Eight-plate epiphysiodesis: are we creating an intra-articular deformity?

Aims: Guided growth using eight-plates is commonly used for correction of angular limb deformities in growing children. The principle is of tethering at the physeal periphery while enabling growth in the rest of the physis. The method is also applied for epiphysiodesis to correct limb-length discrepancy (LLD). Concerns have been raised regarding the potential of this method to create an epiphyseal deformity. However, this has not been investigated. The purpose of this study was to detect and quantify the occurrence of deformities in the proximal tibial epiphysis following treatment with eight-plates. Patients and Methods: A retrospective study was performed including 42 children at a mean age of 10.8 years (3.7 to 15.7) undergoing eight-plate insertion in the proximal tibia for correction of coronal plane deformities or LLD between 2007 and 2015. A total of 64 plates were inserted; 48 plates (34 patients) were inserted to correct angular deformities and 16 plates (8 patients) for LLD. Medical records, Picture Archive and Communication System images, and conventional radiographs were reviewed. Measurements included interscrew angle, lateral and medial plateau slope angles measured between the plateau surface and the line between the ends of the physis, and tibial plateau roof angle defined as 180° minus the sum of both plateau angles. Measurements were compared between radiographs performed adjacent to surgery and those at latest follow-up, and between operated and non-operated plateaus. Statistical analysis was performed using BMDP Statistical Software. Results: Slope angle increased in 31 (49.2%) of operated epiphyses by a mean of 5° (1° to 23°) compared with 29 (31.9%) in non-operated epiphyses (p = 0.043). Roof angle decreased in 29 (46.0%) of operated tibias and in 25 (27.5%) of non-operated ones by a mean of 5° (1° to 18°) (p = 0.028). Slope angle change frequency was similar in patients with LLD, varus and valgus correction (p = 0.37) but roof angle changes were slightly more frequent in LLD (p = 0.059) and correlated with the change in inter screw angles (r = 0.74, p = 0.001). Conclusion: The use of eight-plates in the proximal tibia for deformity correction and limb-length equalization causes a change in the bony morphology of the tibial plateau in a significant number of patients and the effect is more pronounced in the correction of LLD. Cite this article: Bone Joint J 2018;100-B:1112-16.

Trends in the seasonal variation of paediatric fractures.

PURPOSE: The incidence of paediatric fractures is known to peak during the summer as a consequence of unsupervised physical activity. A more sedentary lifestyle is a potential cause for changes in paediatric seasonal fracture frequency and severity. The aim of this study was to evaluate the current seasonal variations of paediatric fractures in order to determine resource allocation in hospitals, community clinics and prevention programs. METHODS: A single institutional review of historical data of all patients aged 0 to 16 years that were diagnosed with fractures between April 2014 and July 2017 in the emergency department of a level 3 orthopaedic trauma centre was conducted. In all, 3484 fractures were reviewed, of which 2991 were included. We stratified fractures according to patients' variants and the hour, day and month with respect to holidays, weekends and weather. RESULTS: While the fracture rate on school days was 6.62 per day, the fracture rate during the summer vacation was 4.45 (p < 0.01). Hot weather was correlated with low fracture rates. The peak hours of admission were 12:00 to 13:00 and 18:00 to 22:00, with more moderate differences during non-school periods. CONCLUSION: The local seasonal variation of paediatric fractures has a bimodal distribution, with similar nadirs during both summer and winter. These rates might reflect a shift to a more sedentary lifestyle during the summer vacation. The presented data can assist in improving the value of injury prevention measures and medical resources allocation. LEVEL OF EVIDENCE: II.

Prenatal ultrasound diagnosis of club foot: outcome and recommendations for counselling and follow-up.

Club foot was diagnosed by ultrasonography in 91 feet (52 fetuses) at a mean gestational age of 22.1 weeks (14 to 35.6). Outcome was obtained by chart review in 26 women or telephone interview in 26. Feet were classified as normal, positional deformity, isolated club foot or complex club foot. At initial diagnosis, 69 feet (40 fetuses) were classified as isolated club foot and 22 feet (12 fetuses) as complex club foot. The diagnosis was changed after follow-up ultrasound scan in 13 fetuses (25%), and the final ultrasound diagnosis was normal in one fetus, isolated club foot in 31 fetuses, and complex club foot in 20 fetuses. At birth, club foot was found in 79 feet in 43 infants for a positive predictive value of 83%. Accuracy of the specific diagnosis of isolated club foot or complex club foot was lower; 63% at the initial ultrasound scan and 73% at the final scan. The difference in diagnostic accuracy between isolated and complex club foot was not statistically significant. In no case was postnatal complex club foot undiagnosed on fetal ultrasound and all inaccuracies were overdiagnoses. Karyotyping was performed in 25 cases. Abnormalities were noted in three fetuses, all with complex club foot and with additional findings on ultrasound.

Immobilisation of forearm fractures in children: extended versus flexed elbow.

Redisplacement of unstable forearm fractures in plaster is common and may be the result of a number of factors. Little attention has been paid to the influence of immobilisation with the elbow extended versus flexed. We prospectively treated 111 consecutive children from two centres with closed forearm fractures by closed reduction and casting with the elbow either extended (60) in China or flexed (51) in Israel. We compared the outcome of the two groups. There was no statistically significant difference in the distribution of the age of the patients, the site of fracture or the amount of angulation and displacement between the groups. During the first two weeks after reduction, redisplacement occurred in no child immobilised with the elbow extended and nine of 51 children (17.6%) immobilised with the elbow flexed. Immobilisation of unstable forearm fractures with the elbow extended appears to be a safe and effective method of maintaining reduction.

Monitoring of the sciatic nerve during hamstring lengthening by evoked EMG.

Traction injury to the sciatic nerve can occur during hamstring lengthening. The aim of this study was to monitor the influence of hamstring lengthening on conduction in the sciatic nerve using evoked electromyography (EMG). Ten children with spastic cerebral palsy underwent bilateral distal hamstring lengthening. Before lengthening, the evoked potential was recorded with the patient prone. During lengthening, it was recorded with the knee flexed to 90 degrees, 60 degrees and 30 degrees, and at the end of lengthening with the hip and knee extended. In all patients, the amplitude of the evoked EMG gradually decreased with increasing lengthening. The mean decrease with the knee flexed to 60 degrees was 34% (10 to 77), and to 30 degrees, 86% (52 to 98) compared with the pre-lengthening amplitude. On hip extension at the end of the lengthening procedure, the EMG returned to the pre-lengthening level. Monitoring of the evoked EMG potential of the sciatic nerve during and after hamstring lengthening, may be helpful in preventing traction injury.

Congenital insensitivity to pain. Orthopaedic manifestations.

We reviewed 13 patients with congenital insensitivity to pain. A quantitative sweat test was carried out in five and an intradermal histamine test in ten. DNA examination showed specific mutations in four patients. There were three clinical presentations: type A, in which multiple infections occurred (five patients); type B, with fractures, growth disturbances and avascular necrosis (three patients); and type C, with Charcot arthropathies and joint dislocations, as well as fractures and infections (five patients, four with mental retardation). Patient education, shoeware and periods of non-weight-bearing are important in the prevention and early treatment of decubitus ulcers. The differentiation between fractures and infections should be based on aspiration and cultures to prevent unnecessary surgery. Established infections should be treated by wide surgical debridement. Deformities can be managed by corrective osteotomies, and shortening by shoe raises or epiphysiodesis. Joint dislocations are best treated conservatively.

Sonographic healing stages of Achilles tendon after tenomuscular lengthening in children with cerebral palsy.

Long-term follow-up of a tenotomized Achilles tendon reveals healing with formation of a tendonlike structure between the cut tendon ends. A prospective study to show sonographic healing stages was designed. Twelve patients (21 Achilles tendons) with the diagnosis of cerebral palsy, spastic diplegia, and shortening of their Achilles tendons underwent a tenomuscular release. The healing process was documented by monthly ultrasound studies. Overall, 102 examinations were done, with a mean follow-up of 5.3 months. The authors found six distinct healing stages, with no significant change occurring in the length of the primary gap at the tenotomy site throughout healing. Based on these results, the authors conclude that sonographic healing stages of the cut Achilles tendon can be defined and staged. They also suggest that transverse tenotomy at the tenomuscular junction is a safe method, with no risk of overlengthening.

Folate copolymer-mediated transfection of cultured cells.

Poly(ethylene glycol) of various sizes was used as a molecular spacer to separate the cell-targeting ligand, folate, from the surface of poly-L-lysine. The resulting ternary macromolecule (pLys-PEG-folate) was investigated in various formulations for its ability to transfect reporter plasmids into receptor-bearing HeLa and IGROV cell lines. Formulations were optimized with respect to DNA content, +/- charge ratio, and the size and amount of PEG substitution off the pLys backbone. Transfection activity was highest 48 h after sample introduction, and PEG 3400 was determined to be the most favorable spacer size tested. pLys-PEG-folate:DNA transfection was also found to be both concentration dependent and saturable; plus, it was blocked by the addition of excess-free folate, indicative of a specific mechanism of uptake. Transfection activity was virtually identical for complexes formed in 10% serum-supplemented media, deionized water, or Hepes buffer. And, cell viability remained greater than 85% at the highest concentrations of pLys-PEG-folate:DNA complexes tested (4.8 microg/mL pLys 331 000; 12 microg/mL DNA). Taken together, these observations provide evidence that pLys-PEG-folate:DNA complexes are taken up specifically by the folate endocytosis pathway, and that the intramolecular spatial distance of the ligand from the pLys backbone dramatically influences transfection.

Immunogenicity of thrombopoietin mimetic peptide GW395058 in BALB/c mice and New Zealand white rabbits: evaluation of the potential for thrombopoietin neutralizing antibody production in man.

Administration of exogenous proteins and peptides as therapeutics carries with it the potential for immune system recognition and the development of neutralizing antibodies to endogenous regulatory proteins. PEGylation of proteins typically reduces their immunogenicity in vivo. GW395058 is a PEGylated peptide thrombopoietin receptor (TPOr) agonist being evaluated for the treatment of chemotherapy-induced thrombocytopenia. Although GW395058 shares no homology with TPO, it does compete with TPO for binding to a common receptor, and a similarity in local structure could result in shared epitopes. Thus GW395058 could elicit TPO-neutralizing antibodies. In this study, we evaluated the immunogenicity of GW395058 in mice, the potential of rabbit antibodies elicited by immunizations with the non-PEGylated parent peptide AF15705 to cross-react with recombinant human (rHu) TPO, and the potential of mouse anti-rHuTPO antibodies elicited by repeated dosing with rHuTPO to cross-react with AF15705. GW395058-dosed mice failed to produce antibodies to AF15705 or rHuTPO. Mouse anti-rHuTPO did not cross-react with AF15705 and rabbit anti-AF15705 antibodies failed to cross-react with rHuTPO. GW395058 caused no immune-mediated lesions in mice, but rHuTPO suppressed megakaryocytopoiesis and caused B-lymphocyte hyperplasia in lymphoid tissues consistent with antigenic stimulation. These data suggest that the potential for an immune response to GW395058 in man would be low.

Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process.

A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

Catalytic activity of the SH2 domain of human pp60c-src; evidence from NMR, mass spectrometry, site-directed mutagenesis and kinetic studies for an inherent phosphatase activity.

During solution structural studies it was apparent that the human recombinant pp60c-src SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[13C,15N]srcSH2 with unlabeled Ac-pYEEIE (I) were examined using 31P and 1H-detected isotope filtered NMR methods. The presence of a high-affinity complex in equimolar solutions of I and U[13C, 15N]-srcSH2 was demonstrated by chemical shift perturbations, line broadening, and the observation of intermolecular nuclear Overhauser effects from the pY and Ile side-chain protons of I to protons on amino acid residues present in the binding pocket of srcSH2. Solutions containing excess I relative to srcSH2 revealed a slow hydrolysis of I to produce Ac-YEEIE and inorganic phosphate. The hydrolysis rate determined from NMR and HPLC-electrospray ionization mass spectrometry data at 30 degrees C for solutions containing excess I was 0.002-0.003 h-1. srcSH2 also catalyzed the hydrolysis of p-nitrophenyl phosphate (pNPP). Isoelectric focusing gels of a number of mutant srcSH2s demonstrated that this activity comigrated with srcSH2. Km, kcat, and kcat/Km were 3.7 +/- 0.4 mM, 3.1 +/- 0.2 x 10(-2) min-1, and 8.4 +/- 0.4 M-1 min-1, respectively, toward pNPP. The C188A mutant of the srcSH2 domain displayed 15% of the activity displayed by wild-type srcSH2, demonstrating that this residue is not absolutely required for activity. Two additional mutations in the known pY binding site, R178K and R158K, also resulted in decreased pNPPase activity, suggesting that the activity resides in or near this site. The inhibitor profile and pH dependence suggest that this is a novel protein Pase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structures of recombinant 19-kDa human fibroblast collagenase complexed to itself.

Collagenase is a member of the matrix metalloproteinase (MMP) family of enzymes. Aberrant regulation of this family has been implicated in pathologies such as arthritis and metastasis. Two crystal forms of the catalytic (19-kDa) domain of human fibroblast collagenase have been determined using collagenase complexed with a peptide-based inhibitor (CPLX) as a starting model [Lovejoy et al. (1994) Science 263, 375]. The first crystal form (CF1) contains one molecule in the asymmetric unit and has been determined at 1.9-A resolution with an R factor of 19.8%. The second crystal form (CF2) contains two molecules (A and B) in the asymmetric unit and has been determined at 2.1-A resolution with an R factor of 19.7%. The catalytic domain of collagenase is spherical with an active site cleft that contains a ligated catalytic zinc ion. Collagenase shares some structural homology with the bacterial zinc proteinase, thermolysin [Matthews et al. (1972) Nature, New Biol. 238, 37], and the crayfish digestive peptidase, astacin [Bode et al. (1992) Nature 358, 164]. The amino terminus (Leu 102 to Gly 105) of CF1 and CF2 molecules A and B differs from the conformation found in CPLX by bending away from the molecule and interacting with the active site cleft of symmetry-related molecules. In this alternative conformation, both the mainchain nitrogen and carbonyl oxygen of Leu 102 ligate the symmetry-related catalytic zinc. Although there are structural differences in the active site clefts of CF1, CF2, and CPLX, a number of complex-stabilizing interactions are conserved. The structure of collagenase will be useful for developing compounds that selectively inhibit individual members of the closely related matrix metalloproteinase family.

Preliminary X-ray diffraction studies of recombinant 19 kDa human fibroblast collagenase.

Crystals of the catalytic domain of human fibroblast collagenase have been grown in the presence and absence of an inhibitor. Crystals of the inhibitor complex grew from 0.2 M ammonium sulfate and 15 to 30% PEG 8000 at 22 degrees C as bipyramids in the space group P6(2) or P6(4). Crystals of the unligated enzyme grew as rods in the space group P4(1)2(1)2 or P4(3)2(1)2 from 1.0 to 2.0 M sodium formate at 4 degrees C. Both crystal forms grew quite slowly over a period of months, but ultimately yielded crystals that diffracted beyond 2.5 A. The collagenase samples used in these studies were heterogeneous at the amino terminus. Three major species (full length, N-1 and N-2) were identified by mass spectrometry and Edman sequencing. Analysis of dissolved crystals revealed the native crystal form selectively crystallized as the N-2 species; however, no selectivity of N-terminal forms was observed for crystals of the inhibitor complex.

Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor.

Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.

High-level expression and purification of mature HIV-1 protease in Escherichia coli under control of the araBAD promoter.

A 1.3-kb segment of Escherichia coli DNA containing the regulatory gene, araC, and the promoter of the araBAD operon was amplified by the polymerase chain reaction (PCR) and cloned into pUC18, resulting in plasmid pKB130 that produced the alpha fragment of beta-galactosidase upon addition of L-arabinose (L-ara). A synthetic gene for human immunodeficiency virus (HIV)-1 preprotease was placed downstream of the ara-BAD promoter in pKB130 to create a translational fusion inducible by addition of L-ara. The fusion protein correctly autoprocessed in vivo to yield a mature 99-amino-acid HIV-1 protease, which was found predominantly in inclusion bodies. This material could be refolded to an active form, which was purified to homogeneity. A small fraction of the protease was expressed in vivo as a soluble active form, which allowed the monitoring of expression during fermentation by a rapid and simple whole cell assay employing an HIV-1 protease-specific fluorogenic substrate.

The detection of anti-Ro/SS-A and anti-La/SS-B activity of human serum monoclonal immunoglobulins (monoclonal gammopathies).

The sera of 340 patients with monoclonal gammopathies were examined for the presence of anti-Ro/SS-A and anti-La/SS-B activity. An enzyme-linked immunosorbent assay (ELISA) technique was employed. Forty-six sera (13.5%) bound to Ro/SS-A, while 79 sera (23.2%) bound to La/SS-B. The anti-Ro/SS-A and anti-La/SS-B antibodies were found in sera of patients with IgG, IgM, and IgA gammopathies. Forty-two of the 46 sera (91.3%) with positive anti-Ro/SS-A activity were found to bind La/SS-B as well, while only 53.2% (42 out of 79) of the sera that had anti-La/SS-B activity also bound Ro/SS-A. The activity against Ro/SS-A and La/SS-B was further confirmed by immunoblotting with purified immunoglobulins. Antibodies to Ro/SS-A and La/SS-B are common in systemic lupus erythematosus, Sjögren's syndrome, and other rheumatic disorders. Yet none of the patients whose serum was found to contain high titres of anti-Ro/SS-A or anti-La/SS-B human monoclonal antibodies presented with symptoms related to such autoimmune diseases. Our results of a high incidence of anti-Ro/SS-A or anti-La/SS-B activity in the sera of patients with monoclonal gammopathies support previous reports of autoantibody properties characteristic of these immunoglobulins.

Induction of calf thymus topoisomerase II-mediated DNA breakage by the antibacterial isothiazoloquinolones A-65281 and A-65282.

A number of quinolones and related antibacterial compounds were screened for activity against calf thymus topoisomerase II by using the P4 unknotting and DNA breakage assays. Several compounds from different structural classes which inhibited DNA unknotting with 50% inhibitory concentrations ranging from 8 to 25 micrograms/ml were identified. Two experimental isothiazoloquinolones from this group, designated A-65281 and A-65282, were also found to induce considerable DNA breakage mediated by calf thymus topoisomerase II, with 32P-end-labeled pBR322 as the substrate. These compounds were nearly as potent as teniposide, with DNA breakage activity evident at concentrations as low as 4 micrograms/ml. However, some differences in DNA cleavage patterns from those with teniposide were evident. These studies have thus identified a new class of agents which have activity against both bacterial and eukaryotic type II topoisomerases. The implications of these data for the selectivity of topoisomerase-directed compounds and the potential toxicity of such compounds developed as antibacterial agents are discussed.

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation.

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.