Primitive neuroectodermal tumor of the diaphragm: a case report.

We present a case of primitive neuroectodermal tumor (PNET) arising from the diaphragm in a neonate. PNETs are rare malignant tumors that belong to the group of small, round, blue-cell neoplasms of childhood. To the best of our knowledge, a PNET originating from the diaphragm has not been previously reported.

Determination of beta-glucosidase enzymatic function of the histoplasma capsulatum H antigen using a native expression system.

The Histoplasma capsulatum H antigen is a major secreted glycoprotein of this pathogenic fungus that is a target of humoral and cell-mediated host responses. Its predicted protein sequence displays homology to beta-glucosidases of other organisms, but a recombinant antigen expressed in a prokaryotic system showed no enzymatic activity. We expressed a recombinant form of the protein carrying a carboxyl-terminus oligohistidine tag in the native fungal background to facilitate proper glycosylation and folding of a product that could then be purified from culture supernatants using nickel affinity chromatography. The recombinant protein was expressed and secreted by a transformant carrying the modified gene under the control of its native promoter. The purified protein from the native expression system showed beta-glucosidase enzymatic activity in substrate gels and quantitative microplate assays. This activity was blocked by glucosidase-specific inhibitors. These results are the first direct demonstration of the function of this protein, and show the utility of expression in a native system to achieve post-translational modification necessary for structural and functional integrity.

Defective running shoes as a contributing factor in plantar fasciitis in a triathlete.

STUDY DESIGN: Case study of a patient who developed plantar fasciitis after completing a triathlon. OBJECTIVES: To describe the factors contributing to the injury, describe the rehabilitation process, including the analysis of defective athletic shoe construction, and report the clinical outcome. BACKGROUND: Plantar fasciitis has been found to be a common overuse injury in runners. Studies that describe causative factors of this syndrome have not documented the possible influence of faulty athletic shoe construction on the symptoms of plantar fasciitis. METHODS AND MEASURES: The patient was a 40-year-old male triathlete who was followed up for an initial evaluation and at weekly intervals up to discharge 4 weeks after injury and at 1 month following discharge. Perceived heel pain, ankle strength, and range of motion were the primary outcome measures. Shoe construction was evaluated to assess the integrity of shoe manufacture and wear of materials by visual inspection of how shoe parts were glued together, if shoe parts were assembled with proper relationship to each other, if the shoe sole was level when resting on a level surface, and if the sole allowed unstable motion. RESULTS: The patient appeared to have a classic case of plantar fasciitis with a primary symptom of heel pain at the calcaneal origin of the plantar fascia. On initial evaluation, right heel pain was a 9 of 10, plantar flexion strength was a 3+/5, and ankle dorsiflexion motion was 10 degrees. One month after discharge, perceived heel pain was 0, plantar flexion strength was 5/5, and dorsiflexion motion was 15 degrees and equal to the uninvolved extremity. The right running shoe construction deficit was a heel counter that was glued into the shoe at an inward leaning angle, resulting in a greater medial tilt of the heel counter compared with the left shoe. The patient was taught how to examine the integrity of shoe manufacture and purchased a new pair of sound running shoes. CONCLUSIONS: A running shoe manufacturing defect was found that possibly contributed to the development of plantar fasciitis. Assessing athletic shoe construction may prevent lower extremity overuse injuries.

Protease inhibitor phenotype BsaskatoonM is not associated with emphysema - a 20-year follow-up study.

OBJECTIVE: To investigate the association between the PiBsaskatoon variant of alpha-1 protease inhibitor (a1 Pi), present in the heterozygous state, and the development of emphysema. DESIGN: Twenty-year follow-up in the third generation of a family with the variant, naturally controlled with regard to both environmental influences and other genetic factors. SETTING: University teaching hospital. POPULATION STUDIED: Ten siblings, five with PiBsaskatoonM phenotype and five with PiM phenotype, 33 to 46 years of age. INTERVENTIONS: Respiratory symptoms and smoking histories; pulmonary function tests, including static lung volumes, dynamic lung volumes before and after salbutamol 200 mg, and diffusing capacity; allergen prick skin tests; serum a1 Pi level, chest radiographs and high resolution computerized tomography lung scans MAIN RESULTS: The two groups of siblings had similar mean ages, smoking histories and prevalence of current mild respiratory symptoms. Pulmonary function data showed normal mean values and no statistically significant differences for all the variables between the two groups. Chest radiographs were normal in all subjects. High resolution computerized tomography scans were normal in eight subjects, and demonstrated mild and very mild centrilobular emphysema in the two subjects with greatest smoking histories (approximately 30 pack-year each); both of these were PiBsaskatoonM phenotype. CONCLUSION: There is no evidence of an association between a1 Pi phenotype PiBsaskatoonM and the development of emphysema.

Histoplasma capsulatum strain variation in both H antigen production and beta-glucosidase activity and overexpression of HAG1 from a telomeric linear plasmid.

The H antigen of the dimorphic fungal pathogen Histoplasma capsulatum was first described over 40 years ago. It is a secreted glycoprotein that is immunogenic during infection. Recent cloning of the H antigen gene (HAG1) indicated sequence homology with genes for fungal beta-glucosidases. To understand the biological role of this immunodominant antigen in H. capsulatum, enzymatic assays were performed to determine whether H. capsulatum contained a beta-glucosidase enzyme activity and whether this activity was encoded by the HAG1 gene. Substrate gels with H. capsulatum culture supernatants revealed beta-glucosidase activity near the predicted mobility of the H antigen. Quantitative microtiter plate assays revealed marked differences in secreted beta-glucosidase activities from three H. capsulatum restriction fragment length polymorphism (RFLP) classes, with RFLP class II strains displaying high levels of enzyme activity, in contrast to the low levels of activity exhibited by class I and III strains. Immunoblotting of culture supernatants with an H antigen-specific antiserum demonstrated differences in H protein expression levels between the H. capsulatum classes, with a correlation between secreted enzyme activity and H protein levels. We took advantage of these class differences to demonstrate multicopy plasmid H gene overexpression by transformation of an HAG1 plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain (G184ASura5-11) transformed with the telomeric overexpression plasmid pMAD401 displayed increased levels of beta-glucosidase enzyme activity and H protein expression compared to the levels in control transformants containing only the single genomic copy of HAG1. This is the first demonstration of telomeric plasmid-mediated protein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a beta-glucosidase.

Constrictive bronchiolitis and ulcerative colitis.

Pulmonary complications occur in an estimated 0.21% of patients with inflammatory bowel disease. The most common presentation of pulmonary manifestations is large airway disease, such as tracheobronchitis, chronic bronchitis or bronchiectasis. Small airway disease, such as constrictive bronchiolitis or bronchiolitis obliterans with organizing pneumonia, is less frequently reported, and is described as occurring in isolation from large airway disease. A case of a postcolectomy ulcerative colitis in a patient who has both large airway involvement, tracheobronchitis and bronchiectasis, and constrictive bronchiolitis is presented.

Mapping the intercellular adhesion molecule-1 and -2 binding site on the inserted domain of leukocyte function-associated antigen-1.

By extensive mutagenic analysis of the inserted domain (I-domain) of the alpha-chain (CD11a) of the leukocyte function-associated antigen-1 (LFA-1), we have defined a putative binding surface for intercellular adhesion molecules 1 and 2 (ICAM-1 and -2). This analysis showed that individually mutating Leu-205 or Glu-241 to alanine completely abolished LFA-1 binding to ICAM-1 or -2 without affecting I-domain structure, as assayed by antibody binding. Mutating Thr-243 to alanine also had a profound effect on LFA-1 binding to ICAM-1 and -2, as seen by complete loss of binding to ICAM-1 and a significant reduction (70% decrease) in binding to ICAM-2. Mutating Glu-146 to alanine reduced LFA-1 binding to ICAM-1 or -2 by 70%, and mutating His-264 or Glu-293 to alanine reduced binding to ICAM-1 or -2 by about 30-40%. Mutating Thr-175 to alanine reduced binding to ICAM-1 by about 30% and binding to ICAM-2 by about 70%. Interestingly, mutating Lys-263 to alanine preferentially abolished LFA-1 binding to ICAM-2. Using these data, we have generated a model of the interface between the LFA-1 I-domain and residues in the first domain of ICAM-1 that have been shown to be critical for this interaction. In addition, this model, together with the ICAM-2 crystal structure, has been used to map residues that are likely to mediate LFA-1 I-domain binding to ICAM-2.

Identification of the binding site in intercellular adhesion molecule 1 for its receptor, leukocyte function-associated antigen 1.

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.

Radiographic appearance of central venous catheters.

Central venous catheters (CVCs) are defined as venous access devices whose tips terminate in the superior or inferior vena cava, regardless of insertion site. CVCs allo reliable, painless, and repeated entry into the venous system and are commonly used for the administration of IV therapy, parenteral nutrition, and blood products as well as for the periodic blood sampling, hemodynamic monitoring, and hemodialysis. Catheter composition and design vary and depend on the duration of intended use and specific functions required. The purpose of this essay is to illustrate commonly used catheters, discuss factors governing catheter selection, and review important catheter-related complications.

The incidence of musculoskeletal injuries in an amateur triathlete racing club.

The sport of triathlon involves three different endurance sports: swimming, biking, and running. Cross-training allows the athlete to train for one sport while resting from another. The repetitive motions required for each sport may lead to overuse and injury. The purpose of this study was to examine musculoskeletal injury incidence in amateur triathletes to determine if these injuries caused lost time from training, racing, working, or daily functioning. Seventy-two recipients responded to survey items that gathered information about demographics, sports participation, and musculoskeletal injury occurrence and interference with sports participation, work, and daily activities. Three-quarters sustained triathlon-related musculoskeletal injuries during training due to overuse. A majority experienced training interruption and interference with daily functioning and sought professional help for their injuries. Little information is available on the treatment of musculoskeletal injuries in triathletes. This survey raises important clinical implications for physical therapists. Further exploration of overuse injury incidence is warranted in this population.

Objective and subjective results of a randomized, double-blind, placebo-controlled trial using cisapride to treat gastroparesis.

Cisapride, a relatively new gastrointestinal prokinetic agent, has been reported to increase gastric emptying and improve symptoms of gastroparesis. We investigated these effects of cisapride in patients with severe idiopathic and diabetic gastroparesis during an eight-week trial. The study design was a two-week single-blind placebo run in period to exclude placebo responders, followed by a six-week randomized, double-blind, placebo-controlled treatment phase. Delayed gastric emptying of solids on radionuclide scan and a minimum symptom intensity score were inclusion criteria. Forty-three patients were entered: four placebo responders and one other patient were excluded, leaving 19 patients randomized to cisapride (20 mg per os three times a day before meals), and 19 patients to placebo. Seven individual symptoms of gastroparesis were scored in a daily diary and reviewed at two-week visits. Sixteen patients in the cisapride group were able to complete the trial compared to 12 on placebo. The gastric emptying study was repeated at the end of treatment or at the time of withdrawal for those who dropped out. Cisapride significantly increased solid gastric emptying relative to baseline (P = 0.005) whereas placebo did not (P > 0.10). Cisapride did not significantly improve any symptom of gastroparesis relative to baseline or to placebo. We conclude that in a population of severe, refractory gastroparetic patients cisapride significantly accelerates gastric emptying of a solid meal without significantly reducing symptoms during a short-term treatment trial compared to placebo. Further trials of cisapride in less advanced and "end-stage" gastroparetics than studied here or combining cisapride with other prokinetic agents or antiemetics, are warranted.

Lipid association, but not the transmembrane domain, is required for tissue factor activity. Substitution of the transmembrane domain with a phosphatidylinositol anchor.

Full-length tissue factor (263 rTF) and three truncated forms have been expressed in human kidney 293 cells; 1) 243 rTF, which lacks the cytoplasmic tail, is fully functional in the chromogenic assay and has a specific activity comparable with that of the full-length molecule, 263 rTF; 2) 219 rTF, which lacks both the transmembrane and cytoplasmic domains, is not functional; 3) the third variant, referred to as TF-PI, is a fusion protein containing the extracellular domain of TF (amino acids 1-219) fused to the last 37 amino acids of decay-accelerating factor which contain a signal for attachment of a phosphatidylinositol membrane anchor (PI). TF-PI is a membrane-bound protein expressed on the cell surface. The PI anchor restores TF activity lost when the transmembrane domain is deleted from the 219 rTF variant. The ability of the PI anchor to restore activity to 219 rTF clearly demonstrates that while the transmembrane domain is not required for TF activity, lipid association is required.

A protocol for the safe administration of debrisoquine in biochemical epidemiologic research protocols for hospitalized patients.

The genetically determined ability to metabolize the antihypertensive drug debrisoquine has been proposed as a genetic risk factor for primary carcinomas of the lung. To test this hypothesis, the metabolism of the drug was evaluated in a case control study. The subjects were characterized by their ability to metabolize debrisoquine after receiving a test dose of the drug followed by the collection of an 8-hour urine sample. They were classified by laboratory analysis into one of the following three groups: extensive, intermediate, and poor metabolizers. Poor metabolizers comprise 10% of the population and are unable to hydroxylate the drug. This group was expected to be at highest risk for deleterious effects from this medication. A protocol was created that included patient education and blood pressure monitoring to administer this medication safely to a group of patients with cancer who were already compromised. Although poor metabolizers showed a small decrease in systolic and diastolic blood pressure, no significant hypotensive episodes or clinical sequelae were observed in any of the groups. These data suggest that debrisoquine can be administered safely in a controlled clinical setting and will be useful for the characterization of lung cancer patients in biochemical epidemiology studies.

Mammalian cell transient expression of tissue factor for the production of antigen.

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.

Cloning and expression of human tissue factor cDNA.

Tissue factor is a membrane protein that plays an essential role in the initiation of blood coagulation. When exposed to the circulation, tissue factor interacts with the serine protease factor VII, and the complex triggers fibrin clot formation by activating both factors IX and X of the coagulation cascade. This report describes the cloning and expression of the complementary DNA (cDNA) for human tissue factor. The cDNA encodes a protein of 263 amino acids preceded by a 32 amino acid signal peptide. The predicted protein sequence contains a potential hydrophobic membrane anchoring domain at its carboxy terminus, and bears no significant homology to any other known protein. Tissue factor mRNA of 2400 nucleotides was detected in adipose, adrenal, small intestine and a number of other tissues by Northern blot hybridization analysis. In order to confirm the identity of the cDNA, an expression vector containing the cloned cDNA was used to transfect cultured mammalian cells. These cells produced active tissue factor which was assayed using purified factors VII and X.