Ursula Felderhoff-Müser

Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury

OBJECTIVE Preterm brain injury is a major cause of disability in later life, and may result in motor, cognitive and behavioural impairment for which no treatment is currently available. The aetiology is considered as multifactorial, and one underlying key player is inflammation leading to white and grey matter injury. Extracellular vesicles secreted by mesenchymal stem/stromal cells (MSC-EVs) have shown therapeutic potential in regenerative medicine. Here, we investigated the effects of MSC-EV treatment on brain microstructure and maturation, inflammatory processes and long-time outcome in a rodent model of inflammation-induced brain injury. METHODS 3-Day-old Wistar rats (P3) were intraperitoneally injected with 0.25mg/kg lipopolysaccharide or saline and treated with two repetitive doses of 1×108 cell equivalents of MSC-EVs per kg bodyweight. Cellular degeneration and reactive gliosis at P5 and myelination at P11 were evaluated by immunohistochemistry and western blot. Long-term cognitive and motor function was assessed by behavioural testing. Diffusion tensor imaging at P125 evaluated long-term microstructural white matter alterations. RESULTS MSC-EV treatment significantly ameliorated inflammation-induced neuronal cellular degeneration reduced microgliosis and prevented reactive astrogliosis. Short-term myelination deficits and long-term microstructural abnormalities of the white matter were restored by MSC-EV administration. Morphological effects of MSC-EV treatment resulted in improved long-lasting cognitive functions INTERPRETATION: MSC-EVs ameliorate inflammation-induced cellular damage in a rat model of preterm brain injury. MSC-EVs may serve as a novel therapeutic option by prevention of neuronal cell death, restoration of white matter microstructure, reduction of gliosis and long-term functional improvement.

Risk for late-onset blood-culture proven sepsis in very-low-birth weight infants born small for gestational age: a large multicenter study from the German Neonatal Network.

Background: It was the aim of this study to assess whether very-low-birth-weight (VLBW) infants born small for gestational age (SGA; birth weight less than 10th percentile) are at increased risk for late-onset sepsis. Methods: This was a prospective, multicenter study of the German Neonatal Network including VLBW infants from 23 to < 32 weeks post menstrual age born 2009–2011. Outcomes were compared between VLBW infants born SGA (birth weight less than tenth percentile according to gestational age and gender) and non-SGA infants. The main outcome measure was at least 1 episode of late-onset sepsis defined as blood-culture–confirmed clinical sepsis occurring at ≥72 hours of age. Results: 5886 VLBW infants were included. In SGA infants (n = 692), an increased incidence of late-onset sepsis was noted compared with non-SGA infants (20.1% vs. 14.3 %, P < 0.001). This difference was only observed among infants with a gestational age of 27 to < 32 weeks and attributed to sepsis episodes with coagulase-negative staphylococci (12.8% vs. 8.3%, P < 0.001). Different treatment modalities (eg more frequent use of central venous lines) and longer duration of invasive therapies (parenteral nutrition, mechanical ventilation, hospitalization) may account for the increased sepsis risk with coagulase-negative staphylococci in our SGA cohort. In a multivariate logistic regression analysis, higher gestational age [per week; odds ratio (OR): 0.75, 95% confidence interval (CI): 0.72–0.78, P< 0.0001], treatment with antenatal steroids (OR: 0.7, 95% CI: 0.53–0.92, P = 0.01), German descendance (OR: 0.76, 95% CI: 0.63–0.91, P = 0.003) and prophylaxis with glycopeptide antibiotics (OR: 0.64, 95% CI: 0.47–0.87, P = 0.005) were shown to be protective against late-onset sepsis. In contrast, longer duration of parenteral nutrition (per day; OR: 1.016, 95% CI: 1.011–1.021, P < 0.0001) and SGA were found to be risk factors (OR: 1.31, 95% CI: 1.02–1.68, P= 0.03). Conclusions: SGA contributes to the risk of late-onset sepsis in VLBW infants. Future studies are needed to investigate the underlying pathophysiology to guide individualized preventive measures in this vulnerable subgroup.

Thrombocytopenia in the First 24 Hours After Birth and Incidence of Patent Ductus Arteriosus

BACKGROUND: Experimental studies suggest that platelet-triggered ductal sealing is critically involved in definite ductus arteriosus closure. Whether thrombocytopenia contributes to persistently patent ductus arteriosus (PDA) in humans is controversial. This was a retrospective study of 1350 very low birth weight (VLBW; <1500 g) infants, including 592 extremely low birth weight (ELBW; <1000 g) infants. METHODS: All infants who had a platelet count in the first 24 hours after birth and an echocardiogram performed on day of life 4 to 5 were included. The incidence of thrombocytopenia was analyzed in infants with and without PDA, and in those who did or did not undergo PDA intervention. The impact of thrombocytopenia, gestational age, birth weight, gender, and sepsis on PDA was determined by receiver operating characteristic curve, odds ratio, and regression analyses. RESULTS: Platelet numbers within the first 24 hours after birth did not differ between VLBW/ELBW infants with and without spontaneous ductal closure. Platelet numbers were not associated with subsequent PDA treatment. Low platelet counts were not related to failure of pharma-cologic PDA treatment and the need for subsequent surgical ligation. Lower gestational age or birth weight, male gender, and sepsis were linked to the presence of PDA in VLBW infants on day of life 4 to 5. CONCLUSIONS: Thrombocytopenia in the first 24 hours after birth was not associated with PDA in this largest VLBW/ELBW infant cohort studied to date. Impaired platelet function, due to immaturity and critical illness, rather than platelet number, might play a role in ductus arteriosus patency.

Dose-dependent effects of erythropoietin in propofol anesthetized neonatal rats

Exposure to Gamma-aminobutyric-acid (GABA)(A)-receptor agonists and N-Methyl-D-Aspartate (NMDA)-antagonists has been demonstrated to induce neurodegeneration in newborn rats. Exogenous erythropoietin (EPO) protects against NMDA antagonist-mediated neuronal death. In this study we evaluated whether EPO is also effective in limiting neurodegeneration of the GABA(A)-mimetic agent propofol in newborn rats. 6 day old rats were randomized to one of four groups and treated with intraperitoneal applications of 3 x 30 mg/kg propofol at 0, 90 and 180 min, propofol in combination with 5000 IU/kg rEPO, propofol in combination with 20,000 IU/kg rEPO or sham injections of PAD II solution as controls. After 24h, brains of the animals were histopathologically examined and a summation score of degenerated cells was calculated for every brain. Propofol increased neuronal degeneration scores from 16,090+/-4336 to 28,860+/-6569 (p<0.01). This effect was completely abolished by low-dose rEPO (14,270+/-4542, p<0.001 versus propofol only; p>0.05 versus controls). In contrast, high-dose rEPO was not protective (23 930+/-8896, p>0.05 versus propofol only). Propofol may cause neuronal death in newborn rat brains, which is prevented by low-dose rEPO but not high-dose rEPO.

Endogenous hypothermic response to hypoxia reduces brain injury: Implications for modeling hypoxic-ischemic encephalopathy and therapeutic hypothermia in neonatal mice

Hypothermia treatment (HT) is the only formally endorsed treatment recommended for hypoxic-ischemic encephalopathy (HIE). However, its success in protecting against brain injury is limited with a number to treat of 7-8. The identification of the target mechanisms of HIE in combination with HT will help to explain ineffective therapy outcomes but also requires stable experimental models in order to establish further neuroprotective therapies. Despite clinical and experimental indications for an endogenous thermoregulatory response to HIE, the potential effects on HIE-induced brain injury have largely been neglected in pre-clinical studies. In the present study we analyzed gray and white matter injury and neurobehavioral outcome in neonatal mice considering the endogenous thermoregulatory response during HIE combined with HT. HIE was induced in postnatal day (PND) 9 C57BL/6 mice through occlusion of the right common carotid artery followed by one hour of hypoxia. Hypoxia was performed at 8% or 10% oxygen (O2) at two different temperatures based on the nesting body core temperature. Using the model which mimics the clinical situation most closely, i.e. through maintenance of the nesting temperature during hypoxia we compared two mild HT protocols (rectal temperature difference 3°C for 4h), initiated either immediately after HIE or with delay of 2h. Injury was determined by histology, immunohistochemistry and western blot analyses at PND 16 and PND 51. Functional outcome was evaluated by Rota Rod, Elevated Plus Maze, Open Field and Novel Object Recognition testing at PND 30-PND 36 and PND 44-PND 50. We show that HIE modeling in neonatal mice is associated with a significant endogenous drop in body core temperature by 2°C resulting in profound neuroprotection, expressed by reduced neuropathological injury scores, reduced loss of neurons, axonal structures, myelin and decreased astrogliosis. Immediately applied post-hypoxic HT revealed slight advantages over a delayed onset of therapy on short- and long-term histological outcome demonstrated by reduced neuropathological injury scores and preservation of hippocampal structures. However, depending on the brain region analyzed neuroprotective effects were similar or even reduced compared to protection by endogenous cooling during HIE modeling. Moreover, long-term neurobehavioral outcome was only partially improved for motoric function (i.e. Rota Rod performance and rearing activity) while cognitive deficits (i.e. novel object recognition) remained unchanged. These findings emphasize the need to maintain the nesting temperature during the initiation of the pathological insult and highlight the urgency to develop and assess new adjuvant therapies for HT in well-defined experimental models.

Regulatory T Cells Ameliorate Intrauterine Growth Retardation in a Transgenic Rat Model For Preeclampsia

Preeclampsia is a multisystemic syndrome during pregnancy that is often associated with intrauterine growth retardation. Immunologic dysregulation, involving T cells, is implicated in the pathogenesis. The aim of this study was to evaluate the effect of upregulating regulatory T cells in an established transgenic rat model for preeclampsia. Application of superagonistic monoclonal antibody for CD28 has been shown to effectively upregulate regulatory T cells. In the first protocol (treatment protocol), we applied 1 mg of CD28 superagonist or control antibody on days 11 and 15 of pregnancy. In the second protocol (prevention protocol), the superagonist or control antibody was applied on days 1, 5, and 9. Superagonist increased regulatory T cells in circulation and placenta from 8.49±2.09% of CD4-positive T cells to 23.50±3.05% and from 3.85±1.45% to 23.27±7.64%, respectively. Blood pressure and albuminuria (30.6±15.1 versus 14.6±5.5 mg/d) were similar in the superagonist or control antibody–treated preeclamptic group for both protocols. Rats treated with CD28 superagonist showed increased pup weights in the prevention protocol (2.66±0.03 versus 2.37±0.05 g) and in the treatment protocol (3.04±0.04 versus 2.54±0.1 g). Intrauterine growth retardation, calculated by brain:liver weight ratio, was also decreased by the superagonist in both protocols. Further analysis of brain development revealed a 20% increase in brain volume by the superagonist. Induction of regulatory T cells in the circulation and the uteroplacental unit in an established preeclamptic rat model had no influence on maternal hypertension and proteinuria. However, it substantially improved fetal outcome by ameliorating intrauterine growth retardation.

Severe H1N1 infection in a pediatric liver transplant recipient treated with intravenous zanamivir: efficiency and complications.

The 2009 emerged pandemic influenza H1N1 is a leading cause for respiratory illness in children. Herein, we report about the first pediatric patient who developed severe bilateral pneumonia with acute respiratory distress syndrome caused by novel influenza H1N1 after liver transplantation. The 2-year-old child received a deceased whole organ liver transplantation because of end-stage Caroli’s disease. Early postoperative course was complicated by portal vein thrombosis, which was successfully treated by thrombectomy 12 hr after transplantation. Immunosuppression was started with cyclosporine A (CSA; 50 mg/m intravenously [IV]; trough blood level 180–230 ng/mL), prednisone (15 mg/ m), and two doses of basiliximab (10 mg on days 0 and 4). Respiratory deterioration occurred 4 days after transplantation. The patient required invasive ventilation and developed adult respiratory distress syndrome (bilateral infiltrates on chest x-ray, PaO2/FiO2 150). Other symptoms were leukopenia and fever. Liver enzymes and function remained stable. Extended bacterial, viral, and fungal diagnostic workup from tracheal secretions revealed positive pandemic H1N1 RNA detected by polymerase chain reaction (PCR). Treatment with oseltamivir (2 30 mg/day) was started immediately after reception of positive H1N1 results at day 3 after the onset of first respiratory symptoms. Because the respiratory situation further deteriorated (PaO2/FiO2 100) and the patient developed gastroparesis, absorption of oseltamivir became questionable. IV zanamivir (2 260 mg/day) was started as “compassionate use” for 5 days from days 10 to 15 after transplantation. Parents gave informed consent, but institutional review board approval could not be achieved because treatment decision was made 2 days before Christmas. The treatment course neither led to negative H1N1 PCR nor to respiratory improvement. CSA was discontinued, and prednisone was reduced to 10 mg/m at day 11 after transplantation. The H1N1 virus tested was susceptible to oseltamivir and zanamivir. Mutations known to be associated with an enhanced pathogenicity were not identified by sequencing analysis. During the treatment with zanamivir, the patient’s liver function worsened as indicated by the increase of serum aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) up to 628 U/L and 193 U/L, respectively. Ultrasound revealed an unexplained retrograde flow of the portal vein but no evidence of portal vein thrombosis. Rejection was ruled out by liver biopsy. Pandemic H1N1 RNA was not detectable in the liver biopsy. To improve the venous outflow of the liver, positive end-expiratory pressure (maximum 16 cm H2O) was gradually reduced without any effect. Portal vein flow normalized 10 days after stopping zanamivir. During zanamivir treatment, serum creatinine level increased from 0.36 mg/dL to a maximum of 0.68 mg/ dL. Kidney function normalized with termination of zanamivir. Oseltamivir was continued for another 10 days. The patient improved slowly and was weaned from mechanical ventilation after 25 days. Pandemic H1N1 RNA in tracheal aspirate became negative 22 days after first detection. Although the majority of cases of pandemic H1N1 infections in children are mild, severe courses have been described, especially in children with comorbidities (1). Until August 2009, 36 deaths had been occurred in children younger than 18 years in the United States (2). Usually treatment of the influenza A virus with the oral preparation oseltamivir is effective to shorten the illness when initiated within 48 hr after first symptoms (3). The neuraminidase inhibitor, zanamivir, developed for IV use is not currently commercially available. In patients with severe illness and a known resistance of pandemic H1N1 to oseltamivir or in patients not tolerating enteral nutrition, this therapy might be an important alternative. Recently, a first case report with successful administration of zanamivir in a child with acute lymphoblastic leukemia has been published (4). In the presented case, zanamivir treatment was initiated as “compassionate use” because of progressive worsening of respiratory symptoms and severe gastroparesis, leading to questionable oseltamivir absorption. However, we were unable to demonstrate any positive effect as neither the symptoms improved nor the PCR became negative. A possible explanation might be the late start of the medication. Conversely, it cannot be excluded that the otherwise unexplained retrograde portal vein flow and kidney injury were caused by the medication. We believe that the coincidence in time of worsening liver function and zanamivir treatment makes a causal relationship at least possible. Duration of treatment with neuraminidase inhibitors was totally 20 days. A prolonged period of treatment is consistent with current published guidelines (5), which recommend treatment in transplant patients until H1N1 PCR becomes negative. Recommendations are based on the fact that viral shedding is prolonged in immunocompromised patients. Conversely, these potential benefits have to outweigh the risk of resistance emergence. Probably, the most important actions for improvement of clinical course in our patient were aggressive ventilatory support and the reduction of immunosuppression, especially the discontinuation of CSA. The presented case also underlines the fact that immunization against pandemic H1N1 should be recommended strongly to the patients on waiting list for organ transplantation (6). Furthermore, vaccination programs for healthcare professionals are emphasized strongly, because our patient was probably infected by a doctor, nurse, or physiotherapist. In conclusion, in other cases, zanamivir has been proven to be effective to shorten pandemic H1N1 influenza illness. If given late in the disease course ( 48 hr after onset of symptoms), caregivers have to balance the reduced efficiency against possible toxic side effects.

Clinical Relevance of Pathogens Detected by Multiplex PCR in Blood of Very-Low-Birth Weight Infants with Suspected Sepsis - Multicentre Study of the German Neonatal Network.

Introduction In the German Neonatal Network (GNN) 10% of very-low-birth weight infants (VLBWI) suffer from blood-culture confirmed sepsis, while 30% of VLBWI develop clinical sepsis. Diagnosis of sepsis is a difficult task leading to potential over-treatment with antibiotics. This study aims to investigate whether the results of blood multiplex-PCR (SeptiFast®) for common sepsis pathogens are relevant for clinical decision making when sepsis is suspected in VLBWI. Methods We performed a prospective, multi-centre study within the GNN including 133 VLBWI with 214 episodes of suspected late onset sepsis (LOS). In patients with suspected sepsis a multiplex-PCR (LightCycler SeptiFast MGRADE-test®) was performed from 100 μl EDTA blood in addition to center-specific laboratory biomarkers. The attending neonatologist documented whether the PCR-result, which was available after 24 to 48 hrs, had an impact on the choice of antibiotic drugs and duration of therapy. Results PCR was positive in 110/214 episodes (51%) and blood culture (BC) was positive in 55 episodes (26%). Both methods yielded predominantly coagulase-negative staphylococci (CoNS) followed by Escherichia coli and Staphylococcus aureus. In 214 BC—PCR paired samples concordant results were documented in 126 episodes (59%; n = 32 were concordant pathogen positive results, n = 94 were negative in both methods). In 65 episodes (30%) we found positive PCR results but negative BCs, with CoNS being identified in 43 (66%) of these samples. Multiplex-PCR results influenced clinical decision making in 30% of episodes, specifically in 18% for the choice of antimicrobial therapy and in 22% for the duration of antimicrobial therapy. Conclusions Multiplex-PCR results had a moderate impact on clinical management in about one third of LOS-episodes. The main advantage of multiplex-PCR was the rapid detection of pathogens from micro-volume blood samples. In VLBWI limitations include risk of contamination, lack of resistance testing and high costs. The high rate of positive PCR results in episodes of negative BC might lead to overtreatment of infants which is associated with risk of mortality, antibiotic resistance, fungal sepsis and NEC.

Sedation monitoring during open muscle biopsy in children by Comfort Score and Bispectral Index – a prospective analysis

Open muscle biopsies in children are generally performed under general anesthesia. Alternatively, deep sedation and analgesia may be required.

Lifesaving liver transplantation for multi-organ failure caused by Bacillus cereus food poisoning.

Bacillus cereus is a spore‐forming, gram‐positive bacterium that causes food poisoning presenting with either emesis or diarrhea. Diarrhea is caused by proteinaceous enterotoxin complexes, mainly hemolysin BL, non‐hemolytic enterotoxin (NHE), and cytotoxin K. In contrast, emesis is caused by the ingestion of the depsipeptide toxin cereulide, which is produced in B. cereus contaminated food, particularly in pasta or rice. In general, the illness is mild and self‐limiting. However, due to cereulide intoxication, nine severe cases with rhabdomyolysis and/or liver failure, five of them lethal, are reported in literature. Here we report the first case of life‐threatening liver failure and severe rhabdomyolysis in this context that could not be survived without emergency hepatectomy and consecutive liver transplantation.