pH-regulated secretion of a glyceraldehyde-3-phosphate dehydrogenase from Streptococcus gordonii FSS2: purification, characterization, and cloning of the gene encoding this enzyme.

Streptococcus gordonii and other viridans streptococci (VS) are primary etiologic agents of infective endocarditis, despite being part of the normal oral microflora. Recently, a surface-bound glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found on the cells of all tested streptococcal species, where it has been implicated as a virulence factor. In contrast, we observed that a soluble extracellular GAPDH was the major secreted protein from S. gordonii FSS2, an endocarditis strain. The biochemical properties and gene sequence of S. gordonii GAPDH are almost identical to those of other streptococcal GAPDHs. Growth at defined pHs showed that secretion of GAPDH is regulated by environmental pH. GAPDH was primarily surface-associated at growth pH 6.5 and shifted to > 90% secreted at growth pH 7.5. Others have identified S. gordonii promoters that are up-regulated by a pH shift similar to that experienced by organisms entering the blood stream (neutral) from the oral cavity (slightly acid). Analysis of our results suggests that secretion of GAPDH may be a similar adaptation by S. gordonii.

Treatment of pressure ulcers in a rehabilitation ward.

This article presents a case study of a patient transferred to a rehabilitation unit for elderly people. The aim of the article is to show that, with a dynamic and committed multidisciplinary team approach, the prevention and treatment of difficult pressure ulcers can be achieved in an ever increasingly frail elderly population. The article will discuss the methods used to treat and tackle the challenges that staff encounter when treating these patients.

Glatiramer acetate (Copaxone) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis.

We examined the effect of glatiramer acetate, a random copolymer of alanine, lysine, glutamic acid, and tyrosine, on antigen-specific T-cell responses in patients with multiple sclerosis (MS). Glatiramer acetate (Copaxone) functioned as a universal antigen, inducing proliferation, independent of any prior exposure to the polymer, in T-cell lines prepared from MS or healthy subjects. However, for most patients, daily injections of glatiramer acetate abolished this T-cell response and promoted the secretion of IL-5 and IL-13, which are characteristic of Th2 cells. The surviving glatiramer acetate-reactive T cells exhibited a greater degree of degeneracy as measured by cross-reactive responses to combinatorial peptide libraries. Thus, it appears that, in some individuals, in vivo administration of glatiramer acetate induces highly cross-reactive T cells that secrete Th2 cytokines. To our knowledge, glatiramer acetate is the first agent that suppresses human autoimmune disease and alters immune function by engaging the T-cell receptor. This compound may be useful in a variety of autoimmune disorders in which immune deviation to a Th2 type of response is desirable.

The effect of palmitic acid on lipoprotein cholesterol levels.

The present study assessed the effect of high versus low palmitic acid intakes of plasma lipoprotein cholesterol levels and on rates for endogenous synthesis of cholesterol in normal and hypercholesterolemic subjects. On day 21 of each diet treatment, a fasting blood sample was drawn for lipoprotein determination and to provide a measure of the background level of deuterium. A priming dose of deuterium was consumed and a second blood sample obtained 24 hours after the first sample. Isotope ratio mass spectrometry was used to determine the incorporation of deuterium into the newly synthesized cholesterol molecule and fractional synthetic rates were calculated. Four diets were formulated to provide combinations of two levels of 16:0 at two levels of 18:2n-6. Subjects received each of the four diet treatments for 21 days, followed by washout periods of 21 days. Serum total cholesterol and LDL-cholesterol was not significantly affected by the high level of 16:0 when diets also contained a high level of 18:2n-6. Fractional synthesis rates of cholesterol observed for each diet treatment did not differ significantly, suggesting no relationship between the endogenous synthesis of cholesterol and dietary 16:0 content. The results indicate that 16:0 has no effect on serum lipoprotein profiles in the presence of recommended intakes for 18:2n-6.

The effect of palmitic acid on lipoprotein cholesterol levels and endogenous cholesterol synthesis in hyperlipidemic subjects.

The present study assesses the effect of high vs. low palmitic acid intakes on plasma lipoprotein cholesterol levels and on rates for endogenous synthesis of cholesterol in healthy and hyperlipidemic subjects. Four diets were formulated to provide combinations of 16:0 at two levels of 18:2n-6. Subjects received each diet treatment for 21 d, followed by washout periods of 21 d. On day 21 of each diet treatment, a fasting blood sample was drawn for lipoprotein determination and to provide a measure of the background level of deuterium. A priming dose of deuterium was consumed and a second blood sample obtained 24 h after the first sample. Isotope ratio mass spectrometry was used to determine the incorporation of deuterium into the newly synthesized cholesterol molecule, and fractional synthetic rates were calculated. Serum total cholesterol and low density lipoprotein-cholesterol was not significantly affected by the high level of 16:0 when diets also contained a high level of 18:2n-6. There was no effect of dietary 16:0 on high density liproprotein-cholesterol at either the high or low levels of intake. The results indicate that 16:0 has no effect on serum lipoprotein profiles in the presence of recommended intakes for 18:2n-6.

Use of deuterium oxide to measure de novo fatty acid synthesis in normal subjects consuming different dietary fatty acid composition1.

The effect of dietary linoleic (C18:2n-6) and palmitic acids (C16:0) on rate of hepatic de novo fatty acid synthesis was assessed in normal subjects. The diet was formulated to provide combinations of high and low levels of C18:2n-6 and C16:0. After 21 days of diet treatment, plasma triacylglycerol level and incorporation of deuterium into the plasma very low density lipoprotein triacylglycerol (VLDL-TG) pool over 24 hours was measured. Plasma triacylglycerol levels were within the normal range. Increasing dietary intake of linoleic acid decreased plasma triacylglycerol level when subjects consumed a low level of dietary palmitic acid. The relative and net amount of de novo synthesized fatty acid in the plasma VLDL-TG pool was not influenced by the diet treatments. A relationship between plasma triacylglycerol level and rate of hepatic de novo fatty acid synthesis was observed.

Palmitic acid effect on lipoprotein profiles and endogenous cholesterol synthesis or clearance in humans.

The effect of palmitic acid (C16:0) on serum lipoprotein cholesterol levels is debatable. If C16:0 is hypercholesterolaemic, then it may increase the endogenous synthesis or decrease clearance of cholesterol. Four diets were formulated to provide combinations of two levels of C16:0 in relation to two levels of PUFA. Healthy male subjects received each of the four diet treatments for 21 days, followed by washout periods of 21 days. On day 21 of each diet treatment, a fasting blood sample was drawn for lipoprotein determination and to provide a measure of the background level of deuterium. A priming dose of deuterium was consumed and a second blood sample obtained 24 hours after the first sample. Isotope Ratio Mass Spectrometry was used to determine the incorporation of deuterium into the newly synthesised cholesterol molecule, and fractional synthetic rates calculated. Serum total cholesterol and LDL-cholesterol was not significantly affected by the high level of C16:0 when diets also contained the high level of PUFA. There was no effect of C16:0 on HDL-cholesterol at either the high or low levels of intake. The fractional synthetic rates of cholesterol observed for each of the diet treatments did not significantly differ from one another, suggesting no relationship between the endogenous synthesis of cholesterol and diet C16:0 content. These results indicate that C16:0 had no effect on serum lipoprotein profiles in the presence of recommended intakes for PUFA, nor did it increase rates of cholesterol synthesis in healthy males.

Digitalis-like factor and digoxin-like immunoreactive factor in diabetic women with preeclampsia, transient hypertension of pregnancy, and normotensive pregnancy.

An endogenous sodium pump inhibitor, or digitalis-like factor (DLF), has been postulated to mediate essential hypertension. It may also play a role in preeclampsia. However, studies of this factor in hypertensive pregnancy have not provided consistent findings. Part of this may be due to the absence of subclassification of pregnant women with pregnancy-induced hypertension (PIH) when assessing these parameters. In this study we explored serum DLF and digoxin-like immunoreactive factor (DLIF) in insulin-dependent diabetic (IDDM) women with normotensive pregnancies or PIH, comparing them to each other and to nondiabetic pregnant women. Our results demonstrated that nondiabetic women with preeclampsia (PE, PIH with proteinuria) had significantly increased serum DLF and DLIF compared to normotensive pregnant women (NL BP). Women with transient hypertension of pregnancy (THP, PIH without proteinuria) had intermediate values (DLF. NL BP: 3.3 +/- 0.6, THP: 4.8 +/- 1.1, PE: 7.6 +/- 1.3% inhibition [Na,K]-ATPase, P < .05 ANOVA; DLIF. NL BP: 0.22 +/- 0.02, THP: 0.28 +/- 0.03, PE: 0.35 +/- 0.02 ng digoxin equivalents/mL, P < .05 ANOVA). Pregnant normotensive IDDM women had significantly higher serum DLF and DLIF activity than their nondiabetic counterparts (DLF. non-IDDM NL BP: 3.3 +/- 0.6 v IDDM NL BP: 8.8 +/- 1.2% inhibition [Na,K]-ATPase, P = .0008; DLIF. non-IDDM NL BP: 0.22 +/- 0.02 v IDDM NL BP: 0.31 +/- 0.02 ng digoxin equivalents/mL, P = .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Percutaneous excimer laser coronary angioplasty of lesions not ideal for balloon angioplasty.

BACKGROUND: Excimer laser coronary angioplasty is a new, investigational technique for treating coronary artery stenoses. Initial reports have demonstrated acute efficacy and relative safety of this procedure, but have not addressed the effect of lesion type on acute success and complication rates. METHODS AND RESULTS: In the first 100 patients undergoing percutaneous excimer laser coronary angioplasty at our institution, acute laser success was obtained in 84% and procedural success was obtained in 94%. There were six acute closures during laser angioplasty and one myocardial infarction. Two patients required emergency coronary bypass surgery. Sixty-five percent of patients had lesions not ideal for balloon angioplasty because of lesion morphology (tubular, diffuse, or chronic total occlusion) or ostial location. There were 10 tubular stenoses, 29 diffuse lesions, 18 chronic total occlusions, and eight ostial lesions, including five aorto-ostial lesions. In this nonideal subgroup, the acute success rate with laser was 86% (72% of chronic total occlusions and 91% of non-totally occluded lesions), and the procedural success rate was 94%. There were three acute occlusions during laser angioplasty but no myocardial infarctions, emergency bypass surgeries, or deaths. One coronary artery perforation occurred without clinical sequelae. Laser angioplasty was successful in four of six lesions (67%) in which balloon angioplasty had failed. Laser success was obtained in 10 of 11 (91%) moderately or heavily calcified stenoses. Eight eccentric lesions and two lesions on bends were successfully treated without dissection or perforation. No side branch occlusions occurred in the 15 patients in whom one or more major branches originated within the lesion treated. Adjunctive balloon angioplasty was performed in 47% of cases, usually to obtain a larger final luminal diameter. Need for adjunctive balloon angioplasty decreased to 36% after a larger (2.0 mm) laser catheter became available. Twenty-eight percent of the 105 lesions treated were American College of Cardiology/American Heart Association classification type A, 47% were type B, and 25% were type C. Laser and procedural successes were obtained in 83% and 97% of type A, 88% and 96% of type B, and 85% and 88% of type C lesions, respectively. CONCLUSIONS: In our initial experience, excimer laser angioplasty was found to be acutely effective and safe therapy for lesions identified as not ideal for balloon angioplasty. This technique may provide a useful adjunct or alternative to balloon angioplasty in selected patients.

Prevalence of unsuspected mitral regurgitation and left ventricular diastolic dysfunction in patients with coronary artery disease and acute pulmonary edema associated with normal or depressed left ventricular systolic function.

To define the prevalence and role of left ventricular (LV) systolic dysfunction, LV diastolic dysfunction and mitral regurgitation (MR) in patients with acute pulmonary edema, 40 patients with coronary artery disease and acute pulmonary edema were prospectively evaluated within 36 hours of presentation. LV ejection fraction and 3 parameters of LV diastolic function were measured with radionuclide ventriculography, whereas MR was assessed with Doppler echocardiography. LV ejection fraction was normal in 11 (27%) and depressed in 29 (73%) patients. Moderate or severe MR without LV diastolic dysfunction was common and equally prevalent in patients with and without LV systolic dysfunction (33 vs 38%; difference not significant). Diastolic dysfunction without MR was less frequent but equally prevalent in patients with and without systolic dysfunction (17 vs 27%; difference not significant). Two (18%) of 11 patients without and 12 (33%) of 36 patients with LV systolic dysfunction had both MR and LV diastolic dysfunction. Furthermore, MR was clinically silent and unsuspected in two-thirds of all patients with MR, regardless of a normal or depressed systolic function. These data show that there is a high prevalence of unrecognized moderate to severe MR in patients with acute pulmonary edema, regardless of the presence or absence of LV systolic dysfunction. Furthermore, the prevalence of LV diastolic dysfunction without MR is relatively low even in patients with normal LV systolic function and pulmonary edema. Thus, unrecognized MR may be an important contributor to the syndrome of acute pulmonary edema in patients with normal or depressed LV systolic function.

Percutaneous excimer laser coronary angioplasty.

To determine the efficacy of percutaneous excimer laser coronary angioplasty as an adjunct or alternative to conventional balloon angioplasty, 55 patients were studied in a multicenter trial. These patients underwent the procedure using a modification of conventional balloon angioplasty technique. A first-generation, 1.6-mm diameter catheter constructed of 12 individual silica fibers concentrically arranged around a guidewire lumen was used. Catheter tip energy density varied from 35 to 50 mJ/mm2. The mean number of pulses delivered at 20 Hz was 1,272 +/- 1,345. Acute success was defined as a greater than or equal to 20% increase in stenotic diameter and a lumen of greater than or equal to 1 mm in diameter after laser treatment. Acute success was achieved in 46 of 55 (84%) patients. Adjunctive balloon angioplasty was performed on 41 patients (75%). The percent diameter stenosis as determined by quantitative angiography decreased from a baseline of 83 +/- 14 to 49 +/- 11% after laser treatment and to 38 +/- 12% in patients undergoing adjunctive balloon angioplasty. The mean minimal stenotic diameter increased from a baseline of 0.5 +/- 0.4 to 1.6 +/- 0.5 mm after laser treatment and to 2.1 +/- 0.5 mm after balloon angioplasty. There were no deaths and no vascular perforations. One patient (1.8%) required emergency coronary bypass surgery. These data suggest that excimer laser energy delivered percutaneously by specially constructed catheters can safely ablate atheroma and reduce coronary stenoses.

Sensitization to epithelial antigens in chronic mucosal inflammatory disease. I. Purification, characterization, and immune reactivity of murine epithelial cell-associated components (ECAC).

To explore the autoantigenic potential of mucosal epithelium that is the site of several chronic, progressive, inflammatory and yet idiopathic diseases, epithelial cell-associated components (designated ECAC) have been isolated in aqueous-soluble form from everted, inflated loops of murine small intestine, initially characterized biochemically and immunologically, and then purified to homogeneity and studied for a disease association through quantitating, by microcytotoxicity assay, their reactivity with patient mononuclear cells and sera. Hydroxyapatite chromatography successfully separated ECAC into several series of major and minor components, with each series (designated 1, 2A, 2B, and 2C) having a unique chemical profile in terms of total carbohydrate, protein, and specific sugars (sialic acid, fucose, galactose). Furthermore, components within each series were shown not to behave as simple blood group substances antigenically, to be free of contaminating intestinal proteases (less than 0.25 micrograms chymotrypsin or equivalent per milliliter) and to possess shared as well as unique antigenic determinants (1 through 4), all of which appeared to be organ-specific for intestine. Gel filtration on Sephacryl S-200 gave a four-peak elution profile for ECAC consistent with a m.w. for components of 3000 to 232,000. Preparative polyacrylamide gel electrophoresis did isolate individual constituents of ECAC, four of which (P1, P2, P4, and P5) were homogeneous and that could, by a hemagglutination inhibition technique, be shown to possess unique organ-specific antigenic determinants. ECAC-specific reactivity of peripheral blood mononuclear cells and sera from patients in the active phase of a chronic inflammatory disorder involving mucosal epithelium indicated autosensitization had occurred, with involvement of several purified epithelium-derived macromolecules. This reactivity appeared to be antibody dependent, occurred at relatively low effector to target ratios, and was not simultaneously directed to control antigens, isolated from kidney in a manner analogous to that used for ECAC. ECAC, isolated by these techniques, may be sufficiently purified to allow an evaluation of their role in the initiation and/or progression of chronic autoaggressive processes occurring in mucosa.

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells.

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.

Goblet cell glycoprotein: an organ-specific antigen for gut. Isolation, tissue localization and immune response.

We report, for the first time, immune responses within two lines of inbred rats to a purified Lewis rat glycoprotein antigen which is organ-specific for intestine. The antigen was prepared by solubilization of gut epithelial cell-associated macromolecules, fractionation in ethanol, and molecular sieve chromatography over Sepharose 2B. Homogeneity of the end product (RGCG-PK1) was supported by results of both double diffusion in agar and SDS polyacrylamide gel electrophoresis. Amino acid analysis and specific sugar determination proved that RGCG-PK1 was not a classical mucin because of its comparatively high tyrosine and low galactosamine + glucosamine content, and the absence of glycosidic linkages to serine and threonine. Organ-specificity was shown by the ability of RGCG (but not liver homogenate) to inhibit precipitation and haemagglutination by heterologous specific sera. Organ-specificity was confirmed by the demonstration of RGCG-PK1-specific immunofluorescence staining of rat small and large intestine, but not esophagus, stomach or liver. RGCG-PK1 determinants within rat and human small bowel were found to be confined to goblet cells and intestinal glycocalyx. Anti-RCGC-PK1 serum showed no reactivity with highly purified xenogeneic mucins nor with syngeneic small bowel mucin. Specific antibody (as well as antibody-dependent cellular cytotoxicity) to RGCG was elicited and detected for up to 10 days in two lines of inbred rats, including the one (Lewis) from which the antigen was isolated. The duration and peak of the humoral immune response were abbreviated compared with that of a xenogeneic control glycoprotein studied in parallel, probably due to immunoregulatory mechanisms operative for self antigens.