Polymorphism, natural selection, and structural modeling of class Ia MHC in the African clawed frog (Xenopus laevis).

In the African clawed frog (Xenopus laevis), two deeply divergent allelic lineages of multiple genes of the class I MHC region have been discovered. For the MHC class I UAA locus, functional differences and the molecular basis for lineages maintenance are unknown. Alleles of linked class I region genes also exhibit strong disequilibrium with specific MHC alleles, but the underlying cause is not clear. We use MHC class Ia sequence data to estimate substitution rates and investigate structural differences between allelic lineages from protein models. Results indicate the operation of natural selection, and differences in the steric properties in the F pocket of the peptide-binding region among lineages. Variability in this pocket likely enables allelic lineages to bind very different sets of peptides and to interact differently with MHC chaperones in the endoplasmic reticulum. These results constitute evidence of the molecular evolutionary basis for 1) the maintenance of allelic lineages, 2) functional differences among lineages, and 3) strong linkage disequilibrium of allelic variants of class I region genes in X. laevis.

Osteoarthritis-like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho).

OBJECTIVE: To investigate whether heterozygosity for a loss-of-function mutation in the gene encoding the alpha1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints. METHODS: Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild-type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP-3) and MMP-13 in knee joints. In 6-month-old animals, fixed-charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique. RESULTS: The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA-like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP-3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP-13 was higher in knee joints from cho/+ mice than in joints from their wild-type littermates, and negative fixed-charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration. CONCLUSION: Heterozygosity for a loss-of-function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.

Efficient recruitment of transfected DNA to a homologous chromosomal target in mammalian cells.

A Chinese hamster ovary cell line hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene was transfected with a plasmid, pAG100, capable of correcting the endogenous aprt mutation by targeted homologous recombination. In some experiments, pAG100 was transfected in combination with one of two 'competitor' plasmids. Competitor pCOMP-A was identical to pAG100 except that the aprt sequence on pCOMP-A had the same mutation as the endogenous aprt gene. Competitor pCOMP-B was identical to pAG100 except for a 763 bp deletion in the aprt sequence encompassing the site of mutation in the endogenous gene. Neither pCOMP-A nor pCOMP-B was capable of correcting the defect in the endogenous aprt gene via gene targeting. We asked whether cotransfection of a 4-fold excess of either competitor DNA molecule with pAG100 would reduce the efficiency of targeted correction of the endogenous aprt gene. We report that while plasmid pCOMP-B did not influence the efficiency of gene targeting by pAG100, plasmid pCOMP-A reduced the number of gene targeting events about 5-fold. These observations indicate that the initial homologous interaction between transfected DNA and a genomic target sequence occurs rapidly and that targeting efficiency is limited by a step subsequent to homologous pairing.

Evaluation of preoperative cultures before second-stage reimplantation of a total knee prosthesis complicated by infection. A comparison-group study.

BACKGROUND: Two-stage reimplantation has proven to be highly successful in the treatment of patients with infection at the site of a total knee arthroplasty. However, up to 20 percent of patients have a recurrence of infection following this treatment. The purpose of our study was to determine whether aspiration of the affected joint and culture of the specimen, performed before reimplantation and after discontinuation of antibiotic therapy, would help to identify patients who might have a recurrent infection. METHODS: We prospectively followed sixty-nine patients who were treated for a culture-proven deep infection at the site of a total knee arthroplasty. Group I consisted of thirty-five patients who were treated with removal of the prosthetic components and irrigation and debridement of the joint, followed by six weeks of antibiotic therapy and reimplantation of a prosthesis. Group II was composed of thirty-four patients who were treated with removal of the components and irrigation and debridement of the joint, six weeks of antibiotic therapy, and then repeat culture four weeks after the antibiotic course had ended. If the culture was negative, the patient was managed with a second-stage reimplantation of a prosthesis. If the culture was positive, the protocol was repeated, beginning with irrigation and debridement. The two groups were similar with regard to male-to-female ratio, age, preoperative Knee Society scores, time since primary surgery, types of infectious organisms, duration of symptoms, duration of follow-up, and number of previous revisions. All of the patients were evaluated clinically with use of the objective scoring system of the Knee Society and were followed with serial radiographs. Success was defined as no infection and a functional prosthesis, with a Knee Society score of at least 75 points at the last (thirty-six-month-minimum) follow-up evaluation. RESULTS: Of the thirty-five patients in Group I, five (14 percent) had recurrence of infection. One of the patients was managed with a successful second-stage revision, three were managed with arthrodesis of the knee, and one continued with chronic antibiotic suppressive treatment. Of the thirty-four patients in Group II, three (9 percent) had a positive culture after the course of antibiotics. The protocol was repeated for all three, and they subsequently had a successful second revision. One other patient (3 percent) in Group II, who had a negative culture, had a recurrent infection and was eventually managed with arthrodesis of the knee. CONCLUSIONS: Prerevision cultures, grown after discontinuation of antibiotic treatment and before reimplantation of the components, helped to identify the patients with infection at the site of a total knee arthroplasty in whom the infection might recur. The performance of aspiration and cultures resulted in a substantial improvement in the clinical outcome.

Characteristics and natural history of transient postoperative pseudosubluxation after total hip arthroplasty.

A series of 13 total hip arthroplasties (THAs) in which immediate postoperative radiographs revealed axial subluxation were reviewed, and the timing, incidence, and subsequent natural history of this phenomenon were determined. Each patient had received perioperative epidural anesthesia and was placed in an abduction splint before transfer to the postoperative care unit. All initial radiographs were taken while the patient was still under the effect of the epidural anesthetic agent (average, 54 minutes; range, 37-80 minutes) postoperatively. In each case, radiographs taken after anesthetic recovery and without limb manipulation revealed reduced components. The balance of the postoperative care and rehabilitation was uncomplicated; patients were followed at least 36 months postoperatively, and in only 1 patient did a subsequent hip dislocation occur. Over a 6-month period, the postoperative films of all THAs were studied, and postoperative subluxation was identified in about 3% of the cases. Postoperative subluxation is likely to be noted when the radiograph is obtained within 1 hour of transfer to the postoperative care unit. It does not appear to have a dramatic effect on the rate of postoperative dislocation, however. This radiographic finding probably is due to the continued effects of regional anesthesia, and reduction occurs as muscle tone returns. If transient subluxation is identified, no change in postoperative care is necessary.

Infected total knee arthroplasty treated by arthroscopic irrigation and débridement.

Sixteen patients with infected total knee arthroplasties (4 postoperative and 12 late hematogenous) were treated by arthroscopic irrigation and débridement. All patients had < or = 7 days of knee symptoms, and there were no radiographic signs of osteitis or prosthetic loosening. Six of the 16 original total knee arthroplasties (38%) did not need prosthesis removal at a mean follow-up of 64 months (range, 36-151 months). Ten other knees were treated with irrigation, débridement, and hardware removal within 7 weeks of the latest procedure used to try to retain components. Two (13%) of these cases ultimately required an arthrodesis for persistent infection. Although we still believe that this method is preferable to resorting immediately to implant removal for acute infections, arthroscopic débridement was less efficacious for most situations when compared with open treatment. We would use arthroscopic irrigation and débridement only under selected circumstances (medically unstable or anticoagulated patients).

Infected total knee arthroplasty treated with arthrodesis using a modular nail.

Failed treatment of infected total knee replacement presents few attractive surgical options. Knee arthrodesis is challenging surgically and can be complicated by nonunion, malunion, or recurrent infection. Recently, a modular titanium intramedullary nail has been used in an attempt to reduce the incidence of nonunion and the rate of complications. In the present study, a review of the results of knee arthrodesis after infected total knee arthroplasty in 21 patients at three large academic institutions was performed. All patients were followed up for a mean of 2.4 years (range, 2-7.5 years). The mean age of the patients was 64 years. The mean number of previous operations was four (range, 2-9 operations). A solid arthrodesis was achieved without additional surgical treatment in 20 of 21 patients (95%). The mean time to fusion was 6.3 months. The one patient who suffered a nonunion achieved fusion after a subsequent bone grafting procedure. Based on the present study, intramedullary arthrodesis with a coupled titanium nail, is a reliable, effective method of achieving fusion after infection of a total knee arthroplasty. This procedure resulted in a high rate of fusion and a lower rate of complications when compared with traditional methods of arthrodesis.

Infections associated with dental procedures in total hip arthroplasty.

Dental procedures may lead to a transient bacteraemia lasting for up to 30 minutes. Of the numerous cases of total hip arthroplasty (THA) reported which have been infected from haematogenous sources, dental procedures have been involved only infrequently. We reviewed the records of 2973 patients after THA. Of the late infections identified in 52 patients, three (6%) were strongly associated with a dental procedure. Infection was diagnosed by culture from the affected joint; Streptococcus viridans was identified in two cases and Peptostreptococcus in one. One patient had diabetes mellitus and another rheumatoid arthritis, both conditions predisposing to infection. The dental operations all lasted for more than 45 minutes and no patient received perioperative antibiotics. Infection of a THA after dental procedures is more common than has been previously suspected. Patients with systemic disease, or who are undergoing extensive procedures, should be considered for prophylactic antibiotic treatment.

Indications, technique, and results of total shoulder arthroplasty in rheumatoid arthritis.

Unconstrained total shoulder arthroplasty is a highly effective and successful operation in the patient with rheumatoid arthritis. Reliable pain relief can be obtained, often with an improvement in range-of-motion and function of the entire extremity. Rheumatoid patients may present with difficult soft-tissue and bony deficiencies, but careful planning and intraoperative procedures help to maximize the outcome and minimize the possible complications.

Stable transfection of mammalian cells by syringe-mediated mechanical loading of DNA.

We show that mammalian cells can be stably transfected by a mechanical loading procedure in which cells are forced through a small opening in the presence of DNA. A suspension of cells and plasmid DNA in growth medium was passed up and down through a 30-gauge needle attached to a 1-ml syringe. Cells were immediately plated at appropriate densities for subsequent selection for stable expression of a marker gene. Two rodent cell lines, Chinese hamster ovary and mouse Ltk- cells, were successfully transfected with an efficiency of about one transfectant per 5 x 10(4) cells. The human HeLa cell line was transfected with a somewhat lower efficiency. Pluronic F-68, a detergent believed to aid in healing of membrane injuries, had no beneficial effect when present during the loading procedure. Successful transfection was accomplished using three different genes as selectable markers. Southern blotting analysis revealed that transfectants contained one or very few copies of the introduced DNA construct integrated into the genome. Several transfectants were demonstrated to remain stable for more than 20 generations of growth in the absence of selection. This procedure is fast, economical, and of general utility.

Total knee arthroplasty infections associated with dental procedures.

Total knee arthroplasties are at risk for hematogenous seeding secondary to procedures that create a transient bacteremia. To define the risk of infection associated with dental surgery, a retrospective review of the records of 3490 patients treated with total knee arthroplasty by the authors between 1982 and 1993 was performed. Sixty-two total knee arthroplasties with late infections (greater than 6 months after their procedure) were identified, and of these, seven infections were associated strongly with a dental procedure temporally and bacteriologically. These seven cases represented 11% of the identified infections or 0.2% of the total knee arthroplasty procedures performed during this period. In addition, among 12 patients referred for infected total knee arthroplasties from outside institutions, two infections were associated with a dental procedure. Five of the nine (56%) patients had systemic risk factors that predisposed them to infection, including diabetes and rheumatoid arthritis. All dental procedures were extensive in nature (average, 115 minutes; range, 75-205 minutes). Eight of the patients received no antibiotic prophylaxis. One patient had only one preoperative dose. Infections associated with dental procedures may be more common than previously suspected. Eight of these patients had no prophylactic antibiotics, and one had inadequate coverage. The authors think that patients with a total knee arthroplasty who have systemic disease that compromises host defense mechanisms against infections and who undergo extensive dental procedures should receive prophylactic antibiotics. A first generation cephalosporin, given 1 hour preoperatively and 8 hours postoperatively would provide the best prophylaxis against the organisms identified in this study.

Multiple irrigation, debridement, and retention of components in infected total knee arthroplasty.

The results of 24 infected total knee arthroplasties (22 patients) that were treated by irrigation, debridement, and retention of the prosthetic components were prospectively studied. Strict criteria were used for the selection of this method of treatment. Patients had to be less than 30 days after index arthroplasty (postsurgical group) or had to have less than 30 days of knee symptoms (hematogenous group). In addition, there had to be no radiographic signs of osteitis or evidence of a loose prosthetic component. Patients had one to three irrigation and debridement procedures depending on systemic signs, knee symptoms, or the results of knee aspirations. All of the immediate postsurgical infections (10 knees) and 10 of the 14 (71%) late hematogenously infected knees retained the prosthesis without further evidence of infection at the final follow-up visit at 48 months (range, 24-140 months). This study shows that in selected circumstances, irrigation, debridement, and retention of the components can result in low morbidity with high success rates.

Ankle fractures. The Lauge-Hansen classification revisited.

Rational treatment of ankle fractures requires knowledge of the extent of bone and soft tissue injury. Although the Lauge-Hansen classification attempts to do this by relating specific fracture patterns to injury mechanism, the experimental underpinning for this classification has not been reexamined rigorously using modern experimental methods. This study examines the hypothesis that the clinically occurring supination and external rotation injury pattern does not result from the mechanism described by Lauge-Hansen. Thirty-two anatomic specimen ankles were mounted on an MTS machine for combined axial loading with external rotation to failure testing. A foot plate supinated the foot 25 degrees. Testing was performed with the ankle at neutral, 25 degrees plantar flexed, 10 degrees to 15 degrees dorsiflexed, and in 6 degrees to 8 degrees leg valgus. Pure supination and external rotation with the ankle in neutral did not result in the Lauge-Hansen supination and external rotation type fractures. This outcome was not altered if the ankle specimens initially were placed in plantar flexion or dorsiflexion. The addition of a valgus load, which pushes the talus laterally against the fibula, resulted in the classic Lauge-Hansen supination and external rotation type fracture. All specimens had an isolated lateral injury or a lateral injury that preceded medial injury.

Enrichment for gene targeting in mammalian cells by inhibition of poly(ADP-ribosylation).

Inhibition of poly(ADP-ribosylation) reduces random genomic integration of transfected DNA and mildly stimulates intrachromosomal homologous recombination in mammalian cells. We investigated the effect of inhibition of poly(ADP-ribosylation) on the efficiency of gene targeting in Chinese hamster ovary (CHO) cell line ATS-49tg. This cell line is hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene and is hypoxanthine phosphoribosyltransferase (hprt) deficient. Plasmid pAG100 contains a portion of the CHO aprt gene sufficient to correct the defect in ATS-49tg cells via gene targeting; pAG100 also contains an Escherichia coli guanine phosphoribosyltransferase (gpt) gene. Following transfection of ATS-49tg cells with pAG100, selection for gpt-positive transfectants allowed recovery of cells that had randomly integrated pAG100 while selection for aprt-positive cells allowed recovery of cells that had undergone gene targeting at the endogenous aprt locus. Treatment of cells with 3 mM 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP-ribose) polymerase, decreased random integration and gene targeting of electroporated pAG100 about 5-fold. In contrast, treatment with 3 mM 3-MB during calcium phosphate transfection could reduce random integration more than 150-fold while reducing gene targeting less than two-fold. Therefore, as much as a 100-fold enrichment for gene targeting was achieved with calcium phosphate transfection.

An axially loaded model of the ankle after pronation external rotation injury.

Using a testing apparatus that allows axial loading and displacement in the sagittal, axial, and coronal planes, 6 ankles were tested under experimental conditions intended to model the Lauge-Hansen pronation external rotation injury. All specimens were rotated through a continuous range of sagittal motion with the ankle under 300 N of axial load as the coupled motion of the ankle in the coronal and axial axes was recorded. Combinations of fibular osteotomy, disruption of the syndesmosis up to 6 cm above the plafond, and deltoid transection were tested to mimic Stages I to III of the pronation external rotation ankle fracture. The effects of stabilization of the fibula and syndesmosis also were examined. Neither fracture of the fibula 4 cm above the plafond nor disruption of the syndesmosis to 6.0 cm resulted in a significant change in coupled motion of the talus. When the superficial deltoid was sectioned, the ankle had increased external rotation in plantar flexion. When the deep deltoid was sectioned, the ankle dislocated in plantar flexion unless the fibula was stabilized. This prevented dislocation but failed to restore normal talar kinematics. This study found no biomechanical support for placement of a syndesmotic screw unless the medial side cannot be stabilized anatomically.