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In regard to cow's milk allergy, the current option of avoiding can be expanded by (re-)introducing milk using a milk ladder. So-called "food ladders" are internationally well known and utilized for both non-IgE-mediated and IgE-mediated cow's milk allergy. Stepping up the stairs from highly processed baked goods with milk via cooked milk products to pasteurized fresh milk reflects the status of acquired tolerance of each level. The allergenicity of milk depends on processing and amount. By implementing the milk ladder, it can enhance the clinical process of tolerance development, lead to meeting nutrient requirements quickly, and involve parents actively in the therapeutical process. The milk ladder, for the first time being published and adapted for Germany, describes a structured framework that might be adapted individually regarding the time period on a certain level or other variations such as preparation/amount of milk products. From a safety perspective, healthcare professionals should pay great attention to patient selection and education prior to implementing the milk ladder. Detailed advice as well as recipes and a graphical presentation can be found in the supplemental material., Competing Interests: The authors declare that there is no conflict of interest., (© Dustri-Verlag Dr. K. Feistle.)

There is a large unmet medical need to develop disease-modifying treatment options for individuals with age-related degenerative diseases of the central nervous system. The sigma-2 receptor (S2R), encoded by TMEM97 , is expressed in brain and retinal cells, and regulates cell functions via its co-receptor progesterone receptor membrane component 1 (PGRMC1), and through other protein-protein interactions. Studies describing functions of S2R involve the manipulation of expression or pharmacological modulation using exogenous small-molecule ligands. These studies demonstrate that S2R modulates key pathways involved in age-related diseases including autophagy, trafficking, oxidative stress, and amyloid-β and α-synuclein toxicity. Furthermore, S2R modulation can ameliorate functional deficits in cell-based and animal models of disease. This review summarizes the current evidence-based understanding of S2R biology and function, and its potential as a therapeutic target for age-related degenerative diseases of the central nervous system, including Alzheimer's disease, α-synucleinopathies, and dry age-related macular degeneration.

Introduction: Volunteering yields valuable benefits to communities, yet globally there is limited published data regarding emergency medical volunteering in communities. Hout Bay Volunteer Emergency Medical Service is thought to be the oldest volunteer ambulance service in Cape Town. The objective of this paper is to quantify the contribution of the community service to the Western Cape Government Health: Emergency Medical Service. This paper describes the inputs, key stakeholder relationships, and the impact of COVID-19 on volunteer input and community needs., Methods: Electronic Computer-Aided Dispatch records were used for analysis. Data extracted included detailed information about all recorded incidents between 1 January 2015 to 31 December 2020. Data were analysed in Google Sheets using Pivot Tables and summary statistics., Results: Between 2015 and 2020 HBVEMS responded to approximately 12% of all call-outs in the Hout Bay area, which equates to 2187.16 h of operational time spent on calls. This excludes standby time, i.e., time spent waiting to be dispatched. There was an expected noticeable difference between response times for ambulances based within Hout Bay, and those from outside Hout Bay. Despite a decline in average call-out rate during the 2020 Level 5 lockdown, the volunteers were able to do more shifts and thus more calls within the community. Call-outs during 2020 were visualised as a ratio of trauma to medical calls. In this period there were noticeably fewer trauma calls., Conclusions: There is a growing need for emergency medical care, and volunteer ambulance services can have a meaningful impact on the continent. The findings support the benefit of developing community-based ambulance services, especially in areas that are remote due to distance or topography. The model can be expanded to other communities across the continent. A key factor for success is actively managing stakeholder relationships which include community-based relationships as well as governmental or formal emergency medical services relationships., Competing Interests: The authors declared no conflicts of interest., (© 2022 The Authors. Published by Elsevier B.V. on behalf of African Federation for Emergency Medicine.)

JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S -> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis., Competing Interests: The authors have declared that no competing interests exist.

This guideline is an update from August 2020 the S2k-guideline "Atopic dermatitis" published in 2015. The reason for updating this chapter of the guideline were the current developments in the field of systemic therapy of atopic dermatitis. The agreed recommendations for systemic treatment in atopic dermatitis of the present guideline are based on current scientific data. Due to the approval of dupilumab for the treatment of moderate to severe atopic dermatitis, which cannot be treated sufficiently with topical drugs alone, this part of the guideline has now been adapted and newly consented. The indication for systemic therapy and the therapeutic response to topical and systemic treatment should be recorded and documented in a suitable form in clinic and practice. A standardized documentation of the indication for system therapy in atopic dermatitis can be recommended and is also part of the updated chapter of this guideline., (© 2021 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin.)

This project's goal was to implement an already validated pediatric discharge toolkit to enhance the effectiveness of transition from hospital to home, thus reducing 30-day readmission rates., Methods: This quality improvement study involved implementing a pediatric discharge planning toolkit to improve upon predetermined outcome measures. Critical elements in the toolkit included: (1) comprehensive patient risk assessment on admission; (2) teach-back curriculum; (3) fax or phone call to the primary care physician; (4) 72-hour follow-up calls; and (5) follow-up appointments, scheduled before discharge, within 2 weeks from discharge from hospital. We used the toolkit to gather data on pediatric patients as they were admitted and then prepare them for discharge from December 2016 until March 2017. The primary outcome measure was the 30-day readmissions to the hospital, and the secondary outcome measure was patient satisfaction scores. Our balancing metrics included follow-up appointments made and length of stay. These measures were compared with preintervention hospital pediatric administrative data collected from December 2015 through March 2016., Results: Data collected during the study period (n = 91) compared to preintervention hospital administrative data collected the year prior (n = 132) showed a 31% reduction in readmissions, 4.8% and 7%, respectively (95% confidence interval 0.68-3.8), P = 0.004. Patient satisfaction scores showed no statistical significance. All patients (100%) in both groups had follow-up appointments made before discharge, and the length of stay showed no statistical difference., Conclusions: This pediatric discharge toolkit improved the efficacy of transition from hospital to home by reducing 30-day readmissions. Patient satisfaction scores were not reduced by utilizing the toolkit., Competing Interests: The authors have no financial interest to declare in relation to the content of this article., (Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.)

The synthesis of stable blue TADF emitters and the corresponding matrix materials is one of the biggest challenges in the development of novel OLED materials. We present six bipolar host materials based on triazine as an acceptor and two types of donors, namely, carbazole, and acridine. Using a tool box approach, the chemical structure of the materials is changed in a systematic way. Both the carbazole and acridine donor are connected to the triazine acceptor via a para- or a meta-linked phenyl ring or are linked directly to each other. The photophysics of the materials has been investigated in detail by absorption-, fluorescence-, and phosphorescence spectroscopy in solution. In addition, a number of DFT calculations have been made which result in a deeper understanding of the photophysics. The presence of a phenyl bridge between donor and acceptor cores leads to a considerable decrease of the triplet energy due to extension of the overlap electron and hole orbitals over the triazine-phenyl core of the molecule. This decrease is more pronounced for the para-phenylene than for the meta-phenylene linker. Only direct connection of the donor group to the triazine core provides a high energy of the triplet state of 2.97 eV for the carbazole derivative CTRZ and 3.07 eV for the acridine ATRZ . This is a major requirement for the use of the materials as a host for blue TADF emitters., (Copyright © 2020 Rodella, Bagnich, Duda, Meier, Kahle, Athanasopoulos, Köhler and Strohriegl.)

Background: The most crucial aspect of a prosthesis is the socket, as it will directly determine gait stability and quality. The current standard of care ischial ramus containment socket is reported to increase coronal stability through gait; however, socket discomfort is the primary complaint among prosthetic users., Objectives: The purpose of this study is to compare ischial ramus containment to alternatives in the transfemoral amputee population. All subjects were fit with three different sockets: traditional ischial ramus containment, a dynamic socket, and a sub-ischial. In this study, authors hypothesized socket skeletal motion would be equivalent across interventions., Study Design: Single-blind, repeated-measures, three-period randomized crossover clinical trial., Methods: Outcome measures were socket comfort score and skeletal motion, viewed coronally with X-ray measuring the position of the skeleton in relationship to the socket in full weight-bearing and full un-loading., Results: The mean age was 38.2 and mean Amputee Mobility Predictor score was 40. Mean vertical movement, horizontal movement, single limb prosthetic stance, mean femoral adduction in swing and stance, and median socket comfort score were not statistically different., Conclusion: The socket design did not significantly effect skeletal motion and socket comfort. All socket designs are suitable depending on the patient-centric preferences and prosthetist skill set., Clinical Relevance: The comfort of the standard of care transfemoral amputation socket has been widely reported as problematic. A comparison of alternative designs in a controlled clinical trial environment will assist the clinician in understanding the impact of design regarding skeletal motion and comfort. Users could benefit from alternatives applied in clinical practice.

Objective: The aim of this study was to synthesize published data regarding long-term effects of ADHD on information learned (measured via achievement tests) and success within the school environment (academic performance). Method: A systematic search identified 176 studies (1980-2012) of long-term (≥2 years) academic outcomes with ADHD. Results: Achievement test outcomes (79%) and academic performance outcomes (75%) were worse in individuals with untreated ADHD compared with non-ADHD controls, also when IQ difference was controlled (72% and 81%, respectively). Improvement in both outcome groups was associated with treatment, more often for achievement test scores (79%) than academic performance (42%), also when IQ was controlled (100% and 57%, respectively). More achievement test and academic performance outcomes improved with multimodal (100% and 67%, respectively) than pharmacological (75% and 33%) or non-pharmacological (75% and 50%) treatment alone. Conclusion: ADHD adversely affects long-term academic outcomes. A greater proportion of achievement test outcomes improved with treatment compared with academic performance. Both improved most consistently with multimodal treatment.

Charge heterogeneity is an important critical quality attribute for the analysis of monoclonal antibodies (mAbs). For this, (imaged) capillary isoelectric focusing ((i)cIEF), ion exchange chromatography (IEC) and, recently, capillary zone electrophoresis (CZE) are the predominantly used techniques. In order to investigate which one is most suitable to answer a specific analytical question, here, the four aforementioned separation techniques were systematically evaluated using NISTmAb and Infliximab as test molecules. The performance parameters (precision, separation efficiency, linearity and sensitivity) were determined under comparable conditions. Moreover, important aspects for daily routine such as speed and ease of use were considered. Each technique has its own pros and cons. The (i)cIEF methodology is distinguished by its excellent separation efficiency. In addition, the native fluorescence mode in icIEF is a good tool to analyze small sample amounts (LOQ: 2.8 mg/l for Infliximab). Nevertheless, high performance liquid chromatography (HPLC) still has superior precision. CZE, and also micellar electrokinetic chromatography (MEKC), have emerged as further interesting alternatives. For all techniques, variations connected to the sample preparation strongly influence precision. Looking at the relative standard deviation (RSD) values of the relative peak areas, all techniques provide acceptable performance (RSD: 0.6-1.6%)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Capillary isoelectric focusing is indispensable for characterizing charge heterogeneity and isoelectric points of biopharmaceuticals. However, there are many influencing parameters and therefore method development is challenging. This study was performed to obtain an in-depth understanding of the imaged CIEF methodology by applying a design of experiments approach. To describe the parameter's effects as objectively as possible, a polynomial regression model was derived for the most important responses. For this purpose, the reference monoclonal antibody suggested by the National Institute of Standards and Technology (NISTmAb) was used as test molecule. The total concentration and the mixing ratio of two types of carrier ampholytes and the added amounts of urea and l-arginine were selected as factors. The effects of these factors on 13 different responses such as resolution or pI values were investigated. In order to reduce the total number of experiments, a d-optimal design with 20 different parameter combinations and six replicates each was chosen. The most significant effects of the four factors were shown for the parameters related to separation efficiency and peak position. In addition, the extent of the factor's effect could be assessed. Depending on the selected factor combination, the pI value can differ up to approximately 0.15 pI units and the resolution value between main peak and adjacent basic peak can range from approximately 1.6 to 2.5, for example., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII-specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-ETA ex vivo. SUMMARY: Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII (FVIII) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII, the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A (ETA) from Pseudomonas aeruginosa for selective elimination of FVIII-specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full-length FVIII protein and the resulting human C2 domain-ETA fusion protein (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested ex vivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-ETA and controls. FVIII-specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody-secreting cells (ASC) and detected by an enzyme-linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC2-ETA reduced the number of C2-specific ASC in a dose-dependent fashion, indicating specific and efficient elimination of C2-specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII-specific B cells in patients with hemophilia A and persisting inhibitors., (© 2018 International Society on Thrombosis and Haemostasis.)

Biopharmaceuticals are increasingly present in pharmaceutical therapy. These highly complex molecules are still challenging for development and quality control. Charge heterogeneity is next to size heterogeneity, glycan analysis, or peptide mapping one main aspect of their thorough characterization. This review focuses on the charge-based analysis of biopharmaceuticals through CE. An overview is given about the three separation techniques cIEF, imaged cIEF and CZE for the analyses of charge variants, emphasizing assays and their precision. Especially the developments in the experimental conditions have been given a high priority: sample preparation, capillary properties, anolytes and catholytes, carrier ampholytes, sacrificing agents, solubility enhancement, isoelectric point marker, mobilization techniques, and detection modes have been discussed. CIEF shows a particularly high heterogeneity in its methods. Therefore, a design space is suggested that states the most important adjustable parameters and their range. This enables an adequate design of experiments for individual sample separations, in order to accelerate and simplify method developments., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this research note, interlaced Size Exclusion Chromatography (SEC) is used to shorten the duration of protein analysis. The applied method has a threefold higher sample throughput than comparable ones using conventional SEC. Further important advantages are the identical area's precision and the preservation of protein stability. Two different applications are presented where a short time interval of analysis is very important. One application is the determination of aggregates of monoclonal antibodies (mAbs). A high number of antibody samples have to be analyzed during the development process. The antibody sample was measured in a series of 60 injections within only 5.5 h instead of 16 h. The second application is the two-dimensional (2 D) separation of protein mixtures. The number of samples typically increases from the first to the second dimension. Thus, interlaced SEC is a reasonable option for the second dimension. This work confirms the functionality of interlaced SEC and widens the applicable range. Interlaced SEC can become a convenient option for method acceleration in the future., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Over the last decade, capillary electrophoresis gained tremendous importance, because it became an indispensible tool for the quality control of biologics, e.g. therapeutic antibodies. Consequently, there has been a continuous development within the CE market. Microchip techniques have been established in the last years. Further trends are complete solutions for specific applications by the usage of reagent kits. Step by step instructions and facilitated handling of the instruments are becoming more common. This work focuses on the sized-based protein analysis with CE-SDS. The instruments CE 7100 by Agilent Technologies, LabChip® GXII Touch HT by PerkinElmer, Maurice S. by Protein Simple and PrinCE NextI870 by Prince Technologies have been evaluated, mainly analyzing protein mixtures of different molecular weights in long series. Published data of the PA 800 plus by SCIEX are also included in the tabled results. Precision, reliability, flexibility, and speed have been identified as the most important performance parameters, others such as resolution, sensitivity, linearity, ease of use and sustainability have also been considered. All tested instruments have shown an excellent performance. Depending on application and necessities, each user can find the most appropriate one., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several studies showed that neutralizing anti-factor VIII (anti-fVIII) antibodies (inhibitors) in patients with acquired hemophilia A (AHA) and congenital hemophilia A (HA) are primarily directed to the A2 and C2 domains. In this study, the frequency and epitope specificity of anti-C1 antibodies were analyzed in acquired and congenital hemophilia inhibitor patients (n = 178). The domain specificity of antibodies was studied by homolog-scanning mutagenesis (HSM) with single human domain human/porcine fVIII proteins and antibody binding to human A2, C1, and C2 domains presented as human serum albumin (HSA) fusion proteins. The analysis with HSA-fVIII domain proteins confirmed the results of the HSM approach but resulted in higher detection levels. The higher detection levels with HSA-fVIII domain proteins are a result of antibody cross-reactivity with human and porcine fVIII leading to false-negative HSM results. Overall, A2-, C1-, and C2-specific antibodies were detected in 23%, 78%, and 68% of patients with AHA (n = 115) and in 52%, 57%, and 81% of HA inhibitor patients (n = 63). Competitive binding of the human monoclonal antibody (mAb) LE2E9 revealed overlapping epitopes with murine C1-specific group A mAbs including 2A9. Mutational analyses identified distinct crucial binding residues for LE2E9 (E2066) and 2A9 (F2068) that are also recognized by anti-C1 antibodies present in patients with hemophilia. A strong contribution of LE2E9- and 2A9-like antibodies was particularly observed in patients with AHA. Overall, our study demonstrates that the C1 domain, in addition to the A2 and C2 domains, contributes significantly to the humoral anti-fVIII immune response in acquired and congenital hemophilia inhibitor patients., (© 2017 by The American Society of Hematology.)

Introduction: The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is currently the most serious complication for patients with haemophilia A undergoing FVIII replacement therapy. Several genetic factors have been acknowledged as risk factors for inhibitor development., Aim: To analyze the influence of genetic factors on the nature of the humoral immune response to FVIII in eight brother pairs with inhibitors., Methods: The domain specificity of FVIII-specific IgG was analysed by antibody binding to FVIII fragments and homologue-scanning mutagenesis (HSM). The FVIII-specific IgG subclasses were measured by direct ELISA., Results: Of the 16 patient analysed with both methods, 12 had A2- and 13 had C2-specific IgG. The presence of A1-, A3- or C1-specific IgG was identified in nine of 14 patients analysed by HSM. IgG1, IgG2 and IgG4 subclasses contributed to the anti-FVIII IgG response, and the amount of FVIII-specific IgG1 (r = 0.66) and IgG4 (r = 0.69) correlated significantly with inhibitor titres. Patients with high concentrations of total anti-FVIII IgG (r = 0.69) or high inhibitor titres (r = 0.52) had a high proportion of FVIII-specific IgG4. Statistical analysis revealed trends/evidence that the subclass distribution (P = 0.0847) and domain specificity to HC/LC (P = 0.0883) and A2/C2 (P = 0.0011) of anti-FVIII IgG were more similar in brothers compared to unrelated subjects., Conclusion: Overall, our data provide a first hint that anti-FVIII IgG characteristics are comparable among haemophilic brothers with inhibitors. Whether genetic factors also influence the nature of patients' antibodies needs to be confirmed in a larger study population., (© 2016 John Wiley & Sons Ltd.)

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance., (© 2016 by The American Society of Hematology.)

Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

Objective: To compare the long-term self-esteem and social function outcomes of individuals with untreated and treated ADHD across childhood, adolescence, and adulthood., Method: A systematic search of 12 databases was performed to identify peer-reviewed, primary research articles, published January 1980 to December 2011, reporting long-term self-esteem and/or social function outcomes (≥2 years; life consequences distinct from symptoms) of individuals with untreated or treated ADHD., Results: Overall, 127 studies reported 150 outcomes. Most outcomes were poorer in individuals with untreated ADHD versus non-ADHD controls (57% [13/23] for self-esteem; 73% [52/71] for social function). A beneficial response to treatment (pharmacological, nonpharmacological, and multimodal treatments) was reported for the majority of self-esteem (89% [8/9]) and social function (77% [17/22]) outcomes., Conclusion: Untreated ADHD was associated with poorer long-term self-esteem and social function outcomes compared with non-ADHD controls. Treatment for ADHD was associated with improvement in outcomes; however, further long-term outcome studies are needed., (© The Author(s) 2013.)

Atopic dermatitis (AD) represents a pruritic, non-contagious, chronic or chronically relapsing, inflammatory skin disease. The course of the disease may be complicated by bacterial or viral superinfections. The first manifestation of the disease and further flare-ups are due to genetic predisposition and also to a variety of further trigger factors. The therapy regimen should be adapted to disease symptoms that are actually present and consider individual features of the disease as reported by the patients or their parents. This short version of the German guideline on AD provides an overview of evidence-based diagnostic and treatment options. All recommendations made here are the result of a consensus of the scientific medical societies, working groups and support groups based on scientific data published to date. Abstracts and details of the studies cited are provided in the long version of this guideline (see: www.awmf.org).

(Reprinted with permission from PLOS One 2015; 10(2):1-19)., (Copyright © 2016 by the American Psychiatric Association.)

Background: Evaluation of treatments for attention-deficit/hyperactivity disorder (ADHD) previously focused on symptom control, but attention has shifted to functional outcomes. The effect of different ADHD treatment periods and modalities (pharmacological, non-pharmacological, and combination) on long-term outcomes needs to be more comprehensively understood., Methods: A systematic search of 12 literature databases using Cochrane's guidelines yielded 403 English-language peer-reviewed, primary studies reporting long-term outcomes (≥2 years). We evaluated relative effects of treatment modalities and durations and effect sizes of outcomes reported as statistically significantly improved with treatment., Results: The highest proportion of improved outcomes was reported with combination treatment (83% of outcomes). Among significantly improved outcomes, the largest effect sizes were found for combination treatment. The greatest improvements were associated with academic, self-esteem, or social function outcomes. A majority of outcomes improved regardless of age of treatment initiation (60%-75%) or treatment duration (62%-72%). Studies with short treatment duration had shorter follow-up times (mean 3.2 years total study length) than those with longer treatment durations (mean 7.1 years total study length). Studies with follow-up times <3 years reported benefit with treatment for 93% of outcomes, whereas those with follow-up times ≥3 years reported treatment benefit for 57% of outcomes. Post-hoc analysis indicated that this result was related to the measurement of outcomes at longer periods (3.2 versus 0.4 years) after treatment cessation in studies with longer total study length., Conclusions: While the majority of long-term outcomes of ADHD improve with all treatment modalities, the combination of pharmacological and non-pharmacological treatment was most consistently associated with improved long-term outcomes and large effect sizes. Older treatment initiation age or longer durations did not markedly affect proportion of improved outcomes reported, but measurement of outcomes long periods after treatment cessation may attenuate results.

The most serious complication in today's treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20-30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2-76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.

Attention-deficit/hyperactivity disorder (ADHD) is a highly prevalent psychiatric disorder in children/adolescents. This study reviews available European-based studies of ADHD-related costs and applies the findings to the Netherlands to estimate annual national costs for children/adolescents from a societal perspective. A systematic literature search was conducted for primary studies in Europe, published January 1, 1990 through April 23, 2013. Per-person cost estimates were converted to 2012 Euros and used to estimate annual national ADHD-related costs based on the Dutch 2011 census, ADHD prevalence rates, family composition, and employment rates. Seven studies met the inclusion criteria. The average total ADHD-related costs ranged from €9,860 to 14,483 per patient and annual national costs were between €1,041 and €1,529 million (M). The largest cost category was education (€648 M), representing 62 and 42 % of the low- and high-value overall national estimates, respectively. By comparison, ADHD patient healthcare costs ranged between €84 M (8 %) and €377 M (25 %), and social services costs were €4.3 M (0.3-0.4 %). While the majority of the costs were incurred by ADHD patients themselves, €161 M (11-15 %) was healthcare costs to family members that were attributable to having an ADHD child/adolescent. In addition, productivity losses of family members were €143-€339 M (14-22 %). Despite uncertainties because of the small number of studies identified and the wide range in the national cost estimates, our results suggest that ADHD imposes a significant economic burden on multiple public sectors in Europe. The limited number of European-based studies examining the economic burden of ADHD highlights the need for more research in this area.

The development of inhibitory anti-FVIII antibodies is currently the most severe complication in the treatment of haemophilia A patients. Inhibitor eradication can be achieved by immune tolerance induction (ITI). Recent findings suggest a correlation between the FVIII-specific IgG subclass distribution and the duration or outcome of ITI. To quantify FVIII-specific IgG subclasses in patients' plasma FVIII-specific IgG standards are required. Here, the isolation of FVIII-specific single chain variable fragments (scFvs) from synthetic phage display libraries and the characterisation of their FVIII domain specificity are described. The isolated scFv 1G10, which binds to the FVIII A2 domain, was cloned into the context of the four human IgG (hIgG) subclasses and expressed in mammalian cells. Purified 1G10-hIgG1, -hIgG2, -hIgG3 and -hIgG4 are used as standards to determine the absolute amounts and relative contribution of the different FVIII-specific IgG subclasses in future studies. The results from these studies will eventually add to understanding the role of the FVIII-specific IgG subclass distribution as prognostic factor for the outcome of ITI.

Objective: Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent mental disorders in children in the United States and often persists into adulthood with associated symptomatology and impairments. This article comprehensively reviews studies reporting ADHD-related incremental (excess) costs for children/adolescents and adults and presents estimates of annual national incremental costs of ADHD., Method: A systematic search for primary United States-based studies published from January 1, 1990 through June 30, 2011 on costs of children/adolescents and adults with ADHD and their family members was conducted. Only studies in which mean annual incremental costs per individual with ADHD above non-ADHD controls were reported or could be derived were included. Per-person incremental costs were adjusted to 2010 U.S. dollars and converted to annual national incremental costs of ADHD based on 2010 U.S. Census population estimates, ADHD prevalence rates, number of household members, and employment rates by age group., Results: Nineteen studies met the inclusion criteria. Overall national annual incremental costs of ADHD ranged from $143 to $266 billion (B). Most of these costs were incurred by adults ($105 B-$194 B) compared with children/adolescents ($38 B-$72 B). For adults, the largest cost category was productivity and income losses ($87 B-$138 B). For children, the largest cost categories were health care ($21 B-$44 B) and education ($15 B-$25 B). Spillover costs borne by the family members of individuals with ADHD were also substantial ($33 B-$43 B)., Conclusion: Despite a wide range in the magnitude of the cost estimates, this study indicates that ADHD has a substantial economic impact in the United States. Implications of these findings and future directions for research are discussed., (Copyright © 2012 American Academy of Child and Adolescent Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: In childhood, attention deficit/hyperactivity disorder (ADHD) is characterized by age-inappropriate levels of inattentiveness/disorganization, hyperactivity/impulsiveness, or a combination thereof. Although the criteria for ADHD are well defined, the long-term consequences in adults and children need to be more comprehensively understood and quantified. We conducted a systematic review evaluating the long-term outcomes (defined as 2 years or more) of ADHD with the goal of identifying long-term outcomes and the impact that any treatment (pharmacological, non-pharmacological, or multimodal) has on ADHD long-term outcomes., Methods: Studies were identified using predefined search criteria and 12 databases. Studies included were peer-reviewed, primary studies of ADHD long-term outcomes published between January 1980 to December 2010. Inclusion was agreed on by two independent researchers on review of abstracts or full text. Published statistical comparison of outcome results were summarized as poorer than, similar to, or improved versus comparators, and quantified as percentage comparisons of these categories., Results: Outcomes from 351 studies were grouped into 9 major categories: academic, antisocial behavior, driving, non-medicinal drug use/addictive behavior, obesity, occupation, services use, self-esteem, and social function outcomes. The following broad trends emerged: (1) without treatment, people with ADHD had poorer long-term outcomes in all categories compared with people without ADHD, and (2) treatment for ADHD improved long-term outcomes compared with untreated ADHD, although not usually to normal levels. Only English-language papers were searched and databases may have omitted relevant studies., Conclusions: This systematic review provides a synthesis of studies of ADHD long-term outcomes. Current treatments may reduce the negative impact that untreated ADHD has on life functioning, but does not usually 'normalize' the recipients.

There is increased global recognition of attention deficit hyperactivity disorder (ADHD) as a serious medical condition with long-term consequences. Although originally conceived of as a childhood disorder, ADHD is being increasingly recognized in adults. Individual geographic regions may have specific interests and objectives for the study of ADHD. A systematic review of long-term outcomes (LTOs) in ADHD was conducted to evaluate research on ADHD LTOs on a global scale. Studies that were at least 2 years in duration were examined. A total of 351 studies were identified in the final analysis. We identified nine outcomes of interest and classified studies by specific geographical regions, age groups studied and study design by region and over time. Published studies of LTOs in ADHD have increased in all geographical regions over the past three decades, with a peak number of 42 publications in 2008. This rise in publications on ADHD LTOs may reflect a rise in global interest and recognition of consequences and impairment associated with ADHD. Although many world regions have published on ADHD LTOs, the majority of studies have emerged from the US and Canada, followed by Europe. While investigators in the US and Canada were predominantly interested in drug addiction as a LTO, European researchers were more interested in antisocial behavior, and Eastern Asian investigators focused on both of these LTOs as well as self-esteem. Geographical differences in the focus of ADHD LTO studies may reflect regional variations in cultural values. Proportionally fewer prospective longitudinal studies and proportionally more retrospective and cross-sectional studies have been published in more recent decades. Finally, more studies focusing on ADHD in adolescents and adults have been conducted in recent years, and particularly adolescents in Eastern Asia. These changes in basic study design may reflect an increase in the recognition that ADHD is a lifetime chronic disorder. This systematic review analysis of publication trends in ADHD LTOs reflects geographically based interests that change over time.

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen.

The histone fold is a structural element that facilitates heterodimerization, and histone fold heterodimers play crucial roles in gene regulation. Here, we investigated the nuclear import of two human histone fold pairs, which belong to the H2A/H2B family: CHRAC-15/CHRAC-17 and p12/CHRAC-17. Our results from in vitro nuclear import assays with permeabilized cells and in vivo cotransfection experiments reveal that importin 13 facilitates nuclear import of both histone fold heterodimers. Using glutathione S-transferase pulldown experiments, we provide evidence that heterodimers are required for efficient binding of importin 13 because the monomers alone do not significantly interact. Mutational analysis shows that stepwise substitution of basic amino acid residues conserved among the histone fold subunits leads to a progressive loss of importin 13 binding and nuclear accumulation of CHRAC-15/CHRAC-17 and p12/CHRAC-17. The distribution of basic amino acid residues among the histone fold subunits essential for nuclear uptake suggests that heterodimerization of the histone fold motif-containing proteins forms an importin 13-specific binding platform.

The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2alpha and NC2beta, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2alpha and NC2beta depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin alpha/beta heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2beta. In contrast, NC2alpha does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2alpha masks the NES in NC2beta, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin alpha/beta-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits.

The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC) is a lysosomal matrix protein that was initially identified in a screen for genes induced by retinoic acid. Recently, it has been spotlighted by several proteome analyses of the lysosomal compartment, but its cellular function and properties remain unknown to date. In this study, Scpep1 from mice was analysed with regard to its intracellular processing into a mature dimer consisting of a 35 kDa N-terminal fragment and a so far unknown 18 kDa C-terminal fragment and the glycosylation status of the mature Scpep1 fragment. Although Scpep1 shares notable homology and a number of structural hallmarks with the well-described lysosomal carboxypeptidase protective protein/cathepsin A, the purified recombinant 55 kDa precursor and the homogenates of Scpep1-overexpressing cells do not show proteolytic activity or increased serine carboxypeptidase activity towards artificial serine carboxypeptidase substrates. Hence, we disrupted the Scpep1 gene in mice by a gene trap cassette, resulting in a Scpep1/beta-galactosidase/neomycin phosphotransferase fusion protein. The fusion protein is devoid of the C-terminal half of Scpep1, including two amino acids of the assumed catalytic triad which is indispensable for its predicted serine carboxypeptidase activity. However, Scpep1-deficient mice were viable and fertile, and did not exhibit either lysosomal storage or reduced lysosomal SC activity under any tested condition.

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented.

The transcriptional activator NF-Y is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC, which specifically binds the CCAAT consensus present in about 30% of eukaryotic promoters. All three subunits contain evolutionarily conserved core regions, which comprise a histone fold motif (HFM) in the case of NF-YB and NF-YC. Our results of in vitro binding studies and nuclear import assays reveal two different transport mechanisms for NF-Y subunits. While NF-YA is imported by an importin beta-mediated pathway, the NF-YB/NF-YC heterodimer is translocated into the nucleus in an importin 13-dependent manner. We define a nonclassical nuclear localization signal (ncNLS) in NF-YA, and mutational analysis indicates that positively charged amino acid residues in the ncNLS are required for nuclear targeting of NF-YA. Importin beta binding is restricted to the monomeric, uncomplexed NF-YA subunit. In contrast, the nuclear import of NF-YB and NF-YC requires dimer formation. Only the NF-YB/NF-YC dimer, but not the monomeric components, are recognized by importin 13 and are imported into the nucleus. Importin 13 competes with NF-YA for binding to the NF-YB/NF-YC dimer. Our data suggest that a distinct binding platform derived from the HFM of both subunits, NF-YB/NF-YC, mediates those interactions.

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133-227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

Bioaccumulation of Cd, Co, Cu, Ni, Pb and Zn in Antarctic gammaridean amphipod collectives, Orchomene plebs (Hurley, 1965), was investigated during a cruise of RV "Polarstern" to the Wedell Sea. With the sole exception of Cd the organisms accumulated metals during exposure and depurated them in uncontaminated seawater. Four independent toxicokinetic experiments and one field study were modelled simultaneously to calculate the following size-dependent bioconcentration factors for organisms with body length 10 mm (BCF(10 mm)): 130 (Co), 4030 (Cu), 190 (Ni), 2900 (Pb), and 5210 (Zn). On the time scale of our experiments the data suggest an increased metal uptake by previously exposed test organisms. The collectives investigated may be regarded as potentially suitable biomonitors for Co, Cu, Ni, Pb and Zn but not for Cd. An approach to evaluate the sensitivity of Orchomene plebs as a biomonitor of waterborne metals in the field indicates minimal increments of the ambient exposure concentrations of 0.01 microg Co l(-1), 0.2 microg Cu l(-1), 0.4 microg Ni l(-1), 0.6 microg Pb l(-1) and 0.3 microg Zn l(-1).

The concentrations of Cd, Co, Cu, Ni, Pb and Zn were determined in the Antarctic copepods Rhincalanus gigas (Brady, 1883), Calanus propinquus (Brady, 1883), Calanoides acutus (Giesbrecht, 1902), Metridia curticauda (Giesbrecht, 1889) and Metridia gerlachei (Giesbrecht, 1902). Samples were taken at seven different stations between 18.01.1999 and 19.02.1999. Metal concentrations in biological tissue were determined by graphite furnace atomic absorption spectroscopy (GFAAS) with Zeeman background correction and by flame AAS (air-acetylene) with deuterium background correction. We found high mean Cd concentrations in the Metridia species of about 10 microg Cd g(-1) and 3-6 microg Cd g(-1) in the other copepods. Co and Pb concentrations were low in all species investigated (<0.1 microg Co g(-1) and <1 microg Pb g(-1)). Zn concentrations were high in M. gerlachei and R. gigas (518 and 430 microg Zn g(-1)). In comparison to copepods from Arctic Seas (Fram Strait, Greenland Sea) and the North Sea, Cd and Cu concentrations appear higher in Antarctic copepods, while Ni and Pb concentrations are similar in both polar regions and Pb concentrations are higher in the North Sea. Variability between species and different regions are discussed.

Bioaccumulation of Cd, Co, Cu, Ni, Pb and Zn in the Antarctic calanoid copepod Calanoides acutus (Giesbrecht, 1902) was investigated during a cruise of RV 'Polarstern' to the Weddell Sea. Main goals were to provide information on accumulation strategies of the organisms tested and to verify toxicokinetic models as a predictive tool. Except for Cd, the organisms accumulated metals upon exposure. It was possible to estimate significant model parameters of two-compartment and hyperbolic models. These models were successfully verified in a second toxicokinetic uptake study. The model verification was extended in a third uptake study with increasing external metal dosing. We found a linear increase of net uptake with external waterborne metal exposures up to 80 microg Pb l(-1), with excellent predictions of the two-compartment model. For Co both models give reasonable predictions up to 20 microg Co l(-1). Regarding Cu, Ni and Zn only hyperbolic model predictions were in good agreement with measured values up to 150 microg Cu l(-1), 80 microg Ni l(-1) and 290 microg Zn l(-1). Due to a decrease of Cd body burden in the experiments, only the hyperbolic model was applicable, leading to reliable predictions up to 20 microg Cd l(-1). These concentrations largely determine the range for which these models may serve as a predictive tool.

Bioaccumulation of Cd, Co, Cu, Ni, Pb and Zn in the Antarctic calanoid copepod Metridia gerlachei (Giesbrecht 1902) was investigated during a cruise of RV 'Polarstern' to the Weddell Sea, primarily to provide information on accumulation strategies for the metals tested. With the sole exception of Cd, the copepod accumulated metals during exposure and depurated them in uncontaminated seawater. The process of uptake and depuration was successfully described by a hyperbolic model, leading to significant estimations of the following experimental bioconcentration factors (BCFs): 210 (Co), 3430 (Cu), 3060 (Ni), 670 (Pb) and 2090 (Zn). Furthermore, we provide an approach to evaluate the sensitivity of Metridia gerlachei as a biomonitor of water-borne metals in the field; the results indicate minimal increments in ambient exposure concentrations of: 0.5 microg Cu l(-1), 0.8 microg Ni l(-1), 0.6 microg Pb l(-1) and 0.2 microg Zn l(-1), suggesting a high sensitivity of M. gerlachei for biomonitoring.

Inflammatory neutrophils elicited by intraperitoneal injection of Corynebacterium parvum, thioglycollate or proteose peptone were capable of lysing different murine and human tumor targets in a short-term chromium-release assay. A single-cell cytotoxicity assay, which evaluated effector-target cell interactions at the single-cell level, confirmed a PMN-mediated tumor-lytic effect. Optimal lysis was achieved by PMNs obtained 6 hr after injection of C. parvum and 16 hr after injection of thioglycollate. In vitro, loss of tumor cell membrane integrity occurred extremely rapidly following conjugation with inflammatory PMNs (beginning within 15 min of the binding step). By 45 min, the lytic event was completed. Addition of catalase or superoxide dismutase to the cytotoxicity assays prevented tumor lysis in a concentration-dependent fashion, indicating that hydrogen peroxide and superoxide, products of the PMN respiratory burst, are mediators of the lytic reaction.

We studied the role of inflammatory neutrophils in the antitumor effects that follow i.p. injection of Corynebacterium parvum (1400 micrograms) into C3HeB/FeJ mice challenged with the murine ovarian teratocarcinoma. Peritoneal neutrophils, obtained from mice 6 hr after injection of C. parvum, exerted significant antitumor effects when injected admixed with murine ovarian terato-carcinoma cells into the peritoneal cavities of normal mice. Treatment of recipient mice with whole-body irradiation or repeated injections of silica prevented the antitumor effects, indicating that neutrophils were activating a second effector mechanism in recipient mice. Peritoneal cells obtained at 24 or 72 hr or at 7 or 11 days following C. parvum injection were considerably less effective in activation of this effector mechanism. Heat-killed C. parvum (6 hr)-induced neutrophils activated antitumor responses, but thioglycolate-induced cells were without effect. Antitumor responses in mice receiving peritoneal neutrophils were not due to simple transfer of C. parvum organisms in the inocula. These results indicate that inflammatory neutrophils, elicited into the peritoneal cavity by injection of C. parvum, play an important role in the activation of subsequent antitumor effects.