Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Invasive electrical stimulation in the treatment of avascular osteonecrosis of the femoral head - mid-term results.

The study aimed to evaluate the outcomes of osteonecrosis of the femoral head (ONFH) in adults after surgical treatment including invasive electromagnetic osteostimulation (E-Stim). Further, the influence of disease stage and several comorbidities on the joint preservation rate should be examined. Sixty patients (66 hip joints) with ONFH were included in this retrospective cross-sectional analysis (mean follow-up: 58 months, 19-110 months). Potential ONFH risk factors and comorbidities (ONFH stage, age, sex, alcohol, smoking, cortisone medication, chemotherapy) were recorded. The influence of specific parameters on the joint preservation rates was evaluated by a multivariate logistic regression analysis. Finally, patients with preserved hip joints underwent an assessment of their last available X-rays. The joint preservation rate depended on the initial ONFH Steinberg stage (I+II: 82.8%, III: 70.8%, ≥ IVa: 38.5%). Initially collapsed ONFH (p ≤ 0.001) and cortisone therapy (p = 0.004) significantly decreased the joint preservation rates. In case of progressed ONFH, the presence of ≥ 2 risk factors resulted in higher THA conversion rates (stage III: OR 18.8; stage ≥IVa: OR 12). In 94% of the available X-rays, the ONFH stage improved or did not progress. No complications could be attributed to the E-Stim device or procedure. The present surgical protocol including minimally invasive E-Stim revealed high joint preservation rates for non-collapsed ONFH after mid-term postoperative follow-up. Especially in progressed ONFH, the-risk profile seems to be crucial and hence, for joint preserving surgery, careful patient selection is recommended.

[Saturated fatty acids and cardiovascular risk : Is a revision of the recommendations on nutrition indicated?] / Gesättigte Fettsäuren und kardiovaskuläres Risiko : Ist eine Revision der Ernährungsempfehlungen angezeigt?

The "fat hypothesis of coronary heart disease", according to which saturated fatty acids (SFA) increase the concentration of low-density lipoprotein-cholesterol (LDL-C) and consequently increase the risk for cardiovascular diseases, influenced the nutritional recommendations over the last 60 years, initially in the USA and later also in Europe. Over the years there accumulated a growing body of evidence from epidemiology and controlled clinical studies that the consumption of SFA per se was not associated with an increased cardiovascular risk and the limitation of consumption of SFA did not show a preventive effect. The focus on the SFA content negated the biologically heterogeneous and sometimes biologically favorable effects of various SFAs. In addition, it was neglected that SFAs in foodstuffs are bound in a variety of complex matrices, which are composed of dozens of nutrients with different structures and concomitant substances and therefore each triggers different biological responses and metabolic effects. Accordingly, such nutrient-based recommendations are principally not very productive and also difficult to realize. In addition, LDL­C is not a suitable marker to assess the effect of lifestyle interventions, such as nutrition or physical activity, on the global cardiovascular risk.

Outcome of Surgical and Conservative Treatment of Patients with Shoulder Impingement Syndrome - a Prospective Comparative Clinical Study.

PURPOSE OF THE STUDY Subacromial impingement is one of the most common reasons for shoulder pain. The surgical management of this condition has recently become the focus of criticism because of the rising number of surgical procedures and the lack of superiority of surgical over conservative treatment. In this prospective comparative study, we compared standardised conservative care with surgical treatment and placed special emphasis on the patients' ability to work. MATERIAL AND METHODS A total of 106 patients (25 women, 81 men; mean age: 45.4 12.3 years) were included in this prospective comparative clinical study. Patients in the non-operative arm (n = 42) received standardised physiotherapy. Patients in the surgical arm (n = 38) underwent arthroscopic subacromial decompression. All patients were followed up at 3, 6 and 12 months. Shoulder function (Constant score), pain (Numerical Rating Scale), and the duration of inability to work were assessed. RESULTS Shoulder function and pain improved significantly with both kinds of treatment. At no time of follow up we detected significant differences between the two treatment options. An analysis of the patients' ability to work showed that conservative treatment was superior to surgical treatment at 3-month follow-up (0.3 versus 5.0 weeks; p < 0.001) and between 4 and 6-month after intervention (0.2 versus 1.6 weeks; p = 0.032). DISCUSSION In the study presented here, significant improvements in function (Constant score) and pain (NRS) were achieved in both the non-operative and the surgical arm. There were no significant differences between the two groups at any time point. These results are similar to those reported by other authors in recent studies. Unlike other research work, however, our study demonstrated a major difference in the development and duration of inability to work. CONCLUSIONS Conservative and surgical treatment of subacromial impingement syndrome led to similar outcomes for shoulder pain and function at 3, 6 and 12 months after intervention. However, patients who were managed conservatively returned to work significantly earlier than patients who underwent surgery. Key words: subacromial impingement, shoulder, constant score, pain, ability to work.

Effects of interfacial micromotions on vitality and differentiation of human osteoblasts.

OBJECTIVES: Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed. METHODS: Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated. RESULTS: Live and dead cell numbers were higher after 25 µm sine and 50 µm triangle micromotions compared with loaded controls. Collagen type I synthesis was downregulated in respective samples. The metabolic activity and osteocalcin expression level were higher in samples treated with 25 µm micromotions compared with the loaded controls. Furthermore, static loading and micromotions decreased the osteoprotegerin/receptor activator of NF-κB ligand ratio. CONCLUSION: Our system enables investigation of the behaviour of bone cells at the bone-implant interface under shear stress induced by micromotions. We could demonstrate that micromotions applied under static pressure conditions have a significant impact on the activity of osteoblasts seeded on collagen scaffolds. In future studies, higher mechanical stress will be applied and different implant surface structures will be considered.Cite this article: J. Ziebart, S. Fan, C. Schulze, P. W. Kämmerer, R. Bader, A. Jonitz-Heincke. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts. Bone Joint Res 2018;7:187-195. DOI: 10.1302/2046-3758.72.BJR-2017-0228.R1.

Mast cells decrease efficacy of anti-angiogenic therapy by secreting matrix-degrading granzyme B.

Resistance towards VEGF-centered anti-angiogenic therapy still represents a substantial clinical challenge. We report here that mast cells alter the proliferative and organizational state of endothelial cells which reduces the efficacy of anti-angiogenic therapy. Consequently, absence of mast cells sensitizes tumor vessels for anti-angiogenic therapy in different tumor models. Mechanistically, anti-angiogenic therapy only initially reduces tumor vessel proliferation, however, this treatment effect was abrogated over time as a result of mast cell-mediated restimulation of angiogenesis. We show that mast cells secrete increased amounts of granzyme b upon therapy, which mobilizes pro-angiogenic laminin- and vitronectin-bound FGF-1 and GM-CSF from the tumor matrix. In addition, mast cells also diminish efficacy of anti-angiogenic therapy by secretion of FGF-2. These pro-angiogenic factors act beside the targeted VEGFA-VEGFR2-axis and reinduce endothelial cell proliferation and angiogenesis despite the presence of anti-angiogenic therapy. Importantly, inhibition of mast cell degranulation with cromolyn is able to improve efficacy of anti-angiogenic therapy. Thus, concomitant mast cell-targeting might lead to improved efficacy of anti-angiogenic therapy.Resistance towards VEGF-centered anti-angiogenic therapy is an important clinical challenge. Here, the authors show that mast cells mediate resistance to anti-angiogenetic inhibitors by altering the proliferative and organizational state of endothelial cells through mobilization of FGF-1 and GM-CSF from the tumor matrix and secretion of FGF-2.

Transplacental transmission of Neospora caninum in moose (Alces alces).

A 5-years-old moose (Alces alces) cow kept in a zoo in the German Federal State of Brandenburg aborted a female foetus of 44cm crown rump length (CRL). Pathohistological analysis revealed several Neospora (N.) caninum infected cells and cysts, as well as multifocal gliosis, necrosis, haemorrhages, dystrophic mineralisation and haemosiderosis in the brain, predominantly in cerebrum and brainstem. In addition, mild lymphocytic meningitis was present. Together with the fresh foetus, a mummified foetus of 16cm CRL was expelled. Neither focal necrosis, nor inflammation was detected in the brain of the mummified foetus. By two polymerase chain reactions (PCR) targeting the pNc5 gene of N. caninum (i.e. an end point PCR and a real-time PCR), by two serological methods (immunofluorescence test and immunoblot), by histological and immunohistochemical analyses, transplacental N. caninum infection was confirmed in the fresh foetus and interpreted as possible cause of abortion. Infection with other agents causing abortion including Bovine Herpesvirus 1 (BHV1), Bluetongue Virus (BTV), Bovine Virus Diarrhoea Virus (BVDV), Brucella spp., Chlamydia spp., Coxiella burnetii and Toxoplasma gondii were excluded. Our findings show that control measures may be necessary to protect captive moose against accidental N. caninum infection. Further studies are needed to explore the importance of neosporosis in wild and captive moose.

Cross-cultural validity of the German version of the Pediatric Evaluation of Disability Inventory (PEDI-G)-a Rasch model application.

AIM: The aim of this study was to evaluate the cross-cultural validity of the German version of the Pediatric Evaluation of Disability Inventory (PEDI-G) when used in Austria, Germany and Switzerland. METHOD: A total of 118 girls and 144 boys participated in this study; 198 of the children (75.6%) had a developmental disability and 64 (24.4%) were without a known disability. The mean age was four years (range 11 months to 10 years and six months, SD 1.91). Item goodness of fit, differential item functioning (DIF) and differential test functioning (DTF) were evaluated by use of a Rasch model. RESULTS: Twenty-four (11.6%) out of 206 items of the Functional Skills Scale and one (5%) out of 20 items of the Caregiver Assistance Scale demonstrated misfit according to the Rasch model. Thirty-four (16.5%) out of 206 items of the Functional Skills Scale and no item from the Caregiver Assistance Scale demonstrated DIF. Almost half (46%) of the items demonstrating misfit also demonstrated DIF, indicating an association between them. The DIF by country only demonstrated a minimal impact on the person measures of the PEDI-G. INTERPRETATION: Even though some items did not meet the statistical and clinical criteria set, the PEDI-G can be used, on a preliminary basis as a valid tool to measure activities of daily living of children with and without a disability in these countries. Further larger studies are needed to evaluate more psychometric item properties of the PEDI-G in relation to context.

[Therapy of a gunshot fracture to the midfoot]. / Therapie einer Schussfraktur am Mittelfußknochen.

We report on a soldier with a gunshot wound to the midfoot. The diaphysis of MT 5 was pounded to pieces and there was a fracture in the diaphysis of MT 4. After debridement and fixation of MT 4 and MT 5 to MT 2 and 3 via two Kirschner wires we conducted a second look procedure with reconstruction of MT 5 via a piece of iliac crest. After eight weeks of unloading and removal of the last wire we started to put successively heavier weight on the foot.

High and novel genetic diversity of Francisella tularensis in Germany and indication of environmental persistence.

In Germany tularemia is a re-emerging zoonotic disease. Therefore, we investigated wild animals and environmental water samples for the presence and phylogenetic diversity of Francisella tularensis in the poorly studied Berlin/Brandenburg region. The phylogenomic analysis of three isolates from wild animals revealed three new subclades within the phylogenetic tree of F. tularensis [B.71 from a raccoon dog (Nyctereutes procyonoides); B.74 from a red fox (Vulpes vulpes), and B.75 from a Eurasian beaver (Castor fiber albicus)]. The results from histological, PCR, and genomic investigations on the dead beaver showed that the animal suffered from a systemic infection. Indications were found that the bacteria were released from the beaver carcass into the surrounding environment. We demonstrated unexpectedly high and novel phylogenetic diversity of F. tularensis in Germany and the fact that the bacteria persist in the environment for at least one climatic season. These findings support a broader host species diversity than previously known regarding Germany. Our data further support the assumption derived from previous serological studies of an underestimated frequency of occurrence of the pathogen in the environment and in wild animals. F. tularensis was isolated from animal species not previously reported as natural hosts in Germany.

Electrical Injuries in Animals: Causes, Pathogenesis, and Morphological Findings.

Electrical injuries in animals occur most often accidentally. They comprise contact to various forms of currents, including alternating, rotary, or direct currents. Depending on various parameters of the current (including the type of circuit, voltage, current and duration of exposure) and conditions of the animal (such as wet or dry hair coat and pathway of current through the body), lesions may be absent or may include early or localized development of rigor mortis, signs of acute circulatory failure, or severe thermoelectrical burns. Such burns may present as external current marks, singed hair or feathers, metallization of the skin, or occasionally internal electroporation injury resulting in muscle necrosis, hemolysis, vascular damage with thrombosis, injury to brain and spinal cord, or skeletal fractures. Furthermore, lightning strikes occur regularly in grazing animals, which have greater risk of death from step potentials (ground current) in addition to direct strike and contact injury. Such cases may have no lesions, external signs of linear or punctate burns, keraunographic markings, or exit burns on the soles of the hooves or the coronary bands. Besides detailed information about the circumstances at the location where the animal was found, electrical injuries in animals require a thorough morphological workup, including additional investigations in conjunction with certain knowledge about the possible lesion spectrum.

Thermal Injuries in Veterinary Forensic Pathology.

Localized thermal injuries in animals may be caused by exposure to fire and radiant heat, contact with hot items including hot liquids or steam, inhalation of hot air, and exposure to cold temperatures. In addition, animal fire victims may have intoxications caused by smoke gas. This article reviews the causes, pathogenetic aspects, morphological findings, additional investigations, differential diagnoses, and causes of death in various forms of thermal injuries. Since these cases do not occur frequently in diagnostic pathology, they represent a challenging task in general but also with respect to forensic or criminal aspects, such as whether a lesion represents an accidental or nonaccidental effect. Besides detailed information about the circumstances at the location, thermal injuries in animals require a thorough morphological evaluation, including additional investigations in conjunction with a profound knowledge about the possible lesion spectrum and suitable additional investigations.

Detection of a Novel Bovine Astrovirus in a Cow with Encephalitis.

Encephalitis can be caused by several infectious agents, including bacteria, fungi, parasites and viruses. In many cases, the causative agent cannot be identified, because the pathogens are unknown or detection methods are not routinely available. In our case, a 15-month-old cow developed central nervous disorders and died within 6 days after the onset of clinical signs. The histopathology revealed an acute encephalitis, predominantly in the brain stem, and a ganglionitis of the trigeminal ganglion with massive neuronal necroses in both the brain and the ganglion. However, a relevant panel of bacterial and viral infections of cattle could be routinely excluded. Therefore, a brain sample from the cow was analysed using a metagenomics approach with next-generation sequencing. A novel bovine astrovirus (BoAstV-BH89/14) could be identified using the analysis pipeline RIEMS, and the finding could be confirmed with a specific BoAstV RT-qPCR. The genome of the bovine astrovirus (BoAstV), belonging to the family Astroviridae in the genus Mamastrovirus, has a length of 6478 bp. Sequence identities between 71% to a sheep astrovirus and 69% to two recently described bovine astroviruses from the USA and Switzerland were ascertained. The latter were also connected to encephalitis cases in cattle. Like these, the new virus described here was detected in different brain sections using the specific BoAstV RT-qPCR and fluorescent in situ hybridization. In conclusion, while astroviruses so far were mainly found in relation to gastroenteritis in animals and humans, recently detected astrovirus infections were also related to encephalitis.

[Evaluation of interest in research among surgically active medical officers in the German Armed Forces]. / Evaluation des Forschungsinteresses unter chirurgisch tätigen Sanitätsoffizieren der Bundeswehr.

BACKGROUND: Research in military medicine and in particular combat surgery is a broad field that has gained international importance during the last decade. In the context of increased NATO missions, this also holds true for the Bundeswehr (German Armed Forces); however, medical officers in surgery must balance research between their clinical work load, missions, civilian and family obligation. MATERIAL AND METHODS: To evaluate engagement with and interest in research, a questionnaire was distributed among the doctors of the surgical departments of the Bundeswehr hospitals by the newly founded working group Chirurgische Forschung der Bundeswehr (surgical research of the Bundeswehr). Returned data were recorded from October 2013 to January 2014 and descriptive statistics were performed. RESULTS: Answers were received from 87 out of 193 military surgeons (45 %). Of these 81 % announced a general interest in research with a predominance on clinical research in preference to experimental settings. At the time of the evaluation 32 % of the participants were actively involved in research and 53 % regarded it as difficult to invest time in research activities parallel to clinical work. Potential keys to increase the interest and engagement in research were seen in the implementation of research coordinators and also in a higher amount of free time, for example by research rotation. CONCLUSION: Research can be regarded as having a firm place in the daily work of medical officers in the surgical departments of the Bundeswehr; however, the engagement is limited by time and structural factors. At the departmental level and in the command structures of the military medical service, more efforts are recommended in the future in order to enhance the engagement with surgical research. This evaluation should be repeated in the coming years as a measuring instrument and data should be compared in an international context.

Genetic polymorphisms of twelve X-STRs of the investigator Argus X-12 kit and additional six X-STR centromere region loci in an Egyptian population sample.

Recently, many researchers have focused on analysis of different X-chromosomal STRs as they bear the potential to efficiently complement the analysis of autosomal and Y-chromosomal STRs in solving special complex kinship deficiency cases. In the current study we examined a sample of 250 unrelated Egyptian males with the Investigator Argus X-12 kit (Qiagen GmbH, Hilden, Germany) which detects 12 X-STR markers distributed over the entire X-chromosome as four closely linked clusters. Microvariant off ladder alleles as well as null alleles have been detected in some loci. Furthermore, discordant results were observed between the Investigator Argus X-12 and the Mentype(®) Argus X-8 kits (Biotype AG, Dresden, Germany). New primers were designed for loci DXS10101, DXS10146 and DXS10148 to correct the allele drop outs observed in these loci with the Investigator Argus X-12 kit. Additionally, DNA sequence analysis revealed the polymorphisms responsible for the allele drop outs. Furthermore, six additional X-STRs (DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165) located in the centromere region at Xp11.21-Xq11.1 were examined in a single multiplex reaction. Allele and haplotype frequencies as well as different forensic statistical parameters of the 18 X-STR loci tested indicated that they are highly informative in different forensic applications in the Egyptian population. However, some modifications still need to be performed on the Investigator Argus X-12 kit before its use in forensic casework is validated.

Tegaserod mimics the neurostimulatory glycan polysialic acid and promotes nervous system repair.

Glycans attached to the cell surface via proteins or lipids or exposed in the extracellular matrix affect many cellular processes, including neuritogenesis, cell survival and migration, as well as synaptic activity and plasticity. These functions make glycans attractive molecules for stimulating repair of the injured nervous system. Yet, glycans are often difficult to synthesize or isolate and have the disadvantage to be unstable in a complex tissue environment. To circumvent these issues, we have screened a library of small organic compounds to search for structural and functional mimetics of the neurostimulatory glycan polysialic acid (PSA) and identified the 5-HT4 receptor agonist tegaserod as a PSA mimetic. The PSA mimicking activity of tegaserod was shown in cultures of central and peripheral nervous system cells of the mouse and found to be independent of its described function as a serotonin (5-HT4) receptor agonist. In an in vivo model for peripheral nerve regeneration, mice receiving tegaserod at the site of injury showed enhanced recovery compared to control mice receiving vehicle control as evidenced by functional measurements and histology. These data indicate that tegaserod could be repurposed for treatment of nervous system injuries and underscores the potential of using small molecules as mimetics of neurostimulatory glycans.

IL7RA haplotype-associated alterations in cellular immune function and gene expression patterns in multiple sclerosis.

Interleukin-7 receptor alpha (IL7RA) is among the top listed candidate genes influencing the risk to develop multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. Soluble IL-7RA (sIL-7RA) protein and mRNA levels vary among the four common IL7RA haplotypes. Here we show and confirm that protective haplotype carriers have three times lower sIL-7RA serum levels than the other three haplotypes. High sIL-7RA concentrations significantly decrease IL-7-mediated STAT5 phosphorylation in CD4(+) T cells. Transcriptome analysis of unstimulated and stimulated CD4(+) T cells of MS patients carrying the different IL7RA haplotypes revealed complex and overlapping patterns in genes participating in cytokine signaling networks, apoptosis, cell cycle progression and cell differentiation. Our findings indicate that genetic variants of IL7RA result in haplotype-associated differential responsiveness to immunological stimuli that influence MS susceptibility not exclusively by varying levels of sIL-7RA.

Probing the energy barriers and magnetization reversal processes of nanoperforated membrane based percolated media.

Magnetization reversal processes in Co/Pt multilayers prepared on nanoperforated templates are probed by magnetization relaxation measurements. The signature of pinning controlled domain wall movement as expected for percolated media is identified. This contrasts with the nucleation-type reversal mechanism of a Co/Pt reference film prepared on a smooth substrate. A zero field energy barrier of 93kBT is determined by fluctuation field measurements and is elucidated by micromagnetic calculations using the nudged elastic band method. This value is sufficiently large to qualify the material as a promising percolated medium.