The humoral immune response more than one year after SARS-CoV-2 infection: low detection rate of anti-nucleocapsid antibodies via Euroimmun ELISA.

PURPOSE: Antibody assays against SARS-CoV-2 are used in sero-epidemiological studies to estimate the proportion of a population with past infection. IgG antibodies against the spike protein (S-IgG) allow no distinction between infection and vaccination. We evaluated the role of anti-nucleocapsid-IgG (N-IgG) to identify individuals with infection more than one year past infection. METHODS: S- and N-IgG were determined using the Euroimmun enzyme-linked immunosorbent assay (ELISA) in two groups: a randomly selected sample from the population of Stuttgart, Germany, and individuals with PCR-proven SARS-CoV-2 infection. Participants were five years or older. Demographics and comorbidities were registered from participants above 17 years. RESULTS: Between June 15, 2021 and July 14, 2021, 454 individuals from the random sample participated, as well as 217 individuals with past SARS-CoV-2 infection. Mean time from positive PCR test result to antibody testing was 458.7 days (standard deviation 14.6 days) in the past infection group. In unvaccinated individuals, the seroconversion rate for S-IgG was 25.5% in the random sample and 75% in the past infection group (P = < 0.001). In vaccinated individuals, the mean signal ratios for S-IgG were higher in individuals with prior infection (6.9 vs 11.2; P = < 0.001). N-IgG were only detectable in 17.1% of participants with past infection. Predictors for detectable N-IgG were older age, male sex, fever, wheezing and in-hospital treatment for COVID-19 and cardiovascular comorbidities. CONCLUSION: N-IgG is not a reliable marker for SARS-CoV-2 infection after more than one year. In future, other diagnostic tests are needed to identify individuals with past natural infection.

Energy-Efficient Decentralized Broadcasting in Wireless Multi-Hop Networks.

Several areas of wireless networking, such as wireless sensor networks or the Internet of Things, require application data to be distributed to multiple receivers in an area beyond the transmission range of a single node. This can be achieved by using the wireless medium's broadcast property when retransmitting data. Due to the energy constraints of typical wireless devices, a broadcasting scheme that consumes as little energy as possible is highly desirable. In this article, we present a novel multi-hop data dissemination protocol called BTP. It uses a game-theoretical model to construct a spanning tree in a decentralized manner to minimize the total energy consumption of a network by minimizing the transmission power of each node. Although BTP is based on a game-theoretical model, it neither requires information exchange between distant nodes nor time synchronization during its operation, and it inhibits graph cycles effectively. The protocol is evaluated in Matlab and NS-3 simulations and through real-world implementation on a testbed of 75 Raspberry Pis. The evaluation conducted shows that our proposed protocol can achieve a total energy reduction of up to 90% compared to a simple broadcast protocol in real-world experiments.

Treosulfan compared with reduced-intensity busulfan improves allogeneic hematopoietic cell transplantation outcomes of older acute myeloid leukemia and myelodysplastic syndrome patients: Final analysis of a prospective randomized trial.

The phase III study was designed to compare event-free survival (EFS) after treosulfan-based conditioning with a widely applied reduced-intensity conditioning (RIC) busulfan regimen in older or comorbid patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) undergoing allogeneic hematopoietic cell transplantation (HCT). A previously reported confirmatory interim analysis of the randomized clinical study including 476 patients demonstrated statistically significant noninferiority for treosulfan with clinically meaningful improvement in EFS. Here, the final study results and pre-specified subgroup analyses of all 570 randomized patients with completed longer-term follow-up are presented. Patients presenting HCT-specific comorbidity index >2 or aged ≥50 years were randomly assigned (1:1) to intravenous (IV) fludarabine with either treosulfan (30 g/m2 IV) or busulfan (6.4 mg/kg IV) after stratification by disease risk group, donor type, and participating institution. The primary endpoint was EFS with disease recurrence, graft failure, or death from any cause as events. EFS of patients (median age 60 years) was superior after treosulfan compared to RIC busulfan: 36-months-EFS rate 59.5% (95% CI, 52.2-66.1) vs. 49.7% (95% CI, 43.3-55.7) with a hazard ratio (HR) of 0.64 (95% CI, 0.49-0.84), p = 0.0006. Likewise, overall survival (OS) with treosulfan was superior compared to busulfan: 36-month-OS rate 66.8% vs. 56.3%; HR 0.64 (95% CI, 0.48-0.87), p = 0.0037. Post hoc analyses revealed that these differences were consistent with the confirmatory interim analysis, and thereby the treosulfan regimen appears particularly suitable for older AML and MDS patients.

Calf Blood Compound (CFC) and Homeopathic Drug Induce Differentiation of Primary Human Skeletal Muscle Cells.

The use of injections to treat structural muscle injuries is controversially discussed. In our controlled in vitro study, we investigated the biological impact of Actovegin and Traumeel alone and in combination on primary human skeletal muscle cells. Cells were characterized by immunofluorescence staining for myogenic factor 5 (Myf5) and MyoD, and cultured with or without Actovegin and / or Traumeel. The effects of these agents were assayed by cell viability and gene expression of the specific markers MyoD, Myf5, neural adhesion molecule (NCAM), and CD31. Myotube formation was determined by myosin staining. Neither Actovegin nor Traumeel showed toxic effects or influenced cell viability significantly. High volumes of Actovegin down-regulated gene expression of NCAM after 3 days but had no effect on MyoD, Myf5, and CD31 gene expression. High volumes of Traumeel inhibited MyoD gene expression after 3 days, whereas after 7 days MyoD expression was significantly up-regulated. The combination of both agents did not significantly influence cell viability or gene expression. This is the first study demonstrating that Actovegin and Traumeel potentially modulate human skeletal muscle cells. The relevance of these in vitro findings has to be highlighted in further in vivo studies.

Exposure to radial extracorporeal shockwaves induces muscle regeneration after muscle injury in a surgical rat model.

The leading cause of training interruption in sport is a muscle injury, for which the standard treatment is nonsteroidal anti-inflammatory drugs (NSAIDs). To find alternative treatments, we investigated whether the radial extracorporeal shockwave application (rESWT) could stimulate muscle regeneration. A lesion with complete rupture (grade III muscle tear) was set in the musculus rectus femoris of 12-week-old Wistar rats, and the NSAID diclofenac, rESWT, or a combined therapy were applied on day 0, 3, and 5 directly following the surgery. Rats were euthanized at 2, 4, and 7 days after surgery and the area of muscle lesion was excised for histological and gene expression analysis to determine the progress in the healing of damaged fibers and tissue regeneration. The best effect on muscle regeneration was observed in the group treated with rESWT alone. Monotherapy by diclofenac showed a smaller but still positive effect and lowest effects were detected when both therapies were applied. rESWT alone demonstrated a significant upregulation of the muscle markers MyoD and myosin. The presence of myosin gene expression indicated newly formed muscle fibers, which was confirmed by hematoxylin and eosin staining. Seven days after injury the amount of mononucleated cell decreased and regenerating fibers could be detected. This effect is most pronounced in the group treated with rESWT alone. In our study, shockwaves demonstrated the best effect on muscle regeneration. Therefore, we recommend prospective clinical studies to analyze the effect of rESWT after sports trauma to improve muscle regeneration and to shorten the rehabilitation.

Effect of bone sialoprotein coating on progression of bone formation in a femoral defect model in rats.

PURPOSE: In orthopedic and trauma surgery, calcium phosphate cement (CPC) scaffolds are widely used as substitute for autologous bone grafts. The purpose of this study was to evaluate bone formation in a femoral condyle defect model in rats after scaffold-coating with bioactive bone sialoprotein (BSP). Our hypothesis was that BSP-coating results in additional bone formation. METHODS: In 20 Wistar rats, defects of 3.0 mm diameter were drilled into the lateral femoral condyles of both legs. BSP-coated scaffolds or uncoated control scaffolds were implanted into the defects. After 4 and 8 weeks, five rats of each group were euthanized, respectively. µCT scans and histological analyses were performed. The ratio of bone volume-total volume (BV/TV) was analyzed and histological sections were evaluated. RESULTS: At week four, bone fraction reached 5.2 ± 1.7% in BSP-coated scaffolds and 4.5 ± 3.2% in the control (p = 0.06). While bone fraction of the BSP-group did not change much between week four and eight [week eight: 5.4 ± 3.8% (p = 0.53)], there was a tendency towards an increase in the control [week eight: 7.0 ± 2.2% (p = 0.08)]. No significant difference in bone fraction were observable between BSP-coated and uncoated scaffolds at week eight (p = 0.08). CONCLUSIONS: A significant superiority of BSP-coated scaffolds over uncoated scaffolds could not be proven. However, BSP-coating showed a tendency towards improving bone ingrowth in the scaffolds 4 weeks after implantation. This effect was only short-lived: bone growth in the control scaffolds tended to outpace that of the BSP-group at week eight.

Effects of losartan and atorvastatin on the development of early posttraumatic joint stiffness in a rat model.

BACKGROUND: After a trauma, exuberant tissue healing with fibrosis of the joint capsule can lead to posttraumatic joint stiffness (PTJS). Losartan and atorvastatin have both shown their antifibrotic effects in different organ systems. OBJECTIVE: The purpose of this study was the evaluation of the influence of losartan and atorvastatin on the early development of joint contracture. In addition to joint angles, the change in myofibroblast numbers and the distribution of bone sialoprotein (BSP) were assessed. STUDY DESIGN AND METHODS: In this randomized and blinded experimental study with 24 rats, losartan and atorvastatin were compared to a placebo. After an initial joint injury, rat knees were immobilized with a Kirschner wire. Rats received either losartan, atorvastatin or a placebo orally daily. After 14 days, joint angle measurements and histological assessments were performed. RESULTS: Losartan increased the length of the inferior joint capsule. Joint angle and other capsule length measurements did not reveal significant differences between both drugs and the placebo. At cellular level both losartan and atorvastatin reduced the total number of myofibroblasts (losartan: 191±77, atorvastatin: 98±58, placebo: 319±113 per counting field, p<0.01) and the percentage area of myofibroblasts (losartan: 2.8±1.8% [p<0.05], atorvastatin: 2.5±1.7% [p<0.01], vs control [6.4±4%], respectively). BSP was detectable in equivalent amounts in the joint capsules of all groups with only a trend toward a reduction of the BSP-stained area by atorvastatin. CONCLUSION: Both atorvastatin and losartan reduced the number of myofibroblasts in the posterior knee joint capsule of rat knees 2 weeks after trauma and losartan increased the length of the inferior joint capsule. However, these changes at the cellular level did not translate an increase in range of motion of the rats´ knee joints during early contracture development.

Evaluation of Bone Sialoprotein Coating of Three-Dimensional Printed Calcium Phosphate Scaffolds in a Calvarial Defect Model in Mice.

The bioactive coating of calcium phosphate cement (CPC) is a promising approach to enhance the bone-healing properties of bone substitutes. The purpose of this study was to evaluate whether coating CPCs with bone sialoprotein (BSP) results in increased bone formation. Forty-five female C57BL/6NRj mice with an average age of six weeks were divided into three groups. Either a BSP-coated or an uncoated three-dimensional plotted scaffold was implanted into a drilled 2.7-mm diameter calvarial defect, or the defect was left empty (control group; no CPC). Histological analyses revealed that BSP-coated scaffolds were better integrated into the local bone stock eight weeks after implantation. Bone volume/total volume (BV/TV) ratios and bone thickness at the bone⁻implant contact were analyzed via micro computed tomography (µCT) after eight weeks. BSP-coated scaffolds and uncoated CPC scaffolds increased bone thickness in comparison to the control (CPC + BSP: 691.1 ± 253.5 µm, CPC: 603.1 ± 164.4 µm, no CPC: 261.7 ± 37.8 µm, p < 0.01). Accordingly, BV/TV was enhanced in both scaffold groups (CPC + BSP: 1.3 ± 0.5%, CPC: 0.9 ± 0.5%, no CPC: 0.2 ± 0.3%, p < 0.01). The BSP coating showed a tendency towards an increased bone thickness (p = 0.18) and BV/TV (p = 0.18) in comparison to uncoated CPC scaffolds. However, a significant increase in bone formation through BSP coating was not found.

Photocrosslinked Dextran-Based Hydrogels as Carrier System for the Cells and Cytokines Induce Bone Regeneration in Critical Size Defects in Mice.

Modified biomaterials have for years been the focus of research into establishing new bone substitutes. In our preceding in vitro study employing different cell cultures, we developed chemically and mechanically characterized hydrogels based on photocrosslinkable dextran derivatives and demonstrated their cytocompatibility and their beneficial effects on the proliferation of osteoblasts and endothelial cells. In the present in vivo study, we investigate photocrosslinked dextran-based hydrogels in critical size defects in mice to evaluate their potential as carrier systems for cells or for a specific angiogenesis enhancing cytokine to induce bone formation. We could demonstrate that, with optimized laboratory practice, the endotoxin content of hydrogels could be reduced below the Food and Drug Administration (FDA)-limit. Dextran-based hydrogels were either loaded with a monoculture of endothelial cells or a co-culture of human osteoblasts with endothelial cells, or with stromal-derived-growth factor (SDF-1). Scaffolds were implanted into a calvarial defect of critical size in mice and their impact on bone formation was assessed by µCt-analyses, histology and immunohistology. Our study demonstrates that promotion of angiogenesis either by SDF-1 or a monoculture of endothelial cells induces bone regeneration at a physiological level. These in vivo results indicate the potential of dextran-based hydrogel composites in bone regeneration to deliver cells and cytokines to the defect site.

Effect of bone sialoprotein coated three-dimensional printed calcium phosphate scaffolds on primary human osteoblasts.

The combination of the two techniques of rapid prototyping 3D-plotting and bioactive surface functionalization is presented, with emphasis on the in vitro effect of Bone Sialoprotein (BSP) on primary human osteoblasts (hOBs). Our primary objective was to demonstrate the BSP influence on the expression of distinctive osteoblast markers in hOBs. Secondary objectives included examinations of the scaffolds' surface and the stability of BSP-coating as well as investigations of cell viability and proliferation. 3D-plotted calcium phosphate cement (CPC) scaffolds were coated with BSP via physisorption. hOBs were seeded on the coated scaffolds, followed by cell viability measurements, gene expression analysis and visualization. Physisorption is an effective method for BSP-coating. Coating with higher BSP concentrations leads to enhanced BSP release. Two BSP concentrations (50 and 200 µg/mL) were examined in this study. The lower BSP concentration (50 µg/mL) decreased ALP and SPARC expression, whereas the higher BSP concentration (200 µg/mL) did not change gene marker expression. Enhanced cell viability was observed on BSP-coated scaffolds on day 3. hOBs developed a polygonal shape and connected in an intercellular network under BSP influence. Quantitative cell morphology analyses demonstrated for BSP-coated CPCs an enhanced cell area and reduced circularity. The strength of the above-mentioned effects of BSP-coated scaffolds in vivo is unknown, and future work is focusing on bone ingrowth and vascularization in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2565-2575, 2018.

Long-Lasting Response to Trabectedin in a Patient with Metastatic Uterine Leiomyosarcoma: A Case Report.

BACKGROUND: Uterine leiomyosarcoma (uLMS) is a rare tumor that accounts for 1% of all uterine malignancies. In spite of adequate surgical resection of uLMS, even in the early stage, patients remain at high risk for local and distant recurrence. Therefore, the treatment of advanced uLMS represents a considerable challenge. METHODS: We report the case of a 47-year-old woman who presented with uLMS with abnormal vaginal bleeding. RESULTS: The patient underwent a total hysterectomy and bilateral adnexectomy, which was followed by 1 year progression-free survival without adjuvant therapy. Thereafter, new lung metastases and local progression at the vaginal stump were observed. Chemotherapy with ifosfamide and doxorubicin was administered. However, after 4 cycles, a CT scan revealed disease progression in the lung metastases. Subsequently, the patient was treated with trabectedin at a dose of 1.5 mg/m2 for 6 cycles resulting in complete remission of the lung metastases as well as partial remission of the mass in the vaginal stump after 9 cycles of trabectedin. The patient is currently on maintenance therapy with trabectedin and has no recurrence. CONCLUSION: Trabectedin seems to be an efficient option for patients with uLMS as demonstrated by a long-lasting response in a pretreated patient with an acceptable safety profile with no signs of cumulative toxicity.

A novel concept for the treatment of couperosis based on nanocrystals in combination with solid lipid nanoparticles (SLN).

For the post laser treatment of couperosis a new dermal formulation was developed combining three actives: vitamin K1, A1 and rutin, where both vitamins were incorporated into solid lipid nanoparticles (SLN) and the poorly soluble antioxidant rutin formulated as nanocrystal. All three formulations were stable over 6 months either on their own or after their incorporation into a hydrogel. Vitamin A1 at 0.3% in emulsions shows local skin irritation due to very rapid release. By forming SLN, prolonged release with less irritation potential but deeper penetration was achieved in porcine ear skin. Due to the nanosized rutin, the new hydrogel showed clearly increased antioxidant activity, representing a stronger protection potential against reactive oxygen species (ROS), compared to marketed anti-redness products with rutin as raw drug powder or water-soluble derivative. In addition, rutin nanocrystals showed up to 5 times pronounced penetration compared to µm-sized raw drug powder. The orientating in-vivo case study revealed a three to six times faster recovery after laser treatment of couperosis by twice daily application of the new hydrogel, regarding scabbed-over areas and erythema. Continued use of the new gel also showed preventive properties against recurrences of veins for at least 8 month.

Surface Functionalization of Orthopedic Titanium Implants with Bone Sialoprotein.

Orthopedic implant failure due to aseptic loosening and mechanical instability remains a major problem in total joint replacement. Improving osseointegration at the bone-implant interface may reduce micromotion and loosening. Bone sialoprotein (BSP) has been shown to enhance bone formation when coated onto titanium femoral implants and in rat calvarial defect models. However, the most appropriate method of BSP coating, the necessary level of BSP coating, and the effect of BSP coating on cell behavior remain largely unknown. In this study, BSP was covalently coupled to titanium surfaces via an aminosilane linker (APTES), and its properties were compared to BSP applied to titanium via physisorption and untreated titanium. Cell functions were examined using primary human osteoblasts (hOBs) and L929 mouse fibroblasts. Gene expression of specific bone turnover markers at the RNA level was detected at different intervals. Cell adhesion to titanium surfaces treated with BSP via physisorption was not significantly different from that of untreated titanium at any time point, whereas BSP application via covalent coupling caused reduced cell adhesion during the first few hours in culture. Cell migration was increased on titanium disks that were treated with higher concentrations of BSP solution, independent of the coating method. During the early phases of hOB proliferation, a suppressive effect of BSP was observed independent of its concentration, particularly when BSP was applied to the titanium surface via physisorption. Although alkaline phosphatase activity was reduced in the BSP-coated titanium groups after 4 days in culture, increased calcium deposition was observed after 21 days. In particular, the gene expression level of RUNX2 was upregulated by BSP. The increase in calcium deposition and the stimulation of cell differentiation induced by BSP highlight its potential as a surface modifier that could enhance the osseointegration of orthopedic implants. Both physisorption and covalent coupling of BSP are similarly effective, feasible methods, although a higher BSP concentration is recommended.

Immunomodulation by interleukin-10 therapy decreases the incidence of relapse and prolongs the relapse-free interval in Psoriasis.

The ability of interleukin-10 therapy to reduce the severity of exacerbated psoriasis has been demonstrated recently. Considering the immunobiologic properties of this cytokine we investigated the effects of long-term interleukin-10 application on the immune system and duration of psoriasis remission. We performed a placebo-controlled, double-blind, phase II trial using interleukin-10 in patients with chronic plaque psoriasis in remission. Patients received subcutaneous injections with either interleukin-10 (10 microg per kg body weight; n = 7) or placebo (n = 10) three times per week until relapse or study termination after 4 months. The treatment was well tolerated. In the placebo group almost all patients (90%) showed a relapse during the observation period. In contrast to this, only two of seven patients (28.6%) relapsed in the interleukin-10-treated group. Kaplan-Meier analysis revealed a significantly lower relapse incidence in the interleukin-10 than in the placebo group (p = 0.02). The mean relapse-free interval time was 101.6 +/- 12.6 d in the interleukin-10 group in comparison with 66.4 +/- 10.4 d in the placebo group. Immunologic activity of interleukin-10 application was indicated by an increase in soluble interleukin-2 receptor plasma levels and higher ex vivo interleukin-4 secretion capacities. Remarkably, a significant negative correlation was demonstrated between the interleukin-4 secretion capacity and Psoriasis Area and Severity Index score (r = -0.36, p < 0.01). Our data suggest that interleukin-10 therapy is immunologic effective, decreases the incidence of relapse and prolongs the disease-free interval in psoriasis. Its value should be further determined in larger trials and for the prevention of re-exacerbation of other inflammatory disorders with a similar immunologic profile.

Application of Parallel Factor Analysis (PARAFAC) to electrophysiological data.

The identification of important features in multi-electrode recordings requires the decomposition of data in order to disclose relevant features and to offer a clear graphical representation. This can be a demanding task. Parallel Factor Analysis (PARAFAC; Hitchcock, 1927; Carrol and Chang, 1970; Harshman, 1970) is a method to decompose multi-dimensional arrays in order to focus on the features of interest, and provides a distinct illustration of the results. We applied PARAFAC to analyse spatio-temporal patterns in the functional connectivity between neurons, as revealed in their spike trains recorded in cat primary visual cortex (area 18). During these recordings we reversibly deactivated feedback connections from higher visual areas in the pMS (posterior middle suprasylvian) cortex in order to study the impact of these top-down signals. Cross correlation was computed for every possible pair of the 16 electrodes in the electrode array. PARAFAC was then used to reveal the effects of time, stimulus, and deactivation condition on the correlation patterns. Our results show that PARAFAC is able to reliably extract changes in correlation strength for different experimental conditions and display the relevant features. Thus, PARAFAC proves to be well-suited for the use in the context of electrophysiological (action potential) recordings.

Impact of a pharmaceutical care program on liver transplant patients' compliance with immunosuppressive medication: a prospective, randomized, controlled trial using electronic monitoring.

INTRODUCTION: Compliance with immunosuppressive therapy plays a major role in the long-term success of organ transplantation. Thus, strategies to promote compliance in posttransplant care are of particular interest. At the pharmacy department of the University Hospital Mainz, a program for pharmaceutical care of organ transplant patients has been developed for the first time ever. The main objective of the presented study was to examine the influence of this program on liver transplant patients' compliance with immunosuppressive therapy. METHODS: To measure compliance, medication event monitoring systems were used. Dosing compliance (DC) was calculated for each patient and the mean DC was compared between the two groups. Further direct and indirect methods of measuring compliance served to confirm the electronic compliance data. RESULTS: Pharmaceutical care of liver transplant patients led to a significant increase in compliance with the immunosuppressive therapy. The mean DC of the intervention group was 90%+/-6% compared with 81%+/-12% in the control group (P=0.015). Only two patients (10%) in the intervention group and nine patients (43%) in the control group showed a DC less than 80% (P=0.032). Furthermore, patients in the intervention group were more likely to achieve target blood levels. CONCLUSION: Patients who received pharmaceutical care with traditional patient care showed significantly better compliance with their immunosuppressive medication than patients who received only traditional patient care. Pharmaceutical care proved to be an effective intervention that should be implemented in posttransplant care.

Potential pitfalls of comparative measurements of reticulocytes with flow cytometry and microscopy in prematures and infants.

Reticulocyte counting by flow cytometry (Bayer H*3, ADVIA 120) in blood of prematures, infants and children > 1 year of age was compared with microscopic counting under research conditions (9000 counted red blood cells per slide). While in children >1 year a good concordance of both methods was observed, 2.3-2.4-fold higher values were obtained in neonates by microscopy (Brilliant Cresyl Blue stain, 0.5%). However, another laboratory found good agreement between H*3-counting and microscopy in samples also obtained from neonates using the same methods. Despite very similar results for all age groups in comparative flow cytometry measurements in both laboratories, counting of smears from neonates differed, showing an approximately 2.3-fold larger amount of reticulocytes in our laboratory. The reason for these observations was a greater enlargement (1250-fold) used routinely in our laboratory compared with 800-fold in the other one. Thus very mature reticulocytes frequently found in neonates could only be detected using a 1250-fold enlargement. Similarly, the low concentration of the colouring matter used in the H*3 (0.0005% oxazin or 0.001% ADVIA 120) is obviously not sufficient for detection of mature reticulocytes. Therefore, it is important to consider this phenomenon and to standardise microscopic enlargement, especially for comparisons in multicentre studies.