Chondral metaplasia in calcific insertional tendinopathy of the Achilles tendon.

OBJECTIVE: To ascertain whether tendon samples harvested from patients with calcific insertional Achilles tendinopathy showed features of failed healing response, and whether abnormal quantities of type II collagen had been produced in that area by these tenocytes. DESIGN: Comparative laboratory study. DESIGN: University teaching hospitals. PATIENTS: Tendon samples were harvested from eight otherwise healthy male individuals (average age 47.5+/-8.4 years, range 38 to 60) who were operated for calcific insertional Achilles tendinopathy and from nine male patients who died of cardiovascular events (mean age 63.1+/-10.9 years) while in hospital. INTERVENTIONS: Open surgery for calcific insertional Achilles tendinopathy. MAIN OUTCOME MEASURE: Semi-quantitative histochemical, immunohistochemical, and immunocytochemical methods to ascertain whether tendinopathic tendons were morphologically different from control tendons, and whether abnormal types of collagen were produced. RESULTS: Tenocytes from tendons from patients with calcific insertional Achilles tendinopathy exhibit chondral metaplasia, and produce abnormally high quantities of collagen type II and III. CONCLUSIONS: The altered production of collagen may be one reason for the histopathological alterations described in the present study. Areas of calcific insertional Achilles tendinopathy have been subjected to abnormal loads. These tendons may be less resistant to tensile forces. Further studies should investigate why some tendons undergo these changes.     

Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy

A deficiency of the protein dystrophin has recently been shown to be the probable cause of Duchenne's muscular dystrophy. We sought to determine the relation between the clinical phenotype and the status of dystrophin in muscle-biopsy specimens from 103 patients with various neuromuscular disorders. We found very low levels (less than 3 percent of normal levels) or no dystrophin in the severe Duchenne phenotype (35 of 38 patients), low concentrations of dystrophin in the intermediate (outlier) phenotype (4 of 7), and dystrophin of abnormal molecular weight in the mild Becker phenotype (12 of 18). Normal levels of dystrophin of normal molecular weight were found in nearly all the patients (38 of 40) with 20 other neuromuscular disorders we studied. These data show the clinical consequences of both quantitative alterations (in Duchenne's and intermediate dystrophy) in a single protein. The biochemical assay for dystrophin should prove helpful in delineating myopathies that overlap clinically with Duchenne's and Becker's dystrophies, and it shows promise as an accurate diagnostic tool.     

High Throughput Fingerprint Analysis of Large-Insert Clones

As part of the Human Genome Project, the Washington University Genome Sequencing Center has commenced systematic sequencing of human chromsome 7. To organize and supply the effort, we have undertaken the construction of sequence-ready physical maps for defined chromosomal intervals. Map construction is a serial process composed of three main activities. First, candidate STS-positive large-insert PAC and BAC clones are identified. Next, these candidate clones are subjected to fingerprint analysis. Finally, the fingerprint data are used to assemble sequence-ready maps. The fingerprinting method we have devised is key to the success of the overall approach. We present here the details of the method and show that the fingerprints are of sufficient quality to permit the construction of megabase-size contigs in defined regions of the human genome. We anticipate that the high throughput and precision characteristic of our fingerprinting method will make it of general utility.     

Theories and applications for sequencing randomly selected clones

Theory is developed for the process of sequencing randomly selected large-insert clones. Genome size, library depth, clone size, and clone distribution are considered relevant properties and perfect overlap detection for contig assembly is assumed. Genome-specific and nonrandom effects are neglected. Order of magnitude analysis indicates library depth is of secondary importance compared to the other variables, especially as clone size diminishes. In such cases, the well-known Poisson coverage law is a good approximation. Parameters derived from these models are used to examine performance for the specific case of sequencing random human BAC clones. We compare coverage and redundancy rates for libraries possessing uniform and nonuniform clone distributions. Results are measured against data from map-based human-chromosome-2 sequencing. We conclude that the map-based approach outperforms random clone sequencing, except early in a project. However, simultaneous use of both strategies can be beneficial if a performance-based estimate for halting random clone sequencing is made. Results further show that the random approach yields maximum effectiveness using nonbiased rather than biased libraries.     

Artificial factor VIII deficient plasma: preparation using monoclonal antibodies and its use in one stage coagulation assays.

Monoclonal antibodies to factor VIII antigen (VIII:Ag) and von Willebrand factor (vWf:Ag) were immobilised on Sephacryl S-1000 and tested for their ability to deplete normal human citrated plasma of factor VIII. A combination of two antibodies to VIII:Ag and one antibody to vWf:Ag was required to produce plasma containing less than 0.01 IU/ml. Its performance in the one stage coagulation assay of VIII:C was equivalent to that of congenital VIII deficient plasma for the assay of normal and haemophilic plasma and factor VIII concentrates. Storage of freeze dried aliquots of this product at -20 degrees C, +4 degrees C, and 37 degrees C showed that it could be used as a substrate for at least six months when stored at temperatures +4 degrees C and below.     

Tenocytes from ruptured and tendinopathic achilles tendons produce greater quantities of type III collagen than tenocytes from normal achilles tendons. An in vitro model of human tendon healing

Type I collagen is the main collagen in tendons; type III collagen is present in small amounts. Ruptured Achilles tendons contain a significantly greater proportion of type Ill collagen, which predisposes them to rupture. We used an in vitro model to determine whether tenocytes from Achilles tendons that were ruptured (N = 22), nonruptured (N = 7), tendinopathic (N = 12), and fetal (N = 8) show different behavior. Samples of Achilles tendon were digested with collagenase and the released tenocytes were collected. Primary tenocyte cultures were established and subsequently cultured onto glass coverslips. Once a confluent monolayer was obtained, the cell populations were "wounded" by scraping a pipette tip along the surface. The cultures were further incubated for either 1, 4, 8, 12, 16, or 24 hours, and production of types I and II collagen was assessed by immunostaining. In cultures from ruptured and tendinopathic tendons, there was increased production of type Ill collagen. Athletic participation places excess stress on the Achilles tendon, which could potentially lead to areas of microtrauma within the tendon. These areas may heal by the production of type III collagen, which is an abnormal healing response. Accumulation of such episodes of microtrauma could resuit in a critical point where the resistance of the tissue to tensile forces is compromised and tendon rupture occurs.     

A Patient with AKI after Cardiac Surgery

Up to 30% of patients undergoing cardiac surgery develop AKI, with 1% requiring RRT. AKI is an independent risk factor for morbidity and mortality. Postoperatively, even minimal changes in serum creatinine are associated with a substantial increase in mortality. No intervention has been definitely proven effective in reducing kidney injury. The successful prevention and management of AKI involves identifying patients at risk for AKI, recognizing subtle abnormalities in a timely manner, performing basic clinical assessments, and responding appropriately to data obtained. With that in mind, in this Attending Rounds, a woman with AKI in the setting of cardiac surgery is presented to highlight the use of history, physical exam, hemodynamic monitoring, laboratory data trends, and urine indices in establishing the correct diagnosis and appropriate management.     

Studies on the Procurement of Blood Coagulation Factor VIII in vitro Studies on Blood Components Prepared in Half-Strength Citrate Anticoagulant

The effect of replacing a standard citrate anticoagulant with one containing half the amount of citrate on the in vitro properties of components prepared from blood donations was investigated. This resulted in a significant improvement in factor VIII stability such that there was little loss during overnight storage, and this was reflected in the factor VIII yield in cryoprecipitate. The quality of cellular components in red cell units stored up to 35 days or platelet concentrates stored up to 7 days was not adversely affected. Although initial levels were similar to those in standard anticoagulant, the extent of fibrinopeptide A generation and complement C3 breakdown in red cell units stored for 35 days in half-strength citrate was somewhat increased.