Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa

Glanzmann thrombasthenia (GT) is the most common inherited disorder of platelets. Most of the molecular defects previously identified in GT have been caused by point (or other small) mutations in the genes for glycoprotein (GP) IIb or GPIIIa. We have used single-strand conformation polymorphism (SSCP) analysis to rapidly identify single- base changes in the GPIIIa gene. Using genomic DNA from normal individuals and patients with GT, each GPIIIa exon and a short stretch of flanking intronic sequence was amplified, heat-denatured, and separated in nondenaturing acrylamide gels. Only those fragments with an abnormal migration pattern were isolated and the nucleotide sequence determined. Using SSCP, we detected the polymorphism in the HPA-1 (P1A) system and all three known silent polymorphisms in the GPIIIa gene. Screening 14 GPIIIa exons from 5 patients with GT, one mutant allele was identified. The nucleotide sequence of the abnormal 240-bp SSCP fragment was determined and a G-->A substitution in the splice donor site of exon iv was identified. Analysis of platelet RNA resulting from this mutation showed two mRNA species: one contained a deletion of exon iv, whereas the other had a 27-bp addition to exon iv due to the use of a cryptic splice site in the downstream intron. Single-base substitutions are the most common mutation in GT and often result in abnormal mRNA splicing. SSCP is a rapid and sensitive technique for identifying mutations or polymorphisms in the GPIIIa gene.     

Functional interleukin-2 receptors are expressed on natural killer-like leukemic cells from a dog with cutaneous lymphoma

We identified a dog with large granular lymphocytic leukemia and cutaneous lymphoma that exhibited constitutive expression of interleukin-2 (IL-2) receptors by the leukemic peripheral blood lymphocytes. The leukemic cells phenotypically resembled natural killer (NK) cells, and their surface IL-2 receptors were functional, as determined by the capacity to bind human recombinant IL-2 with high- affinity resulting in the transduction of proliferation signals and in the development of lymphokine-activated killer cell activity. These cells produced IL-2 spontaneously, and they may have maintained their proliferative state through an IL-2-dependent autocrine growth pathway. Our results indicate that neoplastic lymphocytes of syndromes that involve circulating leukemic cells with dermotropism can originate from NK-like cells. Additionally, the data also suggest that proliferative conditions such as these may be the result of the aberrant production of IL-2. Further, this case illustrates the potential for the use of hematopoietic malignancies in the dog as a suitable animal model for immune targeting of IL-2 receptors as a novel treatment approach for similar malignancies of human beings.     

Recombinant human factor IX: replacement therapy, prophylaxis, and pharmacokinetics in canine hemophilia B

Recombinant human factor IX (rFIX) has been expressed in transduced cultured cell systems since 1985. Because there has been limited in vivo testing of rFIX in hemophilia B subjects, this study was undertaken using the severe hemophilia B canines of the Chapel Hill strain. Three groups of hemophilic dogs received either 50, 100, or 200 IU/kg of rFIX. As a control, a fourth group of hemophilic dogs received 50 IU/kg of a high purity, plasma-derived human FIX (pdFIX). The coagulant and hemostatic effects of rFIX and pdFIX were similar with all comparative dosing regimens. Based on activity data, the elimination half-life of rFIX was 18.9 +/- 2.3 hours and pdFIX was 17.9 +/- 2.1 hours. A prophylactic regimen administering rFIX daily resulted in a continuous therapeutic level of plasma FIX and was accompanied by a two-fold increase in recovery levels by day 5, compared to that observed with administration of a single bolus. The mechanisms of the high to complete recovery of FIX with the prophylactic regimen could depend not only on the degree of saturation of the vascular endothelial binding sites but also on the altered dynamics of the balance of FIX distribution between the intravascular and extravascular compartments. The pharmacokinetic (PK) parameters for rFIX and pdFIX were similar. However, the relative PK values for V1 and V5s of both products on day 5 differed greatly from day 1 and may reflect the changing equilibrium of FIX between compartments with elevated levels of plasma FIX. Neutralizing antihuman FIX antibodies resulting from human FIX antigen being administered to FIX deficient dogs were observed beginning at 14 days. The antigenicity of rFIX and pdFIX appeared to be comparable. Despite the very different procedures used for production of rFIX and pdFIX products, in vivo testing in hemophilia B dogs showed the functional behavior of these products is similar; they are highly effective for replacement therapy and for prophylaxis.     

Residency training in pyloromyotomy: a survey of 331 pediatric surgeons

Purpose

Both pediatric and general surgeons perform pyloromyotomy. Laparoscopic pyloromyotomy (LAP), and changes in referral patterns have affected the training of pediatric surgery fellows and general surgery residents. We surveyed pediatric surgeons regarding these issues.

Methods

We mailed an Institutional Review Board of New Hanover Regional Medical Center-approved survey to 701 members of the American Pediatric Surgical Association within the United States to determine each surgeon's preferred technique for pyloromyotomy (LAP vs Ramstedt or transumbilical procedures [OPEN]), practice setting, involvement with trainees, and opinions regarding pyloromyotomy. Significance was determined using χ² analyses.

Results

A total of 331 (48%) surgeons responded: 197 (60%) performed most or all OPEN, and 85 (26%), most or all LAP. Laparoscopic pyloromyotomy was more likely in academic practices and children's hospitals (P < .05). Residents under surgeons performing LAP were less likely to participate (58% vs 91%; P < .05) or gain competence (22% vs 42%; P < .5). Only 34% of surgeons performing LAP believed that general surgery residents should learn pyloromyotomy, whereas 67% of surgeons performing OPEN believed that residents should learn the procedure (P < .05). A total of 307 (93%) surgeons believed at least 4 OPEN were necessary to become competent, but 126 (44%) reported that their residents performed fewer than 4. Only 104 (31%) surgeons believed that their residents were competent in pyloromyotomy. There were 303 (92%) surgeons who believed that pyloromyotomy should be performed only by pediatric surgeons when possible.

Conclusions

Most general surgical residents are not learning pyloromyotomy, in part because of the adoption of laparoscopic technique, limited operative experience, and the opinion of most pediatric surgeons that the procedure should be performed only by pediatric surgeons.     

Accuracy of three cone-beam CT devices and two software systems in the detection of vertical root fractures

Objectives :The aim of this study was to compare the accuracy of vertical root fracture (VRF) detection using three tomography devices and two software systems in teeth with different endodontic fillings.Methods:The sample consisted of 45 premolars divided into 3 groups: No filling (NF, n=15); Gutta percha (GP, n=15) and Metallic Post (MP, n=15). Cone-beam computed tomography (CBCT) images were acquired in Kodak 9000 3D, Orthopantomography 300 (OP300) and PreXion 3D devices, before and after induced root fractures. Two oral radiologists analyzed all images using InVivoDental and e-Vol DX software systems. The analysis was repeated after 15 days in 30% of the sample. Data analysis compared receiver operating characteristic (ROC) curves, as well the areas under the ROC curves. Accuracy, sensitivity, specificity, positive and negative predictive value were calculated according to each tomographic device and software. Intra- and interexaminer reliability were tested using the Kappa coefficient.Results:The highest accuracy was seen in the image set from the PreXion 3D, using InVivo (0.96) or e-Vol DX (0.92) in image analysis. The OP300 device presented a similar performance of the PreXion 3D in teeth with different endodontic fillings. When using e-Vol DX, the accuracy of Kodak 9000 3D improved from 0.62 to 0.74.Conclusions:The PreXion 3D device is the most accurate when detecting VRF, with a performance similar to the OP300 in endodontic filled teeth. Kodak 9000 3D is indicated for teeth without fillings, with better accuracy using e-Vol DX software.