Enhancing analytical accuracy of intravascular electrochemical oxygen sensors via nitric oxide release using S-nitroso-N-acetyl-penicillamine (SNAP) impregnated catheter tubing.

Implantable medical devices are an integral part of primary/critical care. However, these devices carry a high risk for blood clots, caused by platelet aggregation on a foreign body surface. This study focuses on the development of a simplified approach to create nitric oxide (NO) releasing intravascular electrochemical oxygen (O2) sensors with increased biocompatibility and analytical accuracy. The implantable sensors are prepared by embedding S-nitroso-N-acetylpenacillamine (SNAP) as the NO donor molecule in the walls of the catheter type sensors. The SNAP-impregnated catheters were prepared by swelling silicone rubber tubing in a tetrahydrofuran solution containing SNAP. Control and SNAP-impregnated catheters were used to fabricate the Clark-style amperometric PO2 sensors. The SNAP-impregnated sensors release NO under physiological conditions for 18 d as measured by chemiluminescence. The analytical response of the SNAP-impregnated sensors was evaluated in vitro and in vivo. Rabbit and swine models (with sensors placed in both veins and arteries) were used to evaluate the effects on thrombus formation and analytical in vivo PO2 sensing performance. The SNAP-impregnated PO2 sensors were found to more accurately measure PO2 levels in blood continuously (over 7 and 20 h animal experiments) with significantly reduced thrombus formation (as compared to controls) on their surfaces.

Soft tissue and visceral sarcomas in Irish patients. Interim analysis of data obtained by the Dublin Soft Tissue Tumour Panel.

The Dublin Soft Tissue Sarcoma Panel was established in 1989 with a view to achieving a unified approach to diagnosis and management of soft tissue and visceral sarcomas. This interim report presents data on 265 prospectively-evaluated patients and on a separate retrospective series of 126 patients. The patients in the prospective series were treated by 93 different surgical and medical specialists. Tumours presented in all anatomic sites and ranged in size from 0.2 to 60 cm. Leiomyosarcoma was the commonest tumour type. Eighty-nine tumours were inoperable at clinical presentation. There was a consensus panel diagnosis in over 90%, non-neoplastic reactive lesions and primitive round cell tumours being the most difficult cases diagnostically. Management, including onward referral for chemotherapy or radiation therapy, was inconsistent. The 2-year survival figures were: 43% (1989-91) and 37% (1980-88). These findings should provide a basis for the evaluation of coherent treatment strategies for Irish sarcoma patients.

The Loyola University lung transplant experience.

BACKGROUND: We reviewed our experience with isolated lung transplantation at Loyola University Medical Center, Maywood, Ill. From April 1990 through June 1992, 33 lung transplantations for end-stage pulmonary disease were performed (30 single lung, three bilateral single lung). Recipient diagnoses include chronic obstructive pulmonary disease, alpha 1-antiprotease deficiency, pulmonary fibrosis, primary pulmonary hypertension, Eisenmenger's syndrome, sarcoidosis, cystic fibrosis, bronchiectasis, and bronchiolitis obliterans. METHODS: For patients who underwent transplantation for end-stage obstructive airway disease, we retrospectively reviewed functional indexes before and after transplantation. In addition, the overall survival rate was determined. RESULTS: Successful transplantation resulted in a marked improvement in functional capacity. Single-lung transplantation for end-stage obstructive airway disease resulted in a threefold improvement in the 1-second forced expiratory volume, from 0.49 to 1.64 L. The actual survival for all isolated lung transplant recipients (including both single-lung and bilateral single-lung procedures) was 73%, with a 15% 30-day mortality. CONCLUSION: Isolated lung transplantation can significantly improve functional capacity as well as the quality of life in patients with end-stage lung disease.

Soft tissue and visceral sarcomas in Irish patients. A preliminary report from the Dublin Soft Tissue Tumour Panel.

Sarcomas of the soft tissues and viscera are a diverse group of uncommon neoplasms that often present difficult diagnostic and management problems. The Dublin Soft Tissue Tumour Panel prospectively reviewed pathology material from 137 patients of four Dublin teaching hospitals between January 1989 and August 1990. The prevalence of sarcomas in Irish patients was found to be similar to that estimated for the United States. The most common sarcoma of soft tissues was malignant fibrous histiocytoma (14) and the most common visceral sarcoma was leiomyosarcoma (10). The most problematic diagnoses were 3 peripheral neuroepitheliomas (extraskeletal Ewing's sarcomas) which occurred in patients aged 19-27 years, distinctly younger than the typical adult sarcoma patient. Clinical follow-up is in progress and will be combined with the findings of a linked retrospective study, to facilitate evaluation of all aspects of management.

Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor.

The anterior chamber of the eye is an immunologically privileged site. Over the past 15 years, numerous laboratories documented that the privileged status of this unique site is mediated by multifactorial immunoregulatory processes. Among the participating factors is the aqueous humor that circulates in the anterior chamber and is in contact with most of the tissues in the anterior segment of the eye. Recently, it was found that normal aqueous humor is a powerful inhibitor of antigen-driven T lymphocyte activation, but it spares other important functional properties of T cells. The spectrum of immune inhibitory properties resembles some of the activities of transforming growth factor-beta (TGF-beta), a polypeptide cytokine. Because of this similarity, the authors tried to determine if TGF-beta is present in aqueous humor and whether this cytokine could account for the lymphocyte inhibitory activity of this biologic fluid. They found TGF-beta in aqueous humor by dot-blot analysis. Using the CCL-64 mink lung epithelial cell bioassay for this compound, TGF-beta bioactivity was shown in aqueous humor from several different species, including human. In rabbit and human aqueous humor, most of the biologic activity was due to TGF-beta 2 (80-90%). Dose-response curves generated by using purified porcine TGF-beta showed that aqueous humor contained sufficient concentrations of TGF-beta to account for the observed inhibition in several assays for T cell activation and proliferation. Partial purification of the lymphocyte inhibitor in rabbit aqueous humor by size exclusion in high-performance liquid chromatography demonstrated that several lymphocyte inhibitory fractions contained TGF-beta bioactivity. Finally, neutralizing antisera to TGF-beta 2 were able to reverse most of the lymphocyte inhibitory activity of aqueous humor. It was concluded that TGF-beta was present in high concentration in normal aqueous humor and that this cytokine contributed to the immunosuppressive properties of aqueous humor.

High avidity monoclonal antibody to imidazole ring-opened 7-methylguanine.

A hybridoma (K1A8) secreting a high affinity antibody to imidazole ring-opened 7-methylguanine (N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine) was obtained from spleen cells of a mouse immunized with a conjugate of keyhole-limpet hemocyanin and imidazole ring-opened 7-methylguanylic acid (iro-7mGMP). The antibody recognizes the iro-7-methylguanine (iro-7mG) determinant in the BSA-iro-7mGMP conjugate, in chemically methylated, denatured DNA, and in the ring-opened 7-methylguanosine, 7-methyldeoxyguanosine and 7mGMP haptens. In the competitive ELISA of DNA-iro-7mG, 50% inhibition (I50) was observed at 4 fmol determinant per well (8 x 10(-11) M) using BSA-iro-7mGMP as the immobilized antigen. The lower limit of 7-methylguanine (7mG) detection in DNA is determined by the binding of unmodified DNA per se to the antibody. The intrinsic reaction of DNA with antibody is low; in the competitive ELISA I50 was obtained with 330 micrograms calf thymus DNA per 50 microliters well, equivalent to 4 nmol iro-7mG per mol nucleotide. The 7mG content of calf thymus DNA is 7 nmol per mol nucleotide (approximately 20 amol per micrograms DNA). The limit of detection of 7mG by competitive ELISA is quoted provisionally as 7 nmol iro-7mG per mol nucleotide, where 30% inhibition of antibody binding is obtained in the presence of 105 micrograms DNA per 50 microliters well. Nuclear DNAs of tissue culture cells treated with 0, 0.01 and 0.1 mM N-methyl-N'-nitro-N-nitrosoguanidine contained 0.18, 31 and 320 mumol, respectively, of 7-methylguanine adducts per mol of nucleotides. This report indicates that the K1A8 antibody will serve to quantify DNA alkylation in human populations exposed to low levels of methylating carcinogens.

Pulmonary alveolar proteinosis: primary and secondary, a report of three cases.

Three cases of the rare disorder pulmonary alveolar proteinosis (PAP) presented to our unit over the past thirteen years. Two of the patients conformed to the classical description of idiopathic or "primary" PAP. One patient appeared to have co-existing extrinsic allergic alveolitis with "secondary" PAP, an association not previously described. This patient has required continued steroid therapy, a mode of treatment usually contraindicated in PAP.

Characterization by affinity electrophoresis of an alpha-1,6-glucan-binding protein from Streptococcus sobrinus.

Glucan-binding protein 1 (GBP1), the most abundant glucan-binding protein isolated from culture supernatants of Streptococcus sobrinus 6715-49, has been purified by affinity chromatography on Sephadex G-50 followed by gel permeation chromatography with Bio-Gel P-10. The specificity and affinity of GBP1 for glucans were assessed by affinity electrophoresis. GBP1 did not detectably bind to glucans lacking linear arrays of alpha-1,6 linkages. The association constant for the linear alpha-1,6-glucan Dextran T2000 was 3 x 10(7) M-1. Providing small isomaltosaccharide ligands to compete with this dextran indicated that the binding site maximally accommodated isomaltosaccharides with a degree of polymerization of 8. When glucans produced by purified S. sobrinus glucosyltransferases were tested, GBP1 displayed the highest affinity for the glucan from the soluble-product, primer-independent glucosyltransferase.

Monoclonal antibodies to the extracellular glucosyltransferases from Streptococcus sobrinus 6715.

Murine monoclonal antibodies (MAbs) were raised against the glucosyltransferases (GTFs) of Streptococcus sobrinus 6715. The antibody panels included MAbs raised against the primer-independent, soluble product enzyme (GTF-Si) which did not cross-react with other GTFs, as well as MAbs raised against the primer-dependent, soluble product enzyme (GTF-Sd) which recognized both GTF-Si and GTF-Sd, thus indicating that these catalytically distinct enzymes share epitopes. MAbs raised against GTF-I recognized several forms of GTF-I and did not cross-react with the GTF-S enzymes. None of the MAbs recognized the major glucan-binding protein of S. sobrinus. Two MAbs inhibited glucan synthesis, one blocking primer synthesis by GTF-Si by 89% and the second inhibiting that by GTF-I by 92%.

Epithelial hyperplasia and malignant change in congenital lung cysts.

Patients with congenital lung cysts are at increased risk of developing carcinoma, but the mechanisms concerned are not clear. The case of a young adult who developed a bronchioloalveolar carcinoma associated with a cystic congenital adenomatoid malformation is reported. The adjacent lung showed an unusual intra-alveolar hyperplasia of mucous cells. Two further cases of congenital adenomatoid malformation are also described; both patients presented in infancy and showed similar mucous cell hyperplasia in alveoli surrounding the cysts. In all three cases the staining characteristic of the mucus was identical with that of normal bronchial mucous glands. It is suggested that the benign proliferation represents a premalignant lesion. Its presence in infants shows that it may occur at an early age and reinforces the need for early removal of congenital lung cysts.

Purification and characterization of a primer-independent glucosyltransferase from Streptococcus mutans 6715-13 mutant 27.

Affinity chromatography on Sephadex G-50 and subsequent ion-exchange chromatography on Trisacryl-M-DEAE were used to purify the glucosyltransferase (GTF) enzymes produced by mutant 27 of Streptococcus mutans 6715-13. Complete separation of three types of GTF, including a primer-independent GTF capable of synthesizing a slightly branched, water-soluble glucan (GTF-S), was obtained. The characteristics of this primer-independent GTF-S were compared with those of the normally occurring primer-dependent GTF-S. The Km for sucrose was easily obtained for each enzyme (10(-2) M), but the Km for dextran could only be determined for the primer-dependent GTF-S (5 X 10(-7) M for clinical dextran of molecular weight 60,000 to 90,000). The primer-independent GTF-S did not respond catalytically to the presence of either clinical dextran or the highly branched, water-soluble glucan produced by primer-dependent GTF-S, although it was capable of binding these polysaccharides at a noncatalytic site and of responding to the low-molecular-weight acceptor 1-O-methyl-alpha-D-glucopyranoside. The water-soluble glucan product of primer-independent GTF-S was a superior priming glucan for primer-dependent GTF enzymes as compared with the glucan product of primer-dependent GTF-S. The presence of primer-independent GTF-S in reaction mixtures stimulated glucan synthesis by primer-dependent GTF-S and by GTF synthesizing water-insoluble glucan by at least 10-fold, whereas the presence of similar amounts of primer-dependent GTF-S had no effect on synthesis by GTF synthesizing water-insoluble glucan. Primer-independent GTF-S appears to be a potent source of priming glucan for the primer-dependent GTF enzymes. Its possession of a noncatalytic binding site for glucan, the first observed for the GTF of S. mutans, suggests that it may also serve as a glucan receptor on the S. mutans cell surface.

Renal glomerulopathies associated with Hodgkin's disease.

From a series of 138 patients with Hodgkin's disease seen over a 17-year period, 2 developed the nephrotic syndrome. One patient developed hematuria unrelated to cytotoxic chemotherapy or thrombocytopenia. All three patients had evidence of glomerulopathy on histologic examination of renal tissue, but none had the more usually associated minimal-change glomerular histologic features. The patients with nephrotic syndrome had effective clinical regression with systemic chemotherapy. The significance of renal glomerular disease in association with Hodgkin's disease is discussed.

An enzyme from Streptococcus mutans forms branches on dextran in the absence of sucrose.

An enzyme in glucosyltransferase preparations from Streptococcus mutans catalyzed the transfer of [14C]glucopyranoside from purified isomaltosaccharides, each containing [14C]glucopyranoside at its non-reducing terminus, to acceptor dextran, in the absence of sucrose. Half of the radioactivity present in the resulting [14C]dextrans was resistant to hydrolysis by amylo-1,6-glucosidase. Treatment of the [14C]dextrans with endodextranase resulted in extensive hydrolysis and produced [14C]-labeled limit oligosaccharides containing branch sites. Acetolysis of the [14C]-labeled limit oligosaccharides yielded [14C]nigerose, thus indicating the formation of branch sites on dextran in the absence of sucrose. The enzyme catalyzing this reaction has not been identified but appears to be independent of the major extracellular glucosyltransferases of S. mutans.

The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R.

Soluble dextransucrase activity(ies) was eluted with a solution of clinical dextran from the insoluble dextran--cell complex produced by Streptococcus mutans K1-R grown in the presence of sucrose. Studies of the dextran acceptor-reaction of the soluble enzyme-preparation indicate that it is highly specific for dextran of high molecular weight. Increased dextran synthesis in the presence of dextran acceptor and the apparent inhibition of this stimulation by higher concentrations of dextran result from product modification rather than a direct effect on the level of enzyme activity. The results demonstrate that the potentially water-insoluble structure synthesized by dextransucrase on exogenous, soluble dextran acts as a more-efficient acceptor than the soluble dextran. The role of the acceptor reaction in the biosynthesis of complex dextrans is discussed.

Multiple forms of dextran-binding proteins from Streptococcus mutans.

We have isolated a series of five proteins which appear to possess characteristic individual capacities for synthesizing dextrans and binding dextrans. Our suggestion that these proteins comprise an isozyme-like distribution of lectin and enzyme activities is, of course, very speculative and remains to be rigourously confirmed. However, the very identification of these several dextran binding proteins provides a biochemical basis to explain numerous observations suggesting that more than one mechanism for dextran binding is possessed by S. mutans (for instance: 24-27), especially the observations with mutants (24). These proteins probably are the molecular determinants of host infection by S. mutans and may prove to be potent immunogens for use in a vaccine. The presence of a dextran-binding lectin in S. mutans implicates this bacterial lectin in the earliest stage of infection: Attachment to host tissues. The multiplicity of proteins possessing characteristic dextran-synthesizing and dextran-binding capacities indicates the complexity of the adherence mechanisms evolved in S. mutans. Experiments with other bacteria (10-12, 28) suggest that bacterial lectins, in concert with host tissue carbohydrates, may be the molecular mediators of host recognition and subsequent initial attachment of bacterial cells to host tissues in non-pathogenic as well as pathogenic bacteria.

Specific method for the purification of Streptococcus mutans dextransucrase.

A convenient and rapid method for the purification of Streptococcus mutans dextransucrase is described. Affinity chromatography, on a column containing insoluble dextran purified from a culture of S. mutans 6715-49, gave an almost 300-fold purification, with 76% recovery of enzyme. Subsequent hydrophobic chromatography on butyl-agarose increased the overall enzyme purification to more than 1,000-fold, with a 65% recovery of activity. Two components of the dextransucrase activity were separated during hydrophobic chromatography. Both synthesized insoluble glucan as their major product and were capable of synthesizing soluble glucan in the presence of exogenous soluble dextran. However, the major enzyme component, which coeluted with a catalytically inert, dextran-binding protein, was greatly stimulated by exogenous soluble dextran, whereas the second enzyme component was not.