Pitfalls in diagnosing septic arthritis in Hong Kong children: ten years' experience.

OBJECTIVES: To evaluate the initial presentation of septic arthritis in Hong Kong children with respect to clinical and laboratory findings that can aid making a prompt diagnosis. DESIGN: Retrospective review. SETTING: Five public hospitals in Hong Kong. PATIENTS: Data concerning paediatric patients with septic arthritis were collected from January 2001 to December 2010. Patients with postoperative infections and those without enough retrievable information were excluded. RESULTS: Of 31 patients analysed, on admission only 52% had had a fever of <38.5°C and 71% had raised white blood cell count of <12 x 10(9) /L. In 74% of these patients, Gram stains of blood culture samples yielded no positive findings. The leading causative organism was Staphylococcus aureus (42%), followed by group A Streptococcus (23%). When group A Streptococcus was responsible, five out of seven patients had a complicated clinical course (repeated surgeries, Streptococcus-related organ failure, and chronic joint stiffness). Moreover, in 19% of instances, the empirical antibiotic therapy prescribed on admission did not provide a broad enough spectrum of cover. CONCLUSION: Signs of sepsis such as high fever, raised white blood cell count, and positive Gram smear from blood cultures were only present in around half of these patients with septic arthritis. Furthermore, group A Streptococcus tended to produce many complications. Regrettably, about a quarter of the empirical antibiotic regimens started by frontline staff were deemed not have a broad enough spectrum of cover. Improvement in the initial detection and management of septic arthritis patients is warranted.

Triple coaxial catheter technique for transfacial superior ophthalmic vein approach for embolization of dural carotid-cavernous fistula.

We report a triple coaxial catheter technique to facilitate the venous access to the superior ophthalmic vein during transvenous embolization of dural carotid-cavernous fistula (DCCF) via the transfacial venous route. Two patients with transvenous embolization of DCCFs by coils were treated with transfacial superior ophthalmic vein (SOV) approach by the triple coaxial catheter technique. The triple coaxial catheter system consisted of a 6F guiding catheter as the outer catheter and a 4F guiding catheter as the middle catheter and a microcatheter as the inner catheter to help navigation and manipulation. The DCCFs were completely obliterated in both cases. There were no complications associated with the procedure. The ophthalmic symptoms of the patients had totally resolved at two-month follow-up. The triple coaxial catheter technique can be used with the transfacial SOV approach in embolization of DCCF. This technique has two advantages over the double coaxial catheter technique because it offers additional length and support for the distal navigation of microcatheter into the SOV.

Tensile strength of modified 4-strand cruciate technique for transversely or obliquely lacerated tendons.

PURPOSES: To assess the tensile strength of the modified 4-strand cruciate technique for obliquely lacerated tendons, and to compare the findings with the strength of transversely lacerated tendons repaired at various grasping depths. METHODS: 60 porcine front foot tendons were evenly divided into 4 groups. In groups 1 to 3, tendons were transversely lacerated and repaired with grasping points at both ends away from the laceration by 5 mm, 10 mm, and 15 mm respectively. In group 4, tendons were obliquely lacerated and repaired with a grasping point 5 mm away from the laceration on one end and 15 mm on the other. All tendons were repaired with a modified 4-strand core suture and continuous epitendinous suture, and then tested to failure in a tensile machine. RESULTS: The tensile strength in group 1 was significantly lower than that in the other 3 groups (p<0.005). The tensile strength in group 4 was not significantly different from groups 2 and 3. CONCLUSION: The tensile strength of modified 4-strand cruciate repair configuration is not weakened in obliquely lacerated tendons; the grasping point at one end of the tendon being 15 mm away from laceration provides sufficient strength to compensate for the relatively weak 5-mm end. So long as one grasping point is away from the laceration site by 10 mm, the ultimate tensile strength of the transversely lacerated tendons appears acceptable. The modified 4-strand cruciate repair is safe to use for repairing obliquely lacerated tendons.

A switch in the kinase domain of rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase plays an essential role in the regulation of glucose metabolism by both producing and degrading Fru-2,6-P(2) via its distinct catalytic activities. The 6-PF-2-K and Fru-2,6-P(2)ase active sites are located in separate domains of the enzyme. The kinase domain is structurally related to the superfamily of mononucleotide binding proteins that includes adenylate kinase and the G-proteins. We have determined three new structures of the enzymatic monomer, each with a different ligand in the ATP binding site of the 6-PF-2-K domain (AMP-PNP, PO(4), and water). A comparison of these three new structures with the ATPgammaS-bound 6-PF-2-K domain reveals a rearrangement of a helix that is dependent on the ligand bound in the ATP binding site of the enzyme. This helix motion dramatically alters the position of a catalytic residue (Lys172). This catalytic cation is analogous to the Arg residue donated by the rasGAP protein, and the Arg residue at the core of the GTP or GDP sensing switch motion seen in the heterotrimeric G-proteins. In addition, a succinate molecule is observed in the Fru-6-P binding site. Kinetic analysis of succinate inhibition of the 6-PF-2-K reaction is consistent with the structural findings, and suggests a mechanism for feedback inhibition of glycolysis by citric acid cycle intermediates. Alterations in the 6-PF-2-K kinetics of several proteins mutated near both the switch and the succinate binding site suggest a mode of communication between the ATP- and F6P binding sites. Together with these kinetic data, these new structures provide insights into the mechanism of the 6-PF-2-K activity of this important bifunctional enzyme.

Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities.

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2. 4 A by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.

MHC class II presentation of human acetylcholine receptor in Myasthenia gravis: binding of synthetic gamma subunit sequences to DR molecules.

Embryonic muscle acetylcholine receptor (AChR) is composed of four subunits-alpha, beta, gamma, and delta. Upon innervation, the gamma subunit is substituted by a homologous epsilon subunit. Embryonic muscle AChR might be the original autoantigen in Myasthenia gravis (MG) because it is expressed in the thymus, where the anti-AChR response might start, as well as in adult extrinsic ocular muscles, which are a preferential target in MG. MG patients have antibodies specific for embryonic AChR and CD4+ T cells, which recognize epitopes on the gamma subunit, in association with DR molecules. In the present study, we investigated the binding to several purified DR molecules (DRB1*0101, DRB1*0201, DRB1*0401 and DRB1*0701) of overlapping synthetic peptides, screening the human gamma subunit sequence. Binding of the peptides to the DR molecules were determined from their ability to compete with radiolabelled peptide probes for binding to purified DR molecules. All peptides which were recognized by CD4+ cells bound to the relevant DR molecule. On the other hand, some AChR peptides not recognized by CD4+ cells of MG patients bound well to one or more DR molecules. In terms of relative ability to bind to the DR molecules tested, the gamma subunit behaved like an exogenous antigen: some AChR peptide sequences uniquely bound one DR molecule, others bound several DR alleles, while others did not bind any of the DR molecules tested.

Immunoregulatory CD8+ cells recognize antigen-activated CD4+ cells in myasthenia gravis patients and in healthy controls.

CD8+ cells inhibiting the response of CD4+ cells exist in rodents, recognizing epitopes unique to a CD4+ clone (Ids) or expressed by all activated CD4+ cell (ergotypes). Stimulation of CD8+ cells recognizing ergotypes shared by all Ag-activated CD4+ cells would be useful for treatment of diseases involving undesirable CD4+ responses to ill defined Ags, such as many autoimmune diseases and allergies. As a first step toward demonstrating the existence of anti-ergotype CD8+ immunoregulatory cells in humans, we investigated here whether CD8+ cells recognizing Ag-activated CD4+ cells exist in autoimmune and healthy humans. CD4+ cells specific for human muscle acetylcholine receptor, tetanus, or diphtheria toxoids were propagated from patients with myasthenia gravis patients and healthy controls. Ag-activated CD4+ cells were irradiated and used as Ag to test the response of CD(4+)-depleted CD(8+)-enriched PBMC (CD8+ PBMC) from myasthenic patients and controls and to propagate short-term CD8+ cell lines from CD8+ PBMC. In both patients and controls CD8+ PBMC and CD8+ lines responded vigorously to autologous Ag-activated CD4+ cells. The CD8+ lines responded equally well to the Ag-activated CD4+ cells of different Ag specificity, suggesting that they recognized CD4+ ergotypes. They did not seem to respond to CD4+ cells activated by PHA. The CD8+ cells recognized class I-restricted epitopes, as their response to activated CD4+ cells was suppressed by anti-class I Ab. CD8+ cells recognizing Ag-activated CD4+ were present cells in the controls for 5 to 12 wk after immunization. In myasthenic patients, CD8+ cells recognizing activated anti-acetylcholine receptor CD4+ cells seemed to be always present.

Human acetylcholine receptor presentation in myasthenia gravis. DR restriction of autoimmune T epitopes and binding of synthetic receptor sequences to DR molecules.

Autoimmune Th cells in myasthenia gravis recognize several sequence regions of the human muscle acetylcholine receptor (AChR). Most AChR Th epitopes are presented by HLA class II DR molecules (DR). Four sequence regions of the AChR alpha-subunit form Th epitopes recognized by most myasthenic patients, irrespective of their DR haplotype. In this study we first identified in five myasthenic patients the DR molecule(s) likely to be involved in presentation of T immunodominant AChR sequences. We then investigated the binding to the affinity purified DR molecules thus identified (DR2/w51, DR4/w53, and DR7/w53) and to the DR1 molecule, of a panel of overlapping synthetic peptides screening the human alpha-subunit sequence, previously used to identify AChR Th epitopes in myasthenic patients. The AChR peptides that stimulated anti-AChR autoimmune Th cells all bound the relevant DR molecules. Some AChR peptides never recognized by Th cells of myasthenic patients also bound well to one or more DR molecules. The relative ability to bind to DR molecules of different sequence regions of the AChR, i.e., an autoantigen, agrees well with the results of previous studies on the DR binding of synthetic sequences of exogenous antigens. Some peptide sequences uniquely bound one DR molecule, others bound several DR molecules, and others did not bind any of the DR molecules tested.

T-helper epitopes on human nicotinic acetylcholine receptor in myasthenia gravis.

The synthesis of AChR antibodies requires intervention of AChR-specific Th cells. Because of the paucity of anti-AChR Th cells in the blood of myasthenia gravis (MG) patients, direct studies of these autoimmune cells in the blood are seldom possible. Propagation in vitro of anti-AChR T cells from MG patients by cycles of stimulation with AChR antigens selectively enriches and expands the autoimmune T-cell clones, allowing investigation of their function and epitope specificity. Torpedo electroplax AChR was initially used for propagation of anti-AChR T-cell lines. Those studies demonstrated the feasibility of in vitro propagation of AChR-specific T cells. These are bona fide CD4+ Th cells, which stimulate production in vitro of anti-AChR antibodies by B cells of myasthenic patients and recognize equally well denatured and native AChR, suggesting the usefulness of synthetic human AChR sequences as antigens for propagation of the autoimmune Th cells. We used pools of overlapping synthetic peptides, corresponding to the complete sequences of the human AChR alpha-, beta-, gamma-, and delta-subunits, to propagate AChR-specific Th cells from the blood of MG patients. The AChR sequence regions forming epitopes recognized by the autoimmune T cells were determined by challenging the lines with individual synthetic peptides, 20 residues long, screening the AChR subunit sequences. Although each line had an individual pattern of epitope recognition--as expected from their different HLA-DR haplotype--some peptides were recognized by most of all the CD4+ T-cell lines, irrespective of their DR haplotype. The existence of immunodominant regions of the AChR sequence was verified by investigating the response of unselected CD4+ cells from the blood of a relatively large number of MG patients to the individual peptides screening the human alpha-, gamma-, and delta-subunit sequences. Those studies confirmed that each patient has an individual pattern of peptide recognition. The studies also identified a large number of T epitopes of the human AChR and verified the existence of sequence regions immunodominant for T-helper sensitization, because a limited number of sequence regions, including all those immunodominant for the T-helper lines, were recognized by most patients. Anti-AChR CD4+ T lines could be propagated from some healthy controls only for a brief period of time. They recognized AChR sequences poorly, suggesting a low affinity of their T-cell receptors for the corresponding AChR epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

An assay for simultaneous multiple determinations of peptide binding to MHC class II molecules.

Binding of positively charged radiolabeled synthetic peptides to human major histocompatibility complex class II DR molecules, purified by affinity chromatography from lymphoblastoid B cell lines of different haplotypes, is rapidly, quantitatively, and specifically assayed by selective adsorption of the complexes between peptide and DR molecules onto DEAE-cellulose paper disks. This assay can be used as a revealing system of the ability of unlabeled test peptides to competitively inhibit the binding between the radiolabeled peptide and the DR molecules, thus measuring the binding of the competitor peptides, irrespective of their charge properties, to different DR molecules.

The enhancer of the immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements.

We have located presumptive chromosomal loop anchorage elements within the mouse heavy chain immunoglobulin locus. Analysis of 31 kilobases spanning diversity, joining, enhancer, switch, and the mu and delta constant regions reveals that only a single 1-kilobase segment exhibits specific binding to nuclear matrices. It is of particular significance that the transcriptional enhancer element resides within this matrix association region (MAR). Fine structure mapping indicates that binding is mediated by A+T-rich approximately 350-base pair segments that reside on either side of the enhancer. The MAR sequences residing 5' of the enhancer contain topoisomerase II consensus sequences like the MAR located upstream of the kappa light chain gene enhancer. The heavy chain gene MARs, however, exhibit a lower affinity for matrix association compared to the kappa gene MAR. Significantly, the juxtaposition of enhancer elements with MARs appears to be evolutionarily conserved within the immunoglobulin genes, suggesting that MARs may act as positive and/or negative regulators of enhancer function.