Paradoxical bleeding as a complication of the treatment of hemophilia with factor VIII and factor IX preparations]

Paradoxical bleedings are complications occurring under replacement therapy in haemophiliacs by disturbancies of the primary haemostasis. They have been observed during treatment with factor-VIII- and prothrombin-complex concentrates of long duration and in high dosage. Clinical complications, for example delayed wound healing as well as spontaneous bleedings into the skin and from the mucous membranes, have been observed in one quarter of haemophiliacs under substitution therapy. In one third of these patients pathological parameters of primary haemostasis (prolonged bleeding time, reduced retention, retraction, ADP- and collagen-induced aggregation and the platelet factor 3 release) were found out. The following mechanisms or substances may be the cause for these disturbancies: 1. fibrinogen and factor-VIII split products 2. high content of proteins predominantly fibrinogen and factor-VIII-related antigen 3. antigen-antibody reactions 4. development of inhibitors against the Willebrand factor. For treatment of the paradoxical bleedings freshly prepared cryoprecipitate, prednison and Etamsylatum have been used.     

A Simple Procedure for the Purification and Antiserum Production of Bean Yellow Mosaic Virus

A simple procedure was developed to purify bean yellow mosaic virus from infected faba bean. The procedure included clarification of tissue homogenate by 25% chloroform followed by low-speed centrifugation, virus concentration by polyethylene glycolprecipitation and further purification by agarose-acrylamide gel electrophoresis. The partially purified virus preparation was electrophoresed in 0.5% agarose-2% acrylamide gel for 4 h. Gel bands containing the virus were collected, homogenized, and mixed (1:1) with Freunds adjuvant. Four weekly intramuscular injections and a booster injection four weeks after the fourth injection were given to a rabbit. Antisera collected from the first five bleedings produced high A₄₀₅ readings in ELISA (0.47,5–0.790) with virus-infected faba bean leaves and low readings (0.030–0.065) with healthy tissue. Plates were coated with 5μg/ml of gammaglobulins (IgG) fractionated from the different bleedings of the antiserum prepared and a 1: 1000 dilution ofthe IgG from the third bleeding conjugated to alkaline phosphatase was used.     

The skeletal muscle phenotype of children with Neurofibromatosis Type 1 – A clinical perspective

Neurofibromatosis type 1 (NF1) can affect multiple systems in the body. An under recognised phenotype is one of muscle weakness. Clinical studies using dynamometry and jumping mechanography have demonstrated that children with NF1 are more likely to have reduced muscle force and power. Many children with NF1 are unable to undertake physical activities to the same level as their peers, and report leg pains on physical activity and aching hands on writing. Children and adolescents with NF1 reporting symptoms of muscle weakness should have a focused assessment to exclude alternative causes of muscle weakness. Assessments of muscle strength and fine motor skills by physiotherapists and occupational therapists can provide objective evidence of muscle function and deficits, allowing supporting systems in education and at home to be implemented. In the absence of an evidence base for management of NF1-related muscle weakness, we recommend muscle-strengthening exercises and generic strategies for pain and fatigue management. Currently, trials are underway involving whole-body vibration therapy and carnitine supplementation as potential future management options.     

A mass-length scaling law for modeling muscle strength in the lower limb

Musculoskeletal computer models are often used to study muscle function in children with and without impaired mobility. Calculations of muscle forces depend in part on the assumed strength of each muscle, represented by the peak isometric force parameter, which is usually based on measurements obtained from cadavers of adult donors. The aim of the present study was twofold: first, to develop a method for scaling lower-limb peak isometric muscle forces in typically-developing children; and second, to determine the effect of this scaling method on model calculations of muscle forces obtained for normal gait. Muscle volumes were determined from magnetic resonance (MR) images obtained from ten children aged from 7 to 13yr. A new mass-length scaling law was developed based on the assumption that muscle volume and body mass are linearly related, which was confirmed by the obtained volume and body mass data. Two musculoskeletal models were developed for each subject: one in which peak isometric muscle forces were estimated using the mass-length scaling law; and another in which these parameters were determined directly from the MR-derived muscle volumes. Musculoskeletal modeling and quantitative gait analysis were then used to calculate lower-limb muscle forces in normal walking. The patterns of muscle forces predicted by the model with scaled peak isometric force values were similar to those predicted by the MR-based model, implying that assessments of muscle function obtained from these two methods are practically equivalent. These results support the use of mass-length scaling in the development of subject-specific musculoskeletal models of children.     

Twelve serum protein profiles in children with acute nonbacterial gastroenterocolitis.

In thirty children hospitalized with acute benign, short-duration gastroenterocolitis, no obligate pathogens were isolated from stools. Five bleedings were established from each patient in order to obtain the protein profiles of albumin, orosomucoid, haptoglobin, alpha2-macroglobulin, ceruloplasmin, transferrin, C3-component, C-reactive protein, immunglobulins IgG, IgA, IgM and IgD. The proteins were quantitated by the single radial immunodiffusion method. The initial drop in some of the proteins followed may be related to general protein loss, negative nitrogen balance or hemodilution. The absence of a significant increase in all the investigated immunoglobulin classes contrasted with remarkable increase in haptoglobin and orosomucoid, both reaching normal levels in late convalescence. C-reactive protein could be demonstrated in half of the children showing early normalization with disappearance of clinical symptoms. In contrast to ceruloplasmin and C3- component, alpha2-macroglobulin was not involved in the acute phase protein reaction.     

Gene expression profiles and protein balance in skeletal muscle of burned children after beta-adrenergic blockade

Propranolol, a nonselective beta-blocker, has been shown effective in hypermetabolic burn patients by decreasing cardiac work, protein catabolism, and lipolysis. This study investigates the effect of propranolol on gene and protein expression changes in skeletal muscle of burned children by use of high-density oligonucleotide arrays to establish the genetic profiles and stable isotope technique to quantitate protein synthesis. Thirty-seven children (mean age 9.7 +/- 1.1 yr) were randomized into groups to receive placebo (n = 23) or propranolol (n = 14) titrated to reduce heart rate by 15%. Children had >40% total body surface area burns (mean 43 +/- 5.6%). Protein net balance was determined by stable-isotope infusion technique. Total RNA from muscle biopsies was isolated, labeled, and cRNA hybridized to the HG-U95Av2 Affymetrix array. Mean net balance of protein synthesis and breakdown was -14.3 +/- 12.9 nmol. min-1. 100 ml leg volume-1 for placebo and +69.3 +/- 34.9 nmol. min-1. 100 ml leg volume-1 in the propranolol-treated children (P = 0.012). Comparison of 12,000 genes in burned children receiving placebo showed increased expression of two genes with time, whereas children receiving propranolol showed increased expression of nine genes with a decrease in five genes. We conclude that burned children receiving propranolol showed a significant upregulation in genes involved in muscle metabolism and downregulation of an important enzyme involved in gluconeogenesis and insulin resistance compared with burned children receiving placebo. The upregulation of genes involved in muscle metabolism correlates well with the increase in net protein balance across the leg.     

The morphology of the medial gastrocnemius in typically developing children and children with spastic hemiplegic cerebral palsy

We collected 3D ultrasound images of the medial gastrocnemius muscle belly (MG) in 16 children with spastic hemiplegic cerebral palsy (SHCP) (mean age: 7.8 years; range: 4–12) and 15 typically-developing (TD) children (mean age: 9.5 years; range: 4–13). All children with SHCP had limited passive dorsiflexion range on the affected side with the knee extended (mean ± 1SD: −9.3° ± 11.8). Scans were taken of both legs with the ankle joint at its resting angle (RA) and at maximum passive dorsiflexion (MD), with the knee extended. RA and MD were more plantar flexed (p < 0.05) in children with SHCP than in TD children.

We measured the volumes and lengths of the MG bellies. We also measured the length of muscle fascicles in the mid-portion of the muscle belly and the angle that the fascicles made with the deep aponeurosis of the muscle. Volumes were normalised to the subject’s body mass; muscle lengths and fascicle lengths were normalised to the length of the fibula.

Normalised MG belly lengths in the paretic limb were shorter than the non-paretic side at MD (p = 0.0001) and RA (p = 0.0236). Normalised muscle lengths of the paretic limb were shorter than those in TD children at both angles (p = 0.0004; p = 0.0003). However, normalised fascicle lengths in the non-paretic and paretic limbs were similar to those measured in TD children (p > 0.05). When compared to the non-paretic limb, muscle volume was reduced in the paretic limb (p < 0.0001), by an average of 28%, and normalised muscle volume in the paretic limb was smaller than in the TD group (p < 0.0001).

The MG is short and small in the paretic limb of children with SHCP. The altered morphology is not due to a decrease in fascicle length. We suggest that MG deformity in SHCP is caused by lack of cross-sectional growth.     

Immunochemistry of serum albumin. VI. A dynamic approach to the immunochemical cross reactions of proteins using serum albumins from various species as models.

Antisera against bovine serum albumin were raised in two rabbits. Serial bleedings were obtained at different times after the first immunization, and antisera from these serial bleedings were not mixed but were kept and studied separately. The immunochemical cross-reactions of these antisera with serum albumins from bovine, goat, sheep, porcine, horse, human and chicken were determined by immunoadsorbent studies. These were done by titration so that the values of maximum (plateau) binding by each albumin of radioiodinated antibodies were determined. In each rabbit, the immunochemical cross-reactivity was not static but increased progressievly with time after the first immunization. In the interval 7 days to 398 days the increases in cross-reaction were extremely large. pH dissociation studies revealed that, together with the increase in cross-reactivity of a given albumin with time after immunization, there was a restriction in the antibody heterogeneity towards populations possessing higher affinity. These results provide a rational explanation for the different values of cross-reactivities for a given albumin from different laboratories. The findings are analyzed in relation to the antigenic structure of albumin and their significance in evolutionary studies discussed.     

Quantifying muscle activity in non-ambulatory children with spastic cerebral palsy before and after selective dorsal rhizotomy.

Cerebral palsy is a condition that results in varying degrees of functional deficits. The goal of this study was to develop an objective measure of muscle activity during a prescribed voluntary motor task in non-ambulatory children with spastic cerebral palsy. While performing a simultaneous hip/knee flexion task from the supine position, followed by return to the starting position, electromyographic and kinematic data were obtained from the right leg of eight children before and after selective dorsal rhizotomy and compared with eight age-matched controls. The electromyographic and kinematic data were combined to determine for each muscle of interest (tibialis anterior, soleus, vastus lateralis, biceps femoris) the percentage of the movement cycle for which the muscle was acting concentrically, eccentrically, isometrically or was considered inactive. Averaged over the four muscles, isometric activity decreased by 38% post-op and the time the muscles were inactive increased by 37% following surgery. The percentages of concentric and eccentric activity did not differ significantly between pre- and post-op conditions. Post-operatively, the percentage muscle activity patterns of the children with cerebral palsy more closely resembled that of the control children: averaged across all muscles and contraction types, the difference between the control children and the children with cerebral palsy was reduced by 50% following surgery. This measurement technique indicates promise as a method for quantifying muscle activity during voluntary motor tasks in non-ambulatory children with cerebral palsy.     

Internal, neurologic, psychiatric and psychological characteristics of 25 cases of haemophilia

The medical, neurologic and psychiatric complications in 25 hemophiles were analysed. The medical examinations did not show any changes connected with frequent blood transfusions, and bleedings to different tissues. The only changes found out were the result of local bleedings to joints. The psychological examination of the two persons suggested the brain injury, despite the lack of deviations from the norm at the neurologic examination including electroencephalography.     

Malondialdehyde formation by platelets of hemophiliacs]

The platelets of haemophilic patients produce after stimulation with thrombin (5 NIH-units) a significantly reduced amount of malondialdehyde in comparison to the platelets of healthy children of the same age. There is a positive correlation between the platelet count in citrated whole blood and malondialdehyde production in the group of healthy children, however the same correlation is negative in adults and strongly negative in haemophiliacs. Because of the 24 hour-intervals between the last substitution and investigation in the majority of haemophilic patients, the reduced MDA-production of their platelets seems to be the result of side effects of the administration of plasma fractions. On the other hand, the reduced capacity for the MDA-production of the platelets of haemophiliacs can be explained as the result of the release reaction of platelets after haemostasis activation following bleedings.     

Multi-level personalization of neuromusculoskeletal models to estimate physiologically plausible knee joint contact forces in children

Neuromusculoskeletal models are a powerful tool to investigate the internal biomechanics of an individual. However, commonly used neuromusculoskeletal models are generated via linear scaling of generic templates derived from elderly adult anatomies and poorly represent a child, let alone children with a neuromuscular disorder whose musculoskeletal structures and muscle activation patterns are profoundly altered. Model personalization can capture abnormalities and appropriately describe the underlying (altered) biomechanics of an individual. In this work, we explored the effect of six different levels of neuromusculoskeletal model personalization on estimates of muscle forces and knee joint contact forces to tease out the importance of model personalization for normal and abnormal musculoskeletal structures and muscle activation patterns. For six children, with and without cerebral palsy, generic scaled models were developed and progressively personalized by (1) tuning and calibrating musculotendon units’ parameters, (2) implementing an electromyogram-assisted approach to synthesize muscle activations, and (3) replacing generic anatomies with image-based bony geometries, and physiologically and physically plausible muscle kinematics. Biomechanical simulations of gait were performed in the OpenSim and CEINMS software on ten overground walking trials per participant. A mixed-ANOVA test, with Bonferroni corrections, was conducted to compare all models’ estimates. The model with the highest level of personalization produced the most physiologically plausible estimates. Model personalization is crucial to produce physiologically plausible estimates of internal biomechanical quantities. In particular, personalization of musculoskeletal anatomy and muscle activation patterns had the largest effect overall. Increased research efforts are needed to ease the creation of personalized neuromusculoskeletal models.

     

The effects of repeated bleedings on bone marrow and blood morphology in adult laboratory rats

There were slight and rapidly disappearing changes in the bone marrow and blood morphology of laboratory rats after repeated bleedings on consecutive days, except marked increases in the counts of circulating reticulocytes. However, the significantly elevated reticulocyte count was not accompanied by the similar increase in the count of marrow erythroid nucleated cells. Marked increments could be found in the counts of splenic erythroid nucleated cells and, therefore, it is possible for rat circulating reticulocytes to originate following repeated bleedings from the spleen. The validity of investigations of bone marrow and blood morphology for detecting haemorrhage is discussed with a view to preclinical safety drug evaluations performed on adult laboratory rats.     

Evaluation of musculotendinous stiffness in prepubertal children and adults, taking into account muscle activity.

Musculotendinous (MT) stiffness of the triceps surae (TS) muscle group was quantified in 28 prepubertal children (7-10 yr) by using quick-release movements at different levels of submaximal contractions. Surface electromyograms (EMG) of each part of the TS and of the tibialis anterior were also recorded. A stiffness index, defined as the slope of the angular stiffness-torque relationship (SIMT-Torque), was used to quantify changes in MT stiffness with age. Results showed a significant decrease in SIMT-Torque with age, ranging from 4.02 +/- 0.29 to 2.88 +/- 0.31 rad-1 for the youngest to the oldest children. Because an increase in stiffness with age was expected due to the maturation of elastic tissues, overactivation of the TS was suspected to contribute to the higher SIMT-Torque values found in the youngest children. TS EMG-torque analyses confirmed that neuromuscular efficiency was significantly lower for the 7- or 8-yr-old children compared with 10-yr-old children, notably due to a higher degree of tibialis anterior coactivation found in the youngest children. Thus the stiffness index originally defined as the slope of the angular stiffness-EMG relationship increased significantly with age toward adult values. The results underlined the necessity to take into account the capacities of muscle activation to quantify changes in elastic properties of muscles, when those capacities are suspected to be altered.     

Mitochondrial diseases in children including Leigh syndrome--biochemical and molecular background

Mitochondrial diseases in children are more frequently caused by mutations in nuclear DNA then in mtDNA. Special clinical phenotypes are associated with the mutations in SURF1 gene, in SCO2 gene and with mtDNA depletion syndromes. Leigh syndrome is the most common clinical presentation of various mitochondrial disorders during childhood. Elevation of lactate in blood, cerebrospinal fluid and urine is a simple biochemical marker of mitochondrial disorders but its specificity and sensitivity are low. Biochemical investigation of muscle biopsy and search for mitochondrial mutations remain a gold standard in the diagnosis. The standarized diagnostic criteria to establish level of diagnostic certainty (possible, probable, definite) are proposed to be used in practice; these include clinical features, neuroimaging and muscle biopsy investigations. Further research directions to improve our understanding of mitochondrial pathologies in children are suggested.     

EMG reaction in muscles about the knee to passive velocity,acceleration, and jerk manipulations

Twenty subjects, ten adults and ten children were tested in this study. Each test consisted of applying an ensemble of velocities to the lower limb using a torque motor in such a way that the entire range of motion of the knee was traversed. Eight velocities between 60°/s and 280°/s were reached at 2–3 different acceleration rates and 1–2 different rates of jerk. EMG from three muscles, vastus, rectus, and hamstring were recorded during each move. Regression and correlation coefficients between EMG and kinematic parameters indicated different reactions in both muscle groups and age groups to each of the three kinematic parameters. Adult muscle was dominated by a reaction to the velocity kinematic while children’s muscles were dominated by either acceleration or jerk. The extensor muscles of adults seem to be slightly more sensitive to acceleration and jerk than the flexors. In the muscle responses of children the exact opposite pattern is seen. The small sample size in this study does not allow for a meaningful statistical analysis.     

Evaluation of a method to scale muscle strength for gait simulations of children with cerebral palsy

Cerebral palsy (CP) is a neurological disorder that results in life-long mobility impairments. Musculoskeletal models used to investigate mobility deficits for children with CP often lack subject-specific characteristics such as altered muscle strength, despite a high prevalence of muscle weakness in this population. We hypothesized that incorporating subject-specific strength scaling within musculoskeletal models of children with CP would improve accuracy of muscle excitation predictions in walking simulations. Ten children (13.5 ± 3.3 years; GMFCS level II) with spastic CP participated in a gait analysis session where lower-limb kinematics, ground reaction forces, and bilateral electromyography (EMG) of five lower-limb muscles were collected. Isometric strength was measured for each child using handheld dynamometry. Three musculoskeletal models were generated for each child including a ‘Default’ model with the generic musculoskeletal model’s muscle strength, a ‘Uniform’ model with muscle strength scaled allometrically, and a ‘Custom’ model with muscle strength scaled based on handheld dynamometry strength measures. Muscle-driven gait simulations were generated using each model for each child. Simulation accuracy was evaluated by comparing predicted muscle excitations and measured EMG signals, both in the duration of muscle activity and the root-mean-square difference (RMSD) between signals. Improved agreement with EMG were found in both the ‘Custom’ and ‘Uniform’ models compared to the ‘Default’ model indicated by improvement in RMSD summed across all muscles, as well as RMSD and duration of activity for individual muscles. Incorporating strength scaling into musculoskeletal models can improve the accuracy of walking simulations for children with CP.     

Facial animation in children with Möbius syndrome after segmental gracilis muscle transplant

M?bius syndrome is a complex congenital anomaly involving multiple cranial nerves, including the abducens (VI) and facial (II) nerves, and often associated with limb anomalies. Muscle transplantation has been used to address the lack of facial animation, lack of lower lip support, and speech difficulties these patients experience. The purpose of this study was to investigate the results of bilateral, segmental gracilis muscle transplantation to the face using the facial vessels for revascularization and the motor nerve to the masseter for reinnervation. The outcome of the two-stage procedure was assessed in 10 consecutive children with M?bius syndrome by direct interview, speech assessment, and oral commissure movement. Preoperative data were collected from direct questioning, viewing of preoperative videotapes, notes from prior medical evaluations, and rehabilitation medicine and speech pathology assessments. All of the patients developed reinnervation and muscle movement. The children who described self-esteem to be an issue preoperatively reported a significant posttransplant improvement. The muscle transplants produced a smile with an average commissure excursion of 1.37 cm. The frequency and severity of drooling and drinking difficulties decreased postoperatively in the seven symptomatic children. Speech difficulties improved in all children. Specifically, of the six children with bilabial incompetence, three received complete correction and three had significant improvement. Despite the length and complexity of these procedures, complications were minimal. Muscle transplantation had positive effects in all problematic areas, with a high degree of patient satisfaction and improvement in drooling, drinking, speech, and facial animation. The surgical technique is described in detail and the advantages over regional muscle transfers are outlined. Segmental gracilis muscle transplantation innervated by the motor nerve to the masseter is an effective method of treating patients with M?bius syndrome.     

Effects of ketamine anaesthesia, stress and repeated bleeding on the haematology of vervet monkeys

Haematology values are presented for the vervet monkey (Cercopithecus aethiops), and the relative effects of high dose ketamine anaesthesia, stress of capture and repeated bleedings assessed. Anaesthesia resulted in decreased WBC and RBC values, attributed to depression of cardiovascular function. These effects were the reverse of those of alarm and strenuous exercise (leukocytosis and polycythaemia) during capture. Stress resulted in relatively high white and low red blood cell counts. Opposing effects of stress and anaesthesia led to comparable haematological values for trained, non-anaesthetized vervets and stressed, anaesthetized vervets. Effects of repeated bleedings were opposite in anaesthetized and non-anaesthetized animals. These effects, however, along with those of ketamine anaesthesia and stress, were relatively insignificant compared with the wide variation in haematological values found among individuals. The biological importance of these effects thus appeared to be slight. The concept of 'normal values' is discussed.     

Syngeneic anti-idiotypic antisera to murine monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens

BALB/c mice were immunized with syngeneic anti-HLA class I monoclonal antibodies. The latter included the anti-HLA-A2, A28 monoclonal antibody (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to HLA-B antigens and the MoAb CR10-215 and CR11-115 to the same (or spatially close) monomorphic determinant. Anti-idiotypic antibodies could be detected in bleedings obtained 3 days after the first booster, increased in titer in bleedings obtained after the second booster, and persisted at high levels in subsequent bleedings. The four anti-HLA class I MoAb did not differ in their ability to elicit syngeneic anti-idiotypic antibodies. Cross-blocking studies with a panel of anti-HLA class I, anti-HLA class II, and anti-human melanoma-associated antigen (MAA) MoAb showed that the anti-MoAb CR10-215 and anti-MoAb CR11-115 antisera contain only antibodies to private idiotopes, whereas the anti-HLA MoAb CR11-351 and anti-MoAb Q6/64 antisera also contain antibodies to public idiotopes. The latter are expressed by the anti-HLA class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, and by the anti-HLA class II MoAb Q5/6, Q5/13, 127, and 441. Public idiotopes were not detected on the nine anti-MAA MoAb tested. Public idiotopes do not interfere with the binding of anti-HLA MoAb with the corresponding antigenic determinants. On the other hand private idiotopes are located within the antigen-combining site, because anti-idiotypic antisera specifically inhibit the binding of the corresponding immunizing anti-HLA class I MoAb to cultured human lymphoid cells in a dose-dependent manner. Analysis by isoelectric focusing of the anti-HLA class I MoAb antisera showed that the spectrotype of the anti-MoAb CR11-351 antiserum comprises four components that focus in the pH 6.9 to 6.2 range, the spectrotype of anti-MoAb Q6/64 antiserum comprises three components that focus in the pH 6.5 to 6.1 range, the spectrotype of the anti-MoAb CR10-215 antiserum comprises three components that focus in the pH 6.4 to 6.1 range, and the spectrotype of the anti-MoAb CR11-115 antiserum comprises three components that focus in the pH 6.6 to 6.4 range.