.52, P < 0.0001 vs. rmales = 0.42, P < 0.0001). Moreover, IMAT showed a negative correlation with the glucose infusion rate (GIR; rfemales = −0.43, P = 0.0002 vs. rmales = −0.40, P = 0.0007). Conclusion Quantification of IMAT is possible by standard MR techniques. AT distribution of the lower leg is comparable to the visceral compartment with males having higher IMAT/VAT but lower subcutaneous AT (SCAT). IMAT seems to be involved in the pathogenesis of insulin resistance, as shown by the significant negative correlation with GIR. J. Magn. Reson. Imaging 2009. © 2009 Wiley-Liss, Inc.

In this report, we describe the first case of using the partial phrenic nerve transfer and direct muscular implantation into the deltoid muscle for restoration of the shoulder function and stability. A patient suffering from the partial brachial plexus injury with absent axillary nerve underwent reconstructive surgery by an end-to-end nerve coaptation using two fascicles of the phrenic nerve and two autologous nerve grafts, and direct implantation of nerve grafts into the deltoid muscle. Eighteen months after the procedure, we found a functioning biceps with 90 degrees elbow flexion against gravity and 40 degrees shoulder abduction with satisfactory shoulder stability. Electrophysiology revealed reinnervation potentials in the deltoid and biceps muscle. This case demonstrates a satisfactory result after using transfer of the partial ipsilateral phrenic nerve in combination with muscular implantation to restore shoulder abduction and stability. We recommend the described techniques in cases without other reconstructive options. (c) 2009 Wiley-Liss, Inc. Microsurgery, 2009.

Introduction CD20 is a cell surface glycoprotein, encoded by the MS4A1 gene, which is highly expressed on most B-cells. Although CD20 is suggested to be involved in calcium signaling, its exact function is unknown. The monoclonal antibody rituximab, which targets CD20, has revolutionized the treatment of B-cell lymphoma. Despite the widespread use of CD20 antibodies across lymphoma subtypes, little is known about the endogenous regulation of CD20. The tight correlation between CD20 expression and responsiveness to CD20 antibody suggests that increasing CD20 expression levels could improve rituximab sensitivity. This could be particularly useful for B-cell lymphoma with low CD20 surface expression (e.g. chronic lymphocytic leukemia). The main goal of the project is to dissect and understand the CD20 regulation using an unbiased RNAi approach and to exploit this knowledge to improve the efficacy of anti-CD20 therapy. Methods In order to discover novel regulators of CD20 expression, we performed a genome-wide RNAi screen. The shRNA library targets over 5000 mRNAs, with 5 to 6 shRNAs per target. The CD20 regulatome was studied in a Burkitt lymphoma cell line (Raji) characterized by medium expression of CD20. Results MS4A1 was the most significant screen hit that decreased CD20 surface levels with three shRNAs and served as positive control. Based on stringent selection criteria, we identified hits involved in diverse molecular mechanisms such as signaling, trafficking, chromatin remodeling and cell membrane composition. In total, 21 genes targeted with two non-overlapping shRNAs were selected for validation: 12 decreasing and 9 increasing CD20 levels. For every gene, two shRNAs were synthesized and cloned into shRNA expression vectors. Every shRNA was investigated in separate experiments for its effect on CD20 expression levels on Raji cells. We were able to validate and confirm the influence of those genes on CD20 phenotype on Raji cells, as well as on other B-cell lymphoma cell lines (SuDHL5, BL2 and BL60). The group of genes decreasing CD20 expression can provide an insight into physiological regulation and the machinery involved in CD20 trafficking and processing. More relevant from a therapeutic viewpoint are genes where silencing increases the expression of CD20. In order to investigate a potential therapeutic synergy of our hits with Rituximab, we investigated complement-mediated lysis triggered by Rituximab in vitro after silencing of several hits. In this assay we observed direct correlation between CD20 surface levels with the sensitivity towards Rituximab for our hits: increased CD20 levels led to more Rituximab induced lysis (up to two fold increase), and vice versa. One of the main aims of the screen was the discovery of druggable targets that can be potentially used in combination therapy with Rituximab. Indeed, a chemical inhibitor targeting one of our screen hits was able to increase CD20 expression levels on Raji cells. Conclusion With an unbiased shRNA screen we identified potent regulators of CD20 expression levels. Our approach led to the discovery of novel druggable targets, which modulate CD20 levels. This may provide the rationale for novel combinatory therapies, which might improve the efficiency of anti-CD20 therapy in B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

The surface glycoprotein CD20 is expressed on healthy and malignant B-cells and can be therapeutically exploited by monoclonal antibodies (e.g. Rituximab, Ofatumumab, GA-101). Stem cells and pro-B-cells are lacking CD20 surface protein, which allows the reestablishment of B-cell populations after treatment. Other cell lineages do not express CD20. Rituximab has revolutionized clinical practice in the treatment of malignant B-cell lymphoma. However, refractory lymphoma cells can lose the expression of CD20 after Rituximab exposure and B-cell lymphoma with low CD20 surface expression may be less responsive to treatment. Despite the widespread use of CD20 antibodies across lymphoma subtypes, little is known about the endogenous regulation of CD20. It was shown that several chemical compounds may modulate the surface density of CD20 (e.g. histone deacetylase inhibitors), but the regulatory network controlling CD20 levels is not understood. We aim to dissect the CD20 regulatome and to exploit the findings for novel therapeutic strategies. The CD20 regulatome is studied in a Burkitt lymphoma cell line (Raji) characterized by medium expression of CD20 in comparison to other lymphoma models. As a proof of principle, we were able to down-regulate the surface expression of CD20 assayed by FACS with four independent silencing triggers targeting CD20 mRNA. To discover novel regulators of CD20 expression, we performed an unbiased genome-wide RNAi screen. The screen hits are involved in diverse molecular mechanisms such as signaling, trafficking, chromatin remodeling and membrane composition. The group of genes decreasing the expression of CD20 expression can provide an insight into the physiological and pathological machinery involved in CD20 trafficking and processing. The strongest hit in this group, next to CD20 knockdown, is potentially involved in the direct transport of CD20 to the cell surface. Clinically most relevant is the group of genes where silencing increases the expression of CD20. The two strongest hits in this category are a transcription factor and a gene involved in signaling. Detailed molecular characterization of several chosen hits is currently ongoing. Their inhibition may lead to increased sensitivity to anti-CD20 antibodies and thus provide starting points for rational drug design. Citation Format: Mikolaj Slabicki, Leopold Sellner, Alexander Jethwa, Michael Boettcher, Thorsten Zenz. Modulating therapeutic targets in B-cell lymphoma: Loss-of-function RNAi screen for CD20 regulators. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3159. doi:10.1158/1538-7445.AM2013-3159

Background RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNA's half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool. Results We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNA's half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach. Conclusions In summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.

Extracellular chromatin, for example in the form of neutrophil extracellular traps (NETs), is an important element that propels the pathological progression of a plethora of diseases. DNA drives the interferon system, serves as autoantigen, and forms the extracellular scaffold for proteins of the innate immune system. An insufficient clearance of extruded chromatin after the release of DNA from the nucleus into the extracellular milieu can perform a secret task of moonlighting in immune-inflammatory and occlusive disorders. Here, we discuss (I) the cellular events involved in the extracellular release of chromatin and NET formation, (II) the devastating consequence of a dysregulated NET formation, and (III) the imbalance between NET formation and clearance. We include the role of NET formation in the occlusion of vessels and ducts, in lung disease, in autoimmune diseases, in chronic oral disorders, in cancer, in the formation of adhesions, and in traumatic spinal cord injury. To develop effective therapies, it is of utmost importance to target pathways that cause decondensation of chromatin during exaggerated NET formation and aggregation. Alternatively, therapies that support the clearance of extracellular chromatin are conceivable.

This report describes a case where 4 digits were replanted in a mentally retarded patient with a history of smoking and the inability to follow postoperative arrangements.

Background Rapid and accurate diagnosis of malaria is central to clinical management and the prevention of drug-overuse, which may lead to resistance development, toxicity and economic losses. So far, light microscopy (LM) of Giemsa-stained thick blood smears is the gold standard. Under optimal conditions the procedure is fast and reliable; nevertheless a gain in speed would be a great advantage. Rapid diagnosis tests are an alternative, although they cost more and give qualitative instead of quantitative results. Light-emitting diode (LED) fluorescence microscopy (ledFM 400 ×, 1000 ×) may offer a reliable and cheap alternative, which can be used at the point of care. Methods LedFM and conventional fluorescence microscopy (uvFM) were compared to LM in 210 samples from patients with history of fever in the last 24 hours admitted to the Albert Schweitzer Hospital in Lambaréné, Gabon. Results Sensitivities were 99.1% for ledFM and 97.0% for uvFM, specificities 90.7% for ledFM 400 × and 92.6% for ledFM 1000 × and uvFM. High agreement was found in Bland-Altman-plot and Kappa coefficient (ledFM 1000 ×: 0.914, ledFM 400 × and uvFM: 0.895). The time to diagnosis for both FM methods was shorter compared to LM (LM: 43 min, uvFM: 16 min, ledFM 1000 ×: 14 min, ledFM 400 ×: 10 min). Conclusion ledFM is a reliable, accurate, fast and inexpensive tool for daily routine malaria diagnosis and may be used as a point of care diagnostic tool.