Characterisation of mouse prothymocytes

In this thesis, the study of growth kinetics of thymocytes in lethally irradiated mice as a means to characterise the early T-cell progenitors is described. Normal bone marrow cells were used as the source of hemopoietic cells. The experiments show that it is possible to obtain information on T-cell differentiation from a complex experimental set-up in vivo. The quantitative relation between the thymus regeneration capacity and the CFU-S content of bone marrow cells was established. K"1netk studies of the regeneration process led to formulation of the framework for the lil vivo thymus regeneration assay (Chapter 3). From the study of erythroid and granulocyte-macrophage lineages, it had been established that at least these lineages start from early progenitor cells that are immediate successors of the pluripotent cells, but can be distinguished experimentally from CFU-S (Bol, 1980). However, apart from the cells postulated by Abramson et al. (1977), no such early progenitor cells are known for the T-cell 1"1neage. Our results obtained in thymus repopulation after transplantation of bone marrow from normal mke did not allow a distinction between the T-cell progenitor function of prothymocytes and that of the pluripotent stem cell determined as CFU-

Growth phase of orally administered Lactobacillus strains differentially affects IgG1/IgG2a ratio for soluble antigens: Implications for vaccine development

Lactobacillus strains with probiotic activity are major constituents of numerous common food products. Due to their 'generally regarded as safe'-status (GRAS-status), Lactobacillus strains can also be genetically engineered for use in oral immunotherapeutic applications, such as vaccination and T lymphocyte tolerance induction in autoimmune disease. In the current study, we demonstrate that the growth phase of orally administered individual Lactobacillus strains can differentially affect antigen-specific antibody subclasses IgG1 and IgG2a, which might reflect skewing of systemic activity of T helper cell type 2 (Th2) and T helper cell type 1 (Th1) pathways, respectively. Mice were orally fed different wild type Lactobacillus strains in log phase or stationary phase and immunized intraperitoneally with a T-cell dependent protein antigen. Sera were evaluated for the ratio of antigen-specific IgG1 and IgG2a antibodies. Stationary Lactobacillus murines and Lactobacillus casei cultures, but not two other Lactobacillus strains, evoked significantly higher IgG1/IgG2a ratios than log phase cultures, possibly relating to increased activity of the Th2-pathway. Despite normal variation in antibody responses against TNP-CGG among individual mice, a high correlation was found between the IgG1 and IgG2a responses of mice within experimental groups. This differential antibody response is likely due to growth phase-dependent differences in bacterial cell composition. Since Lactobacillus growth phase dependent skewing of antibody responses possibly reflecting T-cell pathways can inadvertently affect allergic and (auto)-immune responses, the current findings strongly caution against unidimensional views on the oral administration of individual Lactobacillus strains for probiotic or immunotherapeutic purposes, but also suggest additional possibilities for immune modulation

CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis

We investigated the role of CD40-CD40 ligand (CD40L) interactions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Activated helper T cells expressing CD40L (gp39) surface protein were found in MS patient brain sections, but not in brain tissue sections of normal controls or patients with other neurological diseases. CD40L-positive cells were co-localized with CD40-bearing cells in active lesions (perivascular infiltrates). Most of these CD40 bearing cells proved to be of the monocytic lineage (macrophages or microglial cells), and relatively few were B cells. To functionally evaluate CD40-CD40L interactions, EAE was elicited in mice by means of proteolipid-peptide immunization. Treatment with anti-CD40L monoclonal antibody completely prevented the development of disease. Furthermore, administration of anti-CD40L monoclonal antibody, even after disease onset, shortly before maximum disability score was reached led to dramatic disease reduction. The presence of helper T cells expressing CD40L in brain tissue of MS patients and EAE animals, together with the functional evidence provided by successful experimental prevention and therapy in an animal model, indicates that blockade of CD40-CD40L-mediated cellular interactions may be a method for interference in active MS

Intracoronary Heparin Delivery in Humans

Background Inefficacy of systemic drug administration for restenosis prevention may partially relate to insufficient local drug concentration. This study aimed to evaluate the acute feasibility and long-term outcome of using an infusion-perfusion coil balloon, Dispatch. Methods and Results In 22 patients after balloon angioplasty, the coil balloon was studied for (1) feasibility of local heparin delivery, (2) symptoms and signs of ischemia during prolonged deployment compared with angioplasty balloon occlusion, (3) coronary pressure and flow distal to the inflated device, and (4) long-term clinical and angiographic results. During prolonged intracoronary deployment of the coil balloon (29±8 minutes), 5 of 22 patients developed mild chest pain versus 20 of 22 during angioplasty (275±283 seconds). Neither hemodynamic nor vectorcardiographic signs of ischemia were detected, in contrast to angioplasty balloon occlusion. Baseline flow across the coil balloon was 44±31 mL/min, increasing by a factor of 1.8±0.7 during pharmacologically induced hyperemia. A mean volume of 14.2±6.1 mL containing 1416±608 IU of heparin was infused locally at a pressure of 122±54 mm Hg. At 7±1-month follow-up, 1 asymptomatic patient had died, and of the remaining 21, 17 (81%) were asymptomatic. Angiographic follow-up was obtained in 15 of 21 patients (71%), including all 4 symptomatic patients. Mean minimal luminal diameter after the procedure was 2.16±0.49 mm and at follow-up, 1.89±0.45 mm, which corresponds to a restenosis rate (diameter stenosis 50%) of 7% (1/15). Conclusions Intracoronary use of the coil balloon after balloon angioplasty proved to be feasible and subjectively as well as objectively well tolerated during prolonged deployment by virtue of its perfusion properties. High volumes of heparin solution can be infused locally at very low pressure. No unfavorable clinical or angiographic long-term effects were observed

Recovery of left ventricular function after primary angioplasty for acute myocardial infarction.

AIMS: To study recovery of segmental wall thickening (SWT), ejection fraction (EF), and end-systolic volume (ESV) after acute myocardial infarction (AMI) in patients who underwent primary stenting with drug-eluting stents. Additionally, to evaluate the predictive value of magnetic resonance imaging (MRI)-based myocardial perfusion and delayed enhancement (DE) imaging. METHODS AND RESULTS: Twenty-two patients underwent cine-MRI, first-pass perfusion, and DE imaging 5 days after successful placement of a drug-eluting stent in the infarct-related coronary artery. Regional myocardial perfusion and the transmural extent of DE were evaluated. A per patient perfusion score was calculated and consisted of a summation of all segmental scores. Myocardial infarct size was quantified by measuring the volume of DE. At 5 months after AMI, cine-MRI was performed and SWT, EF, and ESV were quantified. EF increased from 48+/-11 to 55+/-9% (P<0.01). SWT at 5 months was inversely related to baseline segmental DE scores (P<0.001) and segmental perfusion scores (P<0.001). EF and ESV at 5 months were related to acute infarct size (R(2)=0.65; P<0.001 and R(2)=0.78; P<0.001, respectively) and the calculated perfusion score (R(2)=0.23; P=0.02 and R(2)=0.14; P=0.09, respectively) at baseline. CONCLUSION: Marked recovery of left ventricular function was observed in patients receiving a drug-eluting stent for AMI. DE imaging appears to be a better prognosticator than perfusion imaging

Reduced experimental autoimmune encephalomyelitis after intranasal and oral administration of recombinant lactobacilli expressing myelin antigen

Oral administration of autoantigens is a safe and convenient way to induce peripheral T-cell tolerance in autoimmune diseases like multiple sclerosis (MS). To increase the efficacy of oral tolerance induction and obviate the need for large-scale purification of human myelin proteins, we use genetically modified lactobacilli expressing myelin antigens. A panel of recombinant lactobacilli was constructed producing myelin proteins and peptides, including human and guinea pig myelin basic protein (MBP) and proteolipid protein peptide 139-151 (PLP139-151). In this study we examined whether these Lactobacillus recombinants are able to induce oral and intranasal tolerance in an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Lewis rats received soluble cell extracts of Lactobacillus transformants intranasally three times prior to induction of EAE. For the induction of oral tolerance, rats were fed live transformed lactobacilli for 20 days. Ten days after the first oral administration EAE was induced. Intranasal administration of extracts containing guinea pig MBP (gpMBP) or MBP72-85 significantly inhibited EAE in Lewis rats. Extracts of control transformants did not reduce EAE. Live lactobacilli expressing guinea pig MBP72-85 fused to the marker enzyme β-glucuronidase (β-gluc) were also able to significantly reduce disease when administered orally. In conclusion, these experiments provide proof of principle that lactobacilli expressing myelin antigens reduce EAE after mucosal (intranasal and oral) administration. This novel method of mucosal tolerance induction by mucosal administration of recombinant lactobacilli expressing relevant autoantigens could find applications in autoimmune disease in general, such as multiple sclerosis, rheumatoid arthritis and uveitis

Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design

Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme's stability while at the same time enhancing the enzyme's activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments