Transphyseal reconstruction of the anterior cruciate ligament in prepubescent children.

We present the results of 17 children of Tanner stage 1 or 2 who underwent reconstruction of the anterior cruciate ligament between 1999 and 2006 using a transphyseal procedure, employing an ipsilateral four-strand hamstring graft. The mean age of the children was 12.1 years (9.5 to 14). The mean follow-up was 44 months (25 to 100). Survival of the graft, the functional outcome and complications were recorded. There was one re-rupture following another injury. Of the remaining patients, all had good or excellent results and a normal International Knee Documentation Committee score. The mean post-operative Lysholm score was 97.5 (SD 2.6) and the mean Tegner activity scale was 7.9 (SD 1.4). One patient had a mild valgus deformity which caused no functional disturbance. No other abnormality or discrepancy of leg length was seen. Measurements with a KT1000 arthrometer showed no significant difference between the normal and the operated legs. In this small series, transphyseal reconstruction of the anterior cruciate ligament appeared to be safe in these young children.

Panton-Valentine leukocidin-secreting Staphylococcus aureus causing severe musculoskeletal sepsis in children. A new threat.

Panton-Valentine leukocidin secreted by Staphylococcus aureus is known to cause severe skin, soft tissue and lung infections. However, until recently it has not been described as causing life-threatening musculoskeletal infection. We present four patients suffering from osteomyelitis, septic arthritis, widespread intravascular thrombosis and overwhelming sepsis from proven Panton-Valentine leukocidin-secreting Staphylococcus aureus. Aggressive, early and repeated surgical intervention is required in the treatment of these patients. The Panton-Valentine leukocidin toxin not only destroys host neutrophils, immunocompromising the patient, but also increases the risk of intravascular coagulopathy. This combination leads to widespread involvement of bone with glutinous pus which is difficult to drain, and makes the delivery of antibiotics and eradication of infection very difficult without surgical intervention.

Update on paediatric ACL injuries.

In the last two decades there has been an increase in the incidence of anterior cruciate ligament (ACL) injuries in children. This may be due to increased awareness, more participation in high demand contact and non-contact sports at an earlier age and better diagnosis as a result of better imaging. A review of the literature suggests that the long-term results of non-operative treatment are poor. While the short to medium-term results of ACL reconstruction in children are encouraging, the long-term results are unknown. In this review, the current trends in the management of paediatric ACL injuries are discussed with particular emphasis on the natural history, surgical techniques, the effect of surgery on the growth plate and complications.

A novel GJA8 mutation is associated with autosomal dominant lamellar pulverulent cataract: further evidence for gap junction dysfunction in human cataract.

PURPOSE: To identify the gene responsible for autosomal dominant lamellar pulverulent cataract in a four-generation British family and characterise the functional and cellular consequences of the mutation. METHODS: Linkage analysis was used to identify the disease locus. The GJA8 gene was sequenced directly. Functional behaviour and cellular trafficking of connexins were examined by expression in Xenopus oocytes and HeLa cells. RESULTS: A 262C>A transition that resulted in the replacement of proline by glutamine (P88Q) in the coding region of connexin50 (Cx50) was identified. hCx50P88Q did not induce intercellular conductance and significantly inhibited gap junctional activity of co-expressed wild type hCx50 RNA in paired Xenopus oocytes. In transfected cells, immunoreactive hCx50P88Q was confined to the cytoplasm but showed a temperature sensitive localisation at gap junctional plaques. CONCLUSIONS: The pulverulent cataract described in this family is associated with a novel GJA8 mutation and has a different clinical phenotype from previously described GJA8 mutants. The cataract likely results from lack of gap junction function. The lack of function was associated with improper targeting to the plasma membrane, most probably due to protein misfolding.

Vitreous-induced modulation of integrins in retinal pigment epithelial cells: effects of fibroblast growth factor-2.

Growth in the presence of vitreous results in transformation of human RPE cells from an epithelioid to a fibroblast-like appearance and leads to an elevation of the expression of alpha(5) and alpha(2) integrins, while the level of alpha(3) integrin is reduced. These changes are inhibited by the presence of FGF-2. Vitreous treatment increases mobility, as does antibody neutralization of FGF-2 or antibody blockade of FGF receptors. The vitreous-induced rise in mobility depends on an increase in alpha(5) integrin expression since it is inhibited by anti-alpha(5) integrin antibodies. Expression of alpha(5) integrin as a result of infection of RPE cells with an alpha(5) integrin-encoding adenovirus induced morphological transformation and an increase in mobility similar to that seen with vitreous. It is concluded that a decrease in FGF-2 plays an important role in vitreous-induced alterations of RPE cell morphology, integrin expression and mobility. High FGF-2 levels prevent at least some of the increased mobility of RPE cells induced by vitreous. This is mediated via extracellular FGF-2 binding to FGF receptor(s) since antibodies to FGF-2 or to its receptor(s) mimic the effects of vitreous. Changes in mobility and morphology involve altered alpha(5) integrin expression since mobility is blocked by antibodies against these proteins while elevated alpha(5) integrin expression increases mobility and leads to morphological changes.

Patello-femoral tracking in the weight-bearing knee: a study of asymptomatic volunteers utilising dynamic magnetic resonance imaging: a preliminary report.

Normal patello-femoral tracking is not well defined, and conventional radiological techniques do not allow imaging in the physiological, weight-bearing stance. A vertical-access open configuration magnetic resonance scanner allows imaging of patello-femoral tracking during weight-bearing and through a wide range of knee flexion. We imaged 40 asymptomatic knees in this way, producing axial scans which were analysed qualitatively and quantitatively using sulcus angle, congruence angle, lateral patello-femoral angle and patellar centralisation, to assess patellar tilt and displacement. Mild lateral tilting in hyperextension with the quadriceps relaxed was seen, but quantitative assessment of this was impeded by internal rotation of the femur in extension. One-half of the knees were slightly laterally displaced in hyper-extension, becoming central during the first 30 degrees of knee flexion. During passive flexion of the knee in a seated position, fewer knees were laterally tilted or displaced, and no consistent change was seen during flexion. These results indicate that mild lateral tilting and displacement can be normal phenomena in the weight-bearing knee in early flexion and should not necessarily be taken as evidence of abnormal tracking in symptomatic patients. Lateral to medial movement of the patella occurs during normal knee flexion. In addition, imaging in the weight-bearing knee can provide valuable information not gained by imaging during passive knee flexion.

Spectrum of dominant mutations in the desmosomal cadherin desmoglein 1, causing the skin disease striate palmoplantar keratoderma.

The adhesive proteins of the desmosome type of cell junction consist of two types of cadherin found exclusively in that structure, the desmogleins and desmocollins, coded by two closely linked loci on human chromosome 18q12.1. Recently we have identified a mutation in the DSG1 gene coding for desmoglein 1 as the cause of the autosomal dominant skin disease striate palmoplantar keratoderma (SPPK) in which affected individuals have marked hyperkeratotic bands on the palms and soles. In the present study we present the complete exon-intron structure of the DSG1 gene, which occupies approximately 43 kb, and intron primers sufficient to amplify all the exons. Using these we have analysed the mutational changes in this gene in five further cases of SPPK. All were heterozygotic mutations in the extracellular domain leading to a truncated protein, due either to an addition or deletion of a single base, or a base change resulting in a stop codon. Three mutations were in exon 9 and one in exon 11, both of which code for part of the third and fourth extracellular domains, and one was in exon 2 coding for part of the prosequence of this processed protein. This latter mutation thus results in the mutant allele synthesising only 25 amino acid residues of the prosequence of the protein so that this is effectively a null mutation implying that dominance in the case of this mutation was caused by haploinsufficiency. The most severe consequences of SPPK mutations are in regions of the body where pressure and abrasion are greatest and where desmosome function is most necessary. SPPK therefore provides a very sensitive measure of desmosomal function.

Interactions within the coiled-coil domain of RetGC-1 guanylyl cyclase are optimized for regulation rather than for high affinity.

RetGC-1, a member of the membrane guanylyl cyclase family of proteins, is regulated in photoreceptor cells by a Ca(2+)-binding protein known as GCAP-1. Proper regulation of RetGC-1 is essential in photoreceptor cells for normal light adaptation and recovery to the dark state. In this study we show that cGMP synthesis by RetGC-1 requires dimerization, because critical functions in the catalytic site must be provided by each of the two polypeptide chains of the dimer. We also show that an intact alpha-helical coiled-coil structure is required to provide dimerization strength for the catalytic domain of RetGC-1. However, the dimerization strength of this domain must be precisely optimized for proper regulation by GCAP-1. We found that Arg(838) within the dimerization domain establishes the Ca(2+) sensitivity of RetGC-1 by determining the strength of the coiled-coil interaction. Arg(838) substitutions dominantly enhance cGMP synthesis even at the highest Ca(2+) concentrations that occur in normal dark-adapted photoreceptor cells. Molecular dynamics simulations suggest that Arg(838) substitutions disrupt a small network of salt bridges to allow an abnormal extension of coiled-coil structure. Substitutions at Arg(838) were first identified by linkage to the retinal degenerative disease, autosomal dominant cone rod dystrophy (adCORD). Consistent with the characteristics of this disease, the Arg(838)-substituted RetGC-1 mutants exhibit a dominant biochemical phenotype. We propose that accelerated cGMP synthesis in humans with adCORD is the primary cause of cone-rod degeneration.

Clinical outcome of congenital talipes equinovarus diagnosed antenatally by ultrasound.

Congenital talipes equinovarus is a common anomaly which can now be diagnosed prenatally on a routine ultrasound scan at 20 weeks of gestation. Prenatal counselling is increasingly offered to parents with affected fetuses, but it is difficult to counsel parents if there is a chance that the fetus may not have talipes. Our study correlates the prenatal ultrasound findings of 14 infants diagnosed as having unilateral or bilateral talipes during their routine 20-week ultrasound scan with their clinical findings at birth and the treatment received. No feet diagnosed as talipes on the ultrasound scan were completely normal at birth and therefore there were no true false-positive results. One foot graded as normal at 20 weeks was found to have a mild grade-1 talipes at birth, but did not require treatment other than simple stretches. A total of 32% of feet required no treatment and so could be considered functional false-positive results on the scan. Serial casting was required by 13% of feet and surgical treatment by 55%. The severity of the talipes is difficult to establish before birth. A number of patients are likely to need surgical treatment, but a proportion will have talipes so mildly that no treatment will be required. In counselling parents at 20 weeks, orthopaedic surgeons need to know whether or not there is a small chance that the ultrasound diagnosis could be wrong and also that the talipes may be so mild that the foot will not require treatment.

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy.

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

The orthopaedic management of peripheral ischaemia in meningococcal septicaemia in children.

Between March 1993 and February 1999, 14 children aged from eight months to 14.75 years were admitted to the paediatric intensive-care unit with meningococcal septicaemia in association with severe peripheral ischaemia. Of these, 13 were operated upon, eight of whom had early fasciotomies. Five children died. Of the nine survivors, one had no amputations while in the other eight 14 limb segments were amputated. We review the case histories and propose a protocol for the early management of these children.

Visual pigments and oil droplets in the retina of a passerine bird, the canary Serinus canaria: microspectrophotometry and opsin sequences.

The visual receptors of the passeriform bird Serinus canaria, the canary, have been examined microspectrophotometrically and the sequences of the opsins determined. Rods have a maximum absorbance (lambda max) at 506 nm. Four spectral classes of single cone are present: long-wave-sensitive (LWS) containing a photopigment with lambda max at 569 nm, middle-wave-sensitive (MWS) with lambda max at 505 nm, short-wave-sensitive (SWS) with lambda max at 442 nm, and ultraviolet-sensitive (UVS) with lambda max at about 366 nm. Double cones possess the 569-nm pigment in both members. Typical combinations of photopigment and oil droplet occur in most cone classes. An ambiguity exists in the oil droplet of the single LWS cones. In some birds, LWS cones are paired with an R-type droplet, whereas in the majority of canaries the LWS pigment is paired with a droplet similar to the P-type of double cones. Mechanisms of spectral tuning within each opsin class are discussed.

Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone-rod dystrophy.

Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone-rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic alpha-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation in vitro and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca(2+) sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca(2+) concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone-rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant in vitro and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone-rod degeneration.

N-terminal deletion in a desmosomal cadherin causes the autosomal dominant skin disease striate palmoplantar keratoderma.

The N-terminal extracellular domain of the cadherins, calcium-dependent cell adhesion molecules, has been shown by X-ray crystallography to be involved in two types of interaction: lateral strand dimers and adhesive dimers. Here we describe the first human mutation in a cadherin present in desmosome cell junctions that removes a portion of this highly conserved first extracellular domain. The mutation, in the DSG1 gene coding for a desmoglein (Dsg1), results in the deletion of the first and much of the second beta-strand of the first cadherin repeat and part of the first Ca2+-binding site, and would be expected to compromise strand dimer formation. It causes a dominantly inherited skin disease, striate palmoplantar keratoderma (SPPK), mapping to chromosome 18q12.1, in which affected individuals have marked hyperkeratotic bands on the palms and soles. In a three generation Dutch family with SPPK, we have found a G-->A transition in the 3" splice acceptor site of intron 2 of the DSG1 gene which segregated with the disease phenotype. This causes aberrant splicing of exon 2 to exon 4, which are in-frame, with the consequent removal of exon 3 encoding part of the prosequence, the mature protein cleavage site and part of the first extracellular domain. This mutation emphasizes the importance of this part of the molecule for cadherin function, and of the Dsg1 protein and hence desmosomes in epidermal function.

Meniscal movement. An in-vivo study using dynamic MRI.

We present the first study in vivo of meniscal movement in normal knees under load. Using an open MR scanner, allowing imaging in physiological positions in near to real-time, 16 young footballers were scanned moving from full extension to 90 degrees flexion in the sagittal and coronal planes. Excursion of the meniscal horns, radial displacement and meniscal height were measured. On weight-bearing, the anterior horn of the medial meniscus moves through a mean of 7.1 mm and the posterior horn through 3.9 mm, with 3.6 mm of mediolateral radial displacement. The height of the anterior horn increases by 2.6 mm and that of the posterior horn by 2.0 mm. The anterior horn of the lateral meniscus moves 9.5 mm and the posterior horn 5.6 mm, with 3.7 mm of radial displacement. The height of the anterior horn increases by 4.0 mm, and that of the posterior horn by 2.4 mm. In non-weight-bearing, the anterior horn of the medial meniscus moves 5.4 mm and the posterior horn 3.8 mm, with 3.3 mm of radial displacement. The anterior horn of the lateral meniscus moves 6.3 mm, and the posterior horn 4.0 mm, with 3.4 mm of radial displacement. The most significant differences between weight-bearing and non-weight-bearing were the movement and vertical height of the anterior horn of the lateral meniscus.

The use of dynamic interventional MRI in developmental dysplasia of the hip.

Conventional methods of imaging in the investigation of developmental dysplasia of the hip all have disadvantages, either in definition or in exposure to radiation. We describe a new open-configuration MR scanner which is unique in that it allows anaesthesia and access to the patient within the imaging volume for surgical procedures and application of casts. We performed 13 scans in eight anaesthetised infants. Dynamic imaging revealed two dislocated hips which were then visualised during reduction. Hip spicas were applied without removing the patient from the scanner. In one hip, an adductor tenotomy was carried out. In all patients, stressing the hips during dynamic imaging allowed an assessment of stability. This was particularly useful in two hips in which an analysis of stability in different positions facilitated the planning of femoral osteotomies. This method of imaging provides new and important information. It has great potential in the investigation of developmental dysplasia of the hip and, with ultrasound, may allow management without the need for radiography.

Clustered cadherin genes: a sequence-ready contig for the desmosomal cadherin locus on human chromosome 18.

We describe the assembly of a cosmid and PAC contig of approximately 700 kb on human chromosome 18q12 spanning the DSC and DSG genes coding for the desmocollins and desmogleins. These are members of the cadherin superfamily of calcium-dependent cell adhesion proteins present in the desmosome type of cell junction found especially in epithelial cells. They provide the strong cell-cell adhesion generated by this type of cell junction for which expression of both a desmocollin and a desmoglein is required. In the autoimmune skin diseases pemphigus foliaceous and pemphigus vulgaris (PV), where the autoantigens are, respectively, encoded by the DSG1 and DSG3 genes, severe areas of acantholysis (cell separation), potentially life-threatening in the case of PV, are evident. Dominant mutations in the DSG1 gene causing striate palmoplantar keratoderma result in hyperkeratosis of the skin on the parts of the body where pressure and abrasion are greatest, viz., on the palms and soles. These genes are also candidate tumor suppressor genes in squamous cell carcinomas and other epithelial cancers. We have screened two chromosome 18-specific cosmid libraries by hybridization with previously isolated YAC clones and DSC and DSG cDNAs, and a whole genome PAC library, both by hybridization with the YACs and by screening by PCR using cDNA sequences and YAC end sequence. The contigs were extended by further PCR screens using STSs generated by vectorette walking from the ends of the cosmids and PACs, together with sequence from PAC ends. Despite screening of two libraries, the cosmid contig still had four gaps. The PAC contig filled these gaps and in fact covered the whole locus. The positions of 45 STSs covering the whole of this region are presented. The desmocollin and desmoglein genes, which are about 30-35 kb in size, are quite well separated at approximately 20-30 kb apart and are arranged in two clusters, one DSC cluster and one DSG cluster, which are transcribed outward from the interlocus region. The order of the genes is correlated with the spatial order of gene expression in the developing mouse embryo, and this, and previous transgenic experiments, suggests that long-range genetic elements that coordinate expression of these genes may be present. The complete bacterial clone contig described in this paper is thus a resource not only for future sequencing but also for investigations into the control of expression of these clustered genes.

Absence of p53 delays apoptotic photoreceptor cell death in the rds mouse.

PURPOSE: This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse model of inherited retinal degeneration is p53 dependent. METHODS: A colony of p53-deficient rds mice were obtained by crossing homozygous rds mice with animals homozygous for a targeted disruption of the p53 gene and genotyping the offspring of the F1 cross. Both parental strains were on a BALB/c background. Age matched p53-deficient rds mice and controls (p53-deficient, rds and BALB/c mice), were sacrificed from day 1 to day 58 after birth. Eyes were paraffin-embedded and a modified terminal dUTP nick-end labeling (TUNEL) technique was used to detect the number of cells displaying DNA fragmentation within the sectioned retina. Eyes were also resin-embedded for semi-thin and ultra-thin sectioning. RESULTS: The peak in photoreceptor apoptosis, which occurs at 16 days in the rds mouse, was delayed by 3 days in p53-deficient rds mice. In addition, there was also a delay in the loss of photoreceptor cells between 16 and 26 days. However, absence of p53 did not prevent retinal degeneration in the rds mouse. The number of photoreceptor cells in p53-deficient rds mice at 35 days was very similar to that in the controls. CONCLUSIONS: We have demonstrated that absence of p53 delays but does not prevent photoreceptor cell loss in the rds mouse. Our results provide evidence for plasticity in the mechanism by which apoptosis proceeds in retinal degeneration.

Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator.

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been associated with a number of male reproductive problems, including testis abnormalities and a reduction in germ cell quality and number. To establish at least one site of functional CFTR expression in the testis, we subjected cultured Sertoli cells to analysis of message, protein, and channel activity for CFTR. With reverse transcription-polymerase chain reaction, we obtained evidence for the presence of CFTR RNA when CFTR primers were used with RNA from cultured Sertoli cells. Western analysis performed with both anti-R and anti-C domain CFTR antibodies revealed immunoreactive material in extracts from primary Sertoli cell cultures that seemed consistent with CFTR previously identified in other cells and tissues. This led us to perform more detailed studies using the whole cell arrangement of the patch-clamp technique. Application of the membrane-soluble cAMP analog, 8-chlorophenyl-thio-cAMP, resulted in the activation of a Cl- current that displayed a permeability sequence of Br- > I- > or = Cl- and was blocked by diphenylamine-2-carboxylate and glibenclamide. In addition, a 13-pS conductance Cl- channel was measured in excised membrane patches exposed to the catalytic subunit of protein kinase A. When taken together, our findings of evidence of CFTR message, immunoreactive material that appeared consistent with CFTR, and Cl- channels with properties similar to those reported for CFTR provide strong evidence that Sertoli cells express a functional CFTR-like protein. The presence of CFTR in these cells may be needed to maintain the specific nutritional and fluid balance in the seminiferous tubule that is vital for normal spermatogenesis.