Investigation of the peak action wavelength of light-activated gene transduction.

Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Previous studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, whereas UVA induces LAGT secondary to reactive-oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage and determine an optimal wavelength to maximize safety and efficacy. UV at 288, 311 and 320 nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4 kJ m⁻² at 311 nm. Consistent with its robust cytotoxicity, 288 nm produced significantly high levels of DNA damage at all doses tested, whereas 311, 320 and 330 nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. Although 288 nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ m⁻² at 311 nm. An in vivo pilot study assessing 311 nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (P<0.05) increase in transduction with insignificant cytotoxicity. In conclusion, 311 nm was found to be the optimal wavelength for LAGT on the basis of its superior efficacy at the peak dose and its broad safety range that is remarkably wider than the other UV wavelengths tested.

CD19 has a potential CD77 (globotriaosyl ceramide)-binding site with sequence similarity to verotoxin B-subunits: implications of molecular mimicry for B cell adhesion and enterohemorrhagic Escherichia coli pathogenesis.

The glycosphingolipid globotriaosyl ceramide (CD77) and other globo-series glycolipids containing terminal galactose (Gal)alpha 1-4Gal residues function as receptors for the verotoxin (Shiga-like toxin) family of Escherichia coli-elaborated toxins. CD77 is also a marker for germinal center B lymphocytes and Burkitt's lymphoma cells. The pan B cell marker CD19 is a 95-kD membrane protein that appears early in B cell differentiation and is only lost upon terminal differentiation to plasma cells. CD19 is involved in signal transduction and has a regulatory role in B cell proliferation and differentiation in response to activation in vitro. However, an endogenous ligand for CD19 has not yet been identified. We report herein that the extracellular domain of CD19 has a potential CD77-binding site with extensive sequence similarity to the verotoxin B-subunits. These B-subunit-like sequences on CD19 are in close proximity following the organization of intervening amino acids into disulfide-linked domains. Cocapping of CD19 and CD77 on Burkitt's lymphoma-derived Daudi cells with anti-CD19 antibodies indicates that CD19 and CD77 are associated on the B cell surface. Cell surface binding of anti-CD19 antibodies is decreased on CD77-deficient mutant Daudi cells, suggesting that CD77 expression influences the surface expression of CD19. Wild-type Daudi cells, but not the CD19/CD77-deficient mutants, bind to matrices expressing the carbohydrate moiety of CD77 or other Gal alpha 1-4Gal containing glycolipids. This binding can be inhibited by anti-CD77 antibodies, the CD77-binding verotoxin B-subunit or anti-CD19 antibodies. Daudi cells exhibit a degree of spontaneous homotypic adhesion in culture while the CD77/CD19-deficient Daudi mutants grow as single cells. The stronger homotypic adhesion that occurs in B cells after antibody ligation of CD19 and that involves, to some extent, the integrin system, is also dramatically lower in the mutant cells relative to the parent cell line. However, reconstitution of mutant cells with CD77 restores the anti-CD19 mAb-induced adhesion to wild-type Daudi cell levels. These studies represent the first time that CD19-mediated signaling has been reconstituted in a low-responder B cell line. These convergent observations provide compelling evidence that CD19/CD77 interactions function in adhesion and signal transduction at a specific stage in B cell development and suggest that such interactions have a role in B lymphocyte homing and germinal center formation in vivo. By targeting CD77+ B cells, verotoxins may suppress the humoral arm of the immune response during infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use.

A new procedure is described which enables gram quantities of adult Onchocerca tissue to be isolated from frozen connective tissue nodules, thus minimizing the risk of enzymatic degradation. Bovine connective tissue nodules containing adult Onchocerca gibsoni worms were obtained from Australia frozen at -70 degrees C and sectioned while still frozen into 3 mm thick slabs. The sections were thawed immediately before use, worm segments removed, rinsed, pelleted, and flash frozen in liquid nitrogen. Quality of the isolated material was demonstrated by the presence of an intact adult epicuticle as determined by electron microscopy, and by the presence of viable uterine larvae and cells. This procedure is applicable to other nodule-forming worms such as Onchocerca volvulus and is suitable for investigations which require the isolation of labile molecules or those present in minute quantities.

A comparison of the glycolipid compositions of cercarial and adult Schistosoma mansoni and their associated hosts.

Glycolipid patterns of cercarial and adult Schistosoma mansoni were determined and compared with those of Biomphalaria glabrata snails and mouse (BALB/c strain) red blood cells by high-performance thin-layer chromatography. Differences in glycolipid content were found between cercariae and adult worms and between these stages and their respective host tissues.

Immunity to Heligmosomoides polygyrus induced by subcutaneous vaccination with post-infection larvae.

The objective of this study was to investigate, using the Heligmosomoides polygyrus (= Nematospiroides dubius)-mouse model, whether live post-infection trichostrongylid larvae recovered from the intestinal wall of donor animals and placed subcutaneously would serve as vaccine protecting against oral challenge by third-stage (infective) larvae (L3). Experiments were conducted to determine the effect of number and age of post-infective larvae as well as age and sex of host on vaccination. Vaccinated BALB/cByJ mice were challenged with 30 L3 and total adult worm burdens compared between vaccinated groups and sham-treated controls (greater than 90% infection rates). All mice subcutaneously vaccinated with either five or 10 larvae harbored significantly fewer challenge parasites in their intestines than did sham-treated controls (P less than 0.001). Both young and mature mice were significantly protected against challenge by the subcutaneous larval vaccine. Adult female mice had significantly (P less than 0.05) fewer parasites than adult male mice. The age of the larvae (indicated as the days between infection and harvesting of the larvae) was important in that day-4 or day-6 larvae (L4) were significantly more protective (P less than 0.001) than day-2 (L3) or day-8 larvae (L5-preadult). Reduction in worm burden for young vaccinated animals ranged from 31 to 39% (P less than 0.001) and for mature animals from 88 to 100% (P less than 0.001). Passive transfer to serum resulted in the reduction of worm burdens by 26-40% (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A technique for estimating the probability of clots in blood/contrast agent mixtures.

Blood that contaminates the contrast agent syringe during angiography may clot and is thus a potential source of emboli. The older, high-osmolality agents prevent clotting. Newer, low-osmolality agents, while possessing other advantages, are less effective in this regard. The time over which such mixtures do not contain clot can be determined by allowing gross clotting to take place, modeling the process mathematically and determining the clot-free time from the model. Blood-contaminated Omnipaque-300 (Winthrop-Breon, New York, NY) (iohexol), Isovue-300 (E.R. Squibb and Sons, New Brunswick, NJ) (iopamidol), Hexabrix (Mallinckrodt, St. Louis, MO) (ioxaglate sodium meglumine), Renografin-76 (E.R. Squibb and Sons, New Brunswick, NJ) (diatrizoate sodium meglumine), and saline were studied in glass and plastic syringes (80 samples of each agent). After deliberate blood contamination, Renografin and Hexabrix showed no clots during the 90-minute study period. There was, however, a 1.3% chance of clotting in Omnipaque (range 0.4%-4.0%) and a 1.9% chance of clotting in Isovue (range 0.6%-5.5%) at 5 minutes after contamination of these contrast agents in plastic syringes. The chance of clotting in glass containers was significantly greater. We conclude that Hexabrix is a substantially stronger anticoagulant than Omnipaque and Isovue. Furthermore, it appears that this method will allow determination of clotting risks for other combinations of contrast agent and container.

Three new low-osmolality contrast agents: a comparative study of patient discomfort.

Relative patient discomfort resulting from carotid injections of three new low-osmolality contrast agents was assessed in 78 patients. Omnipaque-300 (iohexol), Isovue-300 (iopamidol), and hexabrix (ioxaglate) were sequentially injected into both common carotid arteries of each patient. Patients were asked to rank the relative intensities of the three injections on each side. Mean patient rankings revealed that Hexabrix was preferred most often, Omnipaque-300 next, and Isovue-300 the least. The differences are statistically significant. We conclude that while patients usually tolerated all intracarotid low-osmolality contrast agents rather well, the agent preferred most often was Hexabrix.

Elbow injuries in the throwing athlete. Difficult diagnoses and surgical complications.

Elbow injuries in throwing athletes can be challenging from the diagnostic and management perspectives. The stress of repetitive throwing does predispose athletes to certain conditions with which treating clinicians need to be familiar. An understanding of the anatomy of the elbow and the biomechanics of throwing is essential to making the correct diagnosis and instituting proper care. Failure of nonoperative measures often requires surgical intervention. A thorough understanding of the anatomy and the spectrum of conditions that can occur is needed before decisions regarding surgical management can be made. The operative approach to elbow pathology, whether performed open or arthroscopically, should be completed by orthopedists who have experience with the clinical conditions and the appropriate technical facility to provide comprehensive care. This article has reviewed the anatomy, biomechanics, and spectrum of conditions that affect throwing athletes' elbows as well as the potential complications that can be associated with surgical management.

Thin-layer and liquid column chromatographic analyses of the lipids of adult Onchocerca gibsoni.

Lipids were extracted from adult Onchocerca gibsoni with chloroform/methanol and the total lipid content was characterized. Glycolipids were isolated from other lipid classes by Florisil column chromatography and were then fractionated by DEAE-Sephadex ion-exchange chromatography. HPTLC revealed the presence of 9 neutral glycolipid bands and of 15 acidic glycolipid bands that stained for sialic acid with resorcinol. Lipids that contained no carbohydrates were analyzed by a combination of TLC and amino column chromatography. Triacylglycerols, cholesterol, cholesterol esters, and free fatty acids were found to be major components of the neutral lipid fraction, and diacylglycerols and monoacylglycerols were minor components. Phosphatidylethanolamine and phosphatidylcholine were the predominant phospholipids. Phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine were also present in significant amounts, whereas only traces of cardiolipin and phosphatidic acid were detected. Several minor lipids and phospholipids remained unidentified. These results indicate that adult O. gibsoni have a nonglycosylated lipid composition resembling that of other parasitic nematodes as well as a substantial repertoire of glycolipids, including many with the characteristics of gangliosides.

Percutaneous transluminal renal angioplasty in the patient with a solitary functioning kidney.

Since the introduction of percutaneous transluminal renal angioplasty, it has become established as an effective treatment for the correction of renovascular hypertension or the preservation of renal function. However, there has been a general reluctance to use it or other interventional procedures in any patient with a solitary functioning kidney. We attempted renal angioplasty in 11 patients each of whom had a solitary functioning kidney. All 11 had had either a previous contralateral nephrectomy or an autonephrectomy from chronic arterial occlusion. The patients ranged in age from 42 to 81 years. In nine of the 11 patients, a catheter was negotiated across the stenosis, and the lesion was dilated successfully. In two, the lesion could not be crossed. In the group of nine patients whose lesions were dilated successfully, clinical follow-up (2-180 weeks) revealed that hypertension was cured in four and improved in five. Of the two patients in whom angioplasty was not possible, one required emergency renal bypass surgery and the other had no change in renal status. Renal function test results after the procedure were variable and did not correlate with decreases in blood pressure. We conclude that percutaneous renal angioplasty is a satisfactory alternative to surgery for the treatment of renovascular hypertension or the preservation of renal function in a patient with a solitary functioning kidney.

Globotriaosyl ceramide modulates interferon-alpha-induced growth inhibition and CD19 expression in Burkitt's lymphoma cells.

Previous studies have indicated that globotriaosyl ceramide (Gb3 or CD77) plays a role in alpha-interferon signal transduction and CD19-mediated homotypic adhesion in B cell lines derived from Burkitt's lymphoma. These roles for Gb3 may involve the proteins IFNAR-1 (subunit 1 of the interferon-alpha receptor) and CD19, respectively, both of which have potential Gb3-binding sites in their extracellular domains which resemble those of the verotoxin (Shiga toxin and Shiga-like toxin) B subunit. The majority of this work was performed using wild-type Daudi cells and a single, Gb3-deficient mutant cell line, VT500. In the present investigations, these and additional Daudi-derived cells with varying degrees of sensitivity to interferon-alpha were examined for Gb3 expression, interferon-induced growth inhibition and CD19 expression. The degree of interferon-induced growth inhibition and CD19 expression correlated with Gb3 expression in the various cell lines tested. In addition, reconstitution of the VT500 cell line with Gb3 but not other glycolipids partially restored the sensitivity of cells to IFN-induced growth inhibition. The degree to which reconstitution restored sensitivity to growth inhibition was similar to the results of previous studies in which Gb3 reconstitution restored sensitivity to verotoxin-induced cytotoxicity. These results demonstrate that Gb3 is specifically required for IFN-induced growth inhibition in Daudi cells and provide further evidence of a role for Gb3 in CD19 expression and function in these cells.

Adhesive capsulitis of the wrist: arthrographic diagnosis.

Persistent pain and decreased range of motion are disabling complications of wrist trauma. Between 1978 and 1986, in ten patients with persistent pain following trauma, arthrography depicted changes characteristic of adhesive capsulitis. Adhesive capsulitis has been described in the shoulder, hip, and ankle, but little mention has been made of this entity in other joints. Confirmation of this diagnosis requires arthrography, since there are no characteristic findings on plain radiographs. Typical arthrographic findings include decreased capacity, small volar and styloid recesses, and adhesions preventing complete opacification of the joint. The arthrographic diagnosis allows proper institution of appropriate therapy.

Pulmonary arteriography: comparison of cough stimulation effects of diatrizoate and ioxaglate.

Twenty-five patients were observed in a prospective crossover study to determine whether the new low-osmolality contrast agents would be less prone than conventional agents to produce coughing during pulmonary arteriography. Selective left and right pulmonary arteriography (two views of each side) was performed with alternating administrations of diatrizoate sodium meglumine and ioxaglate sodium meglumine. Twenty-one patients had all four injections while four patients received injections on only one side. Sixteen of 25 patients coughed on at least one injection of diatrizoate, with three of these experiencing explosive coughing. One of 25 patients coughed with ioxaglate, and that was only minimally. This difference is statistically significant (P less than .001, on the basis of McNemar chi 2 test for paired data). When no coughing occurred, the quality of the diatrizoate and ioxaglate radiographs was indistinguishable. We conclude that ioxaglate is useful in pulmonary arteriography because of its lack of cough stimulation.

Adhesive capsulitis of the wrist. Diagnosis and treatment.

Adhesive capsulitis occurs as a pathologic entity in the shoulder, hip, and ankle joints. Thickening and contracture of the wrist joint capsule were demonstrated on arthrograms performed on ten patients ranging in age from 20 to 82 years. The patients had pain and limited range of motion in the wrist. The findings included: (1) resistance to the injection of smaller than normal volumes of contrast material, (2) obliteration of recesses, and (3) extravasation of the contrast agent along the needle tract. Closed manipulation under general anesthesia of the wrist on four patients resulted in some improvement in range of motion. A distal ulna fracture in one older patient occurred as a complication of manipulation. Careful technique and judicious patient selection are of paramount importance.

Autoreactivity in women with endometriosis.

Serum samples from 71 patients with laparoscopically staged endometriosis and from 109 age-matched non-pregnant control women were tested for antibodies to nuclear, phospholipid, smooth muscle and sperm antigens. Immunoglobulin G, M and A and complement components C3 and C4 were measured. Prevalence of the following autoantibodies was statistically significantly greater in women with endometriosis than in the control group: antinuclear antibodies, antibodies to ribonucleoproteins, smooth muscle antibodies, lupus anticoagulant and anticardiolipin antibody. Forty-one women with endometriosis (58%) had some type of autoantibody compared with 13 controls (12%). More women in the endometriosis group had IgG and IgM levels above the normal range. This was statistically significant for IgG in women with grade 3 and for IgM in those with grade 4 endometriosis, IgG levels were significantly higher in antibody-positive women with endometriosis than in the control group. Immunoglobulin and complement component levels tended to have a positive correlation with stage of disease.