A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia.

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

[The course and development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannym mutatsiei.

AIM: To study the anamnesis, clinical state, electro-encephalographic and brain MRI characteristics in patients with Rett syndrome (МЕСР2) and epilepsy. MATERIAL AND METHODS: Eleven female patients, aged from 3 to 23 years, with Rett syndrome and MeCP2 mutations were studied. The study continued for 10 years (2006-2015). Assessment of neurological and mental status, night sleep video-EEG monitoring, MRI were performed. RESULTS AND CONCLUSION: Epilepsy was diagnosed in six cases (54.5%). Mean age at onset of epileptic seizures was 3 years 9 month. The following types of seizures were described: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus developed in one patient. Generalized seizures were identified in 56.25%, focal seizures in 43.75%. EEG changes were found in 9 patients (81.8%): slowing of the activity, episodes of periodic regional slowing, regional epileptiform activity and diffuse epileptiform activity, benign focal epileptiform discharges (BFED) of childhood, multiregional epileptiform activity. Five patients were treated with antiepileptic drugs. All of them had improved during treatment: a reduction of frequency of seizures was up to 50% in 4 cases (80%). One patient with resistant epilepsy was treated with the combination of drugs (levetiracetam, topiramate, zonisamide, benzodiazepine) that led to stopping of seizures during night sleep and decrease in the frequency of daytime seizures by 50%. Further research of epilepsy and efficacy of antiepileptic drugs in Rett syndrome is required.

[The course and the development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti techeniia i razvitiia épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannogo mutatsiei.

AIM: Studying data of anamnesis, clinical state, electro-encephalographic, brain MRI in patients with Rett syndrome (МЕСР2). MATERIAL AND METHODS: We studied 11 patients (female) from three to 23 years old with Rett syndrome and MeCP2 mutations. Observation continued 10 years (2006-2015). We analyzed the results of the neurological status, night sleep video-EEG monitoring, MRI. RESULTS AND CONCLUSION: Epilepsy diagnosed in six cases (54, 5%). The overage age of debut of epileptic seizures was 3 years 9 months. There are some types of seizures: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus evolved in one patient. Generalized seizures were 56, 25%, focal seizures - 43, 75%. EEG changing marked in nine patients (81, 8%): slowdown back activity, episodes of periodic regional slowdown, regional epileptiform activity, and diffuse epileptiform activity like benign focal epileptiform discharges (BFED). five patients took antiepileptic drugs. All of them had improved during treatment. There were reducing of frequency of the seizures up 50% - 4 cases (80%). one patients with resistant epilepsy was taken combination of drugs (levetirecetam, topiromat, zonisamide, benzodiazepine) with stopping of seizures in the night sleep and decreasing of frequency of daytime seizures to 50%. We believe there is very important of study epilepsy in patients with Rett syndrome and improvement of its treatment.

Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Simulator training in fetoscopic laser surgery for twin-twin transfusion syndrome: a pilot randomized controlled trial.

OBJECTIVE: To evaluate the effect of a newly developed training curriculum on the performance of fetoscopic laser surgery for twin-twin transfusion syndrome (TTTS) using an advanced high-fidelity simulator model. METHODS: Ten novices were randomized to receive verbal instructions and either skills training using the simulator (study group; n = 5) or no training (control group; n = 5). Both groups were evaluated with a pre-training and post-training test on the simulator. Performance was assessed by two independent observers and comprised a 52-item checklist for surgical performance (SP) score, measurement of procedure time and number of anastomoses missed. Eleven experts set the benchmark level of performance. Face validity and educational value of the simulator were assessed using a questionnaire. RESULTS: Both groups showed an improvement in SP score at the post-training test compared with the pre-training test. The simulator-trained group significantly outperformed the control group, with a median SP score of 28 (54%) in the pre-test and 46 (88%) in the post-test vs 25 (48%) and 36 (69%), respectively (P = 0.008). Procedure time decreased by 11 min (from 44 to 33 min) in the study group vs 1 min (from 39 to 38 min) in the control group (P = 0.69). There was no significant difference in the number of missed anastomoses at the post-training test between the two groups (1 vs 0). Subsequent feedback provided by the participants indicated that training on the simulator was perceived as a useful educational activity. CONCLUSIONS: Proficiency-based simulator training improves performance, indicated by SP score, for fetoscopic laser therapy. Despite the small sample size of this study, practice on a simulator is recommended before trainees carry out laser therapy for TTTS in pregnant women.

Random nanolasing in the Anderson localized regime.

The development of nanoscale optical devices for classical and quantum photonics is affected by unavoidable fabrication imperfections that often impose performance limitations. However, disorder may also enable new functionalities, for example in random lasers, where lasing relies on random multiple scattering. The applicability of random lasers has been limited due to multidirectional emission, lack of tunability, and strong mode competition with chaotic fluctuations due to a weak mode confinement. The regime of Anderson localization of light has been proposed for obtaining stable multimode random lasing, and initial work concerned macroscopic one-dimensional layered media. Here, we demonstrate on-chip random nanolasers where the cavity feedback is provided by the intrinsic disorder. The strong confinement achieved by Anderson localization reduces the spatial overlap between lasing modes, thus preventing mode competition and improving stability. This enables highly efficient, stable and broadband wavelength-controlled lasers with very small mode volumes. Furthermore, the complex interplay between gain, dispersion-controlled slow light, and disorder is demonstrated experimentally for a non-conservative random medium. The statistical analysis shows a way towards optimizing random-lasing performance by reducing the localization length, a universal parameter.

Phenotypic protein profiling of different B cell sub-populations using antibody CD-microarrays.

Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.

[Twin-twin transfusion syndrome--front line in fetal medicine]. / Tvilling-tvillingtransfusionssyndrom--frontlinje i fostermedicin.

Twin-twin transfusion develops in 10-20% of monochorionic twin pregnancies. As far as we know the underlying disturbance is an unbalanced anastomosis between the two fetuses on the placental surface. Without treatment mortality is as high as 80% if diagnosed before viability. One of this article's authors spent two months in Hamburg at the "Allgemeine Krankenhaus", Barmbeck in Germany and describes the technique used there to coagulate these anastomoses via fetoscope. The outcome seems very promising with overall survival of 80% using this method. In the article pathogenesis and alternative treatments are also discussed.

[Twin-twin transfusion syndrome--front line in fetal medicine]. / Tvilling--tvillingtransfusionssyndrom--frontlinje i fostermedicin.

Twin-twin transfusion develops in 10-20% of monochorionic twin pregnancies. As far as we know the underlying disturbance is an unbalanced anastomosis between the two fetuses on the placental surface. Without treatment mortality is as high as 80% if diagnosed before viability. One of this article's authors spent two months in Hamburg at the 'Allgemeine Krankenhaus', Barmbeck in Germany and describes the technique used there to coagulate these anastomoses via fetoscope. The outcome seems very promising with overall survival of 80% using this method. In the article pathogenesis and alternative treatments are also discussed.

New device for hand-assisted laparoscopic surgery.

Laparoscopic surgery has undergone a rapid evolution since the first laparoscopic cholecystectomy of Erich Mühe in 1985. Many surgeons felt that further technological success would be related not only to increasing experience and skill of surgeons, but also technological advances which would enable surgeons to perform increasingly more difficult and complex tasks. Progress has been rapid for some, but broad acceptance by surgeons has been slow.

Enlargement of cisterna magna as an indicator of chromosomal abnormalities in a low-risk Asian population.

AIMS: The aims of this study was to invent the prevalence of cisterna magna (CM) enlargement in a low-risk population and relate this to chromosomal abnormalities and initial delivery outcome. STUDY DESIGN: 11,145 patients having routine ultrasound scan at 21st week of pregnancy were screened for abnormalities. Cases with CM enlargement were traced and outcome retrieved either from case notes or from the patients. RESULTS: In our low-risk population six fetuses were found to have CM enlargement. Only one had a chromosomal abnormality, 47XY + 18, and this fetus also presented with other malformations. Four were healthy at birth and one, also with other malformations, is showing signs of mental retardation. All cases were male. CONCLUSION: Isolated CM enlargement does not seems to be an indicator for chromosomal abnormalities, especially if the fetus is a male. Still, it should alert the examiner of the possibility of other malformations, which may be of importance.

Screening for fetal growth restriction: a mathematical model of the effect of time interval and ultrasound error.

OBJECTIVE: We estimated the effect of ultrasound error and time interval between examinations on the false-positive rate for detecting fetal growth restriction (FGR). METHODS: Using published growth curves for the fetal abdominal circumference and a coefficient of variation for ultrasound error of 5%, computer simulation was used to estimate false-positive rates in relation to the time interval between ultrasound examinations. Growth restriction was diagnosed when there was no apparent growth in fetal abdominal circumference between two consecutive examinations. In separate studies, the false-positive rate was plotted against gestational age at the first ultrasound examination. RESULTS: There was a dramatic increase in false-positive rates as the time interval between examinations was reduced. When the initial scan was performed at 32 weeks, the false-positive rate increased from 3.2% for an interval of 4 weeks to 30.8% for an interval of 1 week. At a 2-week interval, the error was 16.9%. There was a significant increase in the false-positive rate as the gestational age at the initial ultrasound was increased. At 28 weeks, the false-positive rate with a 2-week interval was 11.8%, increasing to 24.1% at 38 weeks. By varying the coefficient of variation of the ultrasound error, the false-positive rate increased from 0.8% at an error of 2% to 31.9% at an error of 10%. CONCLUSION: Ultrasound scanning at 2-week intervals is associated with false-positive rates for growth restriction in excess of 10%, increasing to much higher rates late in the third trimester. Improved screening performance should be attainable by increasing the interval between scans and reducing measurement errors.

Lactate compared with pH analysis at fetal scalp blood sampling: a prospective randomised study.

OBJECTIVE: Fetal scalp blood sampling is a widely used method for assessing fetal condition in the event of ominous fetal heart rate patterns. The purpose of this randomised trial was to compare the value of fetal scalp blood lactate and pH management in cases of abnormal intrapartum fetal heart rate tracings. METHODS: Of 341 cases of ominous fetal heart rate patterns, 169 were randomly assigned to pH analysis, and 172 to lactate measurements. Lactate was measured using a lactate card requiring 5 microL of blood and yielding the result within 60 seconds. pH analysis was performed with an ABL 510 acid-base analyser requiring 35 microL of blood and yielding the results within 47 seconds. RESULTS: Unsuccessful fetal blood sampling procedures (no result or an unreliable result) occurred significantly more often in the pH subgroup than in the lactate subgroup (OR 16.1 with 95% CI 5.8-44.7). In the pH subgroup the failure rate was inversely related to the degree of cervical dilatation. Compared with the pH subgroup, the lactate subgroup was characterised by fewer fetal scalp incisions per blood sampling attempt (median 1.0 [interquartile range (IQR) 1-1] vs 2.0 [IQR 1-2]), and significantly less time required for the sampling procedure (median 120 s [IQR 90-147] vs 230 s [IQR 180-300]). The groups did not differ in mode of delivery, neonatal outcome and umbilical artery acid-base balance and lactate levels. CONCLUSION: This trial showed the levels of lactate and pH in fetal scalp blood to be comparable in predicting perinatal outcome, but the procedure to measuring lactate was more successful than that for pH. Owing to its simplicity of performance, lactate analysis is an attractive alternative for intrapartum fetal monitoring.

Tissue distribution of transplanted fetal liver cells in the human fetal recipient.

OBJECTIVE: Our purpose was to study the tissue distribution and concentrations of transplanted fetal liver cells in the human fetus. STUDY DESIGN: Radiolabeled indium 111 fetal liver cells were injected in vivo under ultrasonographic guidance into 10 normal fetuses (13 to 17 weeks of gestation) before a prostaglandin abortion. Six fetuses were injected intraperitoneally and four intracardially. Another two fetuses serving as controls were injected with indium-labeled maternal plasma. The fetuses were all alive, at least until 6 hours before expulsion. After expulsion the fetuses were dissected, and radioactivity was measured in various fetal tissues. Results for each tissue were expressed as percentages of the total injected dose. RESULTS: Significantly greater uptake of fetal liver cells in the liver, spleen, thymus, kidney, lung, and placenta was obtained with intracardiac than with intraperitoneal injection. Skeletal uptake did not differ in relation to mode of administration. With intracardiac injection uptake was greater in such parenchymal organs as the liver, spleen, and thymus (4.9%, 4.0%, and 3.9%, respectively). Uptake in the rib, clavicle, humerus, and sternum was 2.7%, 1.8%, 2.1%, and 1.1%, respectively. Placental uptake was 0.1%. The intracardiac route yielded a higher concentration of cells in different fetal organs than did injection of only radiolabeled maternal plasma, suggesting an active uptake of cells in different fetal hematopoietic organs. CONCLUSION: The mode of administration of fetal liver cells seems to be a major determinant of donor cell concentration in the transplanted human fetus and may be a significant determinant of the rate of successful engraftment.

Influence of ingesting a solution of branched-chain amino acids on perceived exertion during exercise.

On two occasions, seven male endurance-trained cyclists performed exhaustive exercise on a cycle ergometer in the morning after they had performed a bout of exercise the preceding evening in an attempt to lower the muscle glycogen stores. The subjects exercised at a work rate corresponding to approximately 70% of their maximal oxygen uptake for 60 min, followed by another 20 min of maximal exercise. During exercise the subjects were given either a solution of branched-chain amino acids (BCAAs) or flavoured water (placebo). Every 10 min during exercise the subjects rated their perceived exertion and mental fatigue on two different Borg scales. During the 60 min exercise at a given work rate the subjects ratings of perceived exertion when they were given BCAAs were 7% lower, and their ratings of mental fatigue were 15% lower than when they were given placebo. In addition, the performance in the colour task of Stroops Colour Word Test performed after exercise was improved when BCAAs had been ingested during exercise, compared with the results from the placebo trial. There was no difference in the physical performance between the two trials measured as the amount of work done during the last 20 min of exercise when the subjects performed at their maximum. The plasma concentration ratio of free tryptophan/BCAAs, which increased by 45% during exercise and by 150% 5 min after exercise in the placebo trial, remained unchanged or even decreased when BCAAs were ingested.

In utero haematopoietic stem cell transplantation: current perspectives and future potential.

In utero transplantation (IUT) of haematopoietic cells is a new therapeutic option for families with increased risk of having a child with an inherited disorder. Immunological naiveté and the rapidly expanding haematopoietic system in the first trimester human fetus, make therapeutic intervention by IUT a real possibility for those disorders which can be diagnosed early in gestation. Fewer cells are required than in postnatal BMT and therapy can be offered before the pathological sequelae of a disorder become manifested. However, only a few cases of IUT have been performed in humans and it is imperative that consensus is reached quickly on issues such as cell numbers/cell types so that the benefits of this approach to treatment can be realised. This review presents the current status of IUT, the cases thus far recorded and offers a prospective view of developments in this rapidly expanding area.

Cytokine stimulation of human fetal hematopoietic cells.

The effects of interleukins 3 and 6, stem cell factor, and granulocyte-macrophage colony-stimulating factor on human fetal hematopoietic, bone marrow, and cord blood cells were studied on the basis of the colony-forming capacity. Fetal hematopoietic cells from 28 elective abortions, three bone marrow samples, and three cord blond samples were incubated with cytokines and investigated for the presence of BFU-E (burst-forming units--erythroid), CFU-GM (colony-forming units--granulocytes, macrophages), and CFU-GEMM (colony-forming units--granulocytes, erythrocytes, macrophages, megakaryocytes). Single and combined cytokines and preincubation versus adding cytokines in culture were investigated. Interleukin-6 alone had the most pronounced effect on BFU-E formation. All four cytokines in combination yielded the highest scores for CFU-GM (p < 0.05) and CFU-GEMM (p < 0.05), whereas BFU-E was not enhanced. The mode of cytokine exposure was not a determinant of colony formation.