Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats.

Graft-versus-host disease (GvHD), a major complication of allogeneic bone marrow transplantation, has been ascribed to mature T cells in the graft. Because T cells play an important role in engraftment of the bone marrow and decrease the probability of relapse of leukemia, a treatment strategy was developed to preserve the benefits of T cells in the graft and to control the severe complications of GvHD. This can be accomplished by the genetic modification of donor T cells with a suicide gene that allows their selective in vivo elimination and subsequently the abrogation of GvHD. For clinical benefit the alloreactivity of herpes simplex virus thymidine kinase (HSV-TK) gene-transduced T cells should be retained. Therefore, we investigated the influence of gene transduction and the selection procedure on T cells. We demonstrated that activation and culturing of T cells reduce their capacity to induce lethal GvHD in an allogeneic rat bone marrow transplantation model. Furthermore, positive immunomagnetic selection of gene-transduced T cells resulted in loss of the GvHD-inducing capacity of HSV-TK(+) T cells directly after MACS (magnetic cell sorting) selection; this loss could be recovered by a 1-day expansion of the selected T cells. No effect on alloreactivity was observed to be caused by the gene transduction procedure. Our study resulted in the development of an optimized culture and gene transduction protocol with preservation of T cell alloreactivity. Treatment of transplanted rats with ganciclovir resulted in a rapid reduction in the number of HSV-TK(+) T cells in the peripheral blood and in increased survival of the animals.     

Effect of ionenes on structure and catalytic activity of cobaltphthalocyanine, 1. Visible light spectroscopic investigations

The structural behaviour of cobalt(II)phthalocyanine-tetrasodium sulfonate (CoPc(NaSO₃)₄) ( 1 ), a catalyst used in thiol oxidation, was investigated in the presence of ionenes (poly-quaternary ammonium salts), ( 2 ) using visible light spectroscopy. It was observed that ionenes, which have been shown to promote the catalytic activity, strongly enhance the aggregation of the cobalt complex. No cobalt site isolation occurred up to N⁺/Co = 10⁵. Furthermore, a stoichiometric CoPc(NaSO₃)₄/ionene complex appeared to be formed with an N⁺/Co-ratio of 4:1. This ratio was found to hold for ionenes with M?_n varying from 6 100 to 22 000 and charge densities from 0,8 to 1,5. As the charge density was increased, the observed CoPc(NaSO₃)₄ spectrum was more affected. Experiments with oxygen in alkaline solution revealed that the complex was resistant toward oxygen adduct formation in the presence of ionenes. This phenomenon is discussed in relation to the catalytic activity of the system.     

Effects of isometric exercise on blood cell adrenoceptors in essential hypertension

The effect of handgrip (HG) isometric exercise on plasma catecholamines, alpha 2-adrenoceptors on platelets and beta 2-adrenoceptors on lymphocytes was studied in normotensive subjects (NT) and essential hypertensive subjects (HT). Whereas systolic blood pressure (SBP) increases were similar in NT and HT subjects, diastolic blood pressure (DBP) and heart rate (HR) increased more in the former group. Baseline values and changes in plasma epinephrine (E) and norepinephrine (NE) did not differ between both groups. No differences were apparent in alpha 2-adrenoceptor density and affinity between NT and HT subjects before or after the test. HG isometric exercise induced a similar increase in beta 2-adrenoceptors on lymphocytes of 22 +/- 7 and 13 +/- 5% in NT and HT subjects, respectively. Affinity to the beta 2-adrenoceptors under baseline conditions was somewhat lower in HT (8.1 +/- 0.4 pM) than in NT subjects (6.5 +/- 0.5 pM), and this difference persisted during the test. Our results indicate that there are no differences in alpha 2- and beta 2-adrenoceptor densities either at baseline conditions or after HG isometric exercise between NT and HT subjects. Small differences noted in affinity to the beta 2-adrenoceptors require further investigation.     

De-novo expression of vascular ecto-5′-nucleotidase and down-regulation of glomerular ecto-ATPase in experimental chronic renal transplant failure

Ischemic injury plays an important role in chronic renal transplant failure (CRTF). Down-regulation of ecto-adenosine triphosphatase (ATPase) in combination with up-regulation of ecto-5'-nucleotidase is a hallmark of ischemic injury. We studied the expression of renal ecto-5'-nucleotidase and ecto-ATPase in experimental renal transplantation. Fisher 344-to-Lewis allografted rats were either treated with an angiotensin-converting enzyme inhibitor (ACEi) or left untreated. Lewis-to-Lewis syngrafted rats served as controls. Untreated allografted rats developed proteinuria, glomerulosclerosis, and mild intimal hyperplasia. ACEi completely prevented focal and segmental glomerulosclerosis (FGS) and proteinuria, but significantly enhanced intimal hyperplasia. Untreated allografted rats revealed marked vascular ecto-5'-nucleotidase activity, which increased with ACEi. Vascular ecto-5'-nucleotidase activity was absent in syngrafted animals. Ecto-5'-nucleotidase activity correlated well with intimal hyperplasia. Glomerular ecto-ATPase expression was significantly reduced in untreated allografted rats compared to syngrafted rats and correlated well with the extent of FGS. ACEi prevented reduction in glomerular ecto-ATPase. We found de-novo expression of ecto-5'-nucleotidase at sites of renal intimal hyperplasia. Glomerular ecto-ATPase expression was markedly reduced in allografted rats and was prevented by ACEi. These enzyme expression patterns suggest local ischemic damage in experimental CRTF.     

Long-term Results of Primary Stent Placement to Treat Infrarenal Aortic Stenosis

OBJECTIVE: To determine the safety and the long-term results of primary stent placement for localized distal aortic occlusive disease. DESIGN: Retrospective observational study. PATIENTS AND METHODS: From July 1998 to July 2005 17 patients (14 female and 3 men, mean age 57 years (39-80)) were treated for intermittent claudication. Five of these patients underwent additional endovascular treatment of focal iliac lesions. RESULTS: Technical success defined as residual stenosis of less than 50% or a trans-stenotic systolic pressure gradient <10% was achieved in 14 of 17 (82%) patients. Major complications included dissection at the puncture site in one patient and thrombosis of additional iliac stents in another patient. Both of these complications were successfully treated. During a mean follow-up of 27 months (range 1-86), four patients had recurrence of symptoms due to in-stent restenoses (n=2), femoral (n=1) or iliac occlusion (n=1), respectively. By Kaplan-Meier analysis, primary aortic hemodynamic patency was 83% at 3 years. Secondary aortic hemodynamic patency was 100%. The primary clinical patency was 68% at 3 years. CONCLUSION: Primary stent placement for distal aortic stenoses is an alternative to surgical treatment because of its high patency and relatively low complication rates.     

The influence of protein solubilisation, conformation and size on the burst release from poly(lactide-co-glycolide) microspheres.

Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for alpha-helical proteins released from freeze-dried microspheres in aqueous buffer, with dramatic loss of structure for a beta-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released.