Approaches to managing volunteer marrow donor registry HLA data. Algorithms for directing donor center-initiated HLA-DR typing of selected donors

The German bone marrow donor center (DKMS) hasrecruited over 732 500 donors during the first 9 years of its existence. Initially, donors were typed for HLA-A and B, and DR typing was only done on request for a patient-initiated search. In 1994, a project was started which led to the donor center-initiated DR typing (DCI-DRT) of >35,000 donors. These donors were selected by donor-specific criteria (age, sex, height and weight) and according to HLA-A and B phenotypes. The latter was done to avoid unnecessary DR typing of the most common A, B phenotypes With a follow up of >6 years, this strategy has led to a number of confirmatory typings (CT) (n=4588) and stem cell harvests (n=568), which is at least comparable to those ensuing after patient-initiated HLA-DR typing (126 000 DR typings, 8,213 CTs, 888 resulting in stem-cell donation). DCI-DRT seems to be a cost-effective strategy which may help to reduce search times and improve search outcome, and improve the overall efficiency of donor center operations     

Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis

Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost "on top" of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.     

Effect of exercise on coronary endothelial function in patients with coronary artery disease

BACKGROUND: Studies of the cardioprotective effects of exercise training in patients with coronary artery disease have yielded contradictory results. Exercise training has been associated with improvement in myocardial perfusion even in patients who have progression of coronary atherosclerosis. We therefore conducted a prospective study of the effect of exercise training on endothelial function in patients with coronary artery disease. METHODS: We randomly assigned 19 patients with coronary endothelial dysfunction, indicated by abnormal acetylcholine-induced vasoconstriction, to an exercise-training group (10 patients) or a control group (9 patients). To reduce confounding, patients with coronary risk factors that could be influenced by exercise training (such as diabetes, hypertension, hypercholesterolemia, and smoking) were excluded. In an initial study and after four weeks, the changes in vascular diameter in response to the intracoronary infusion of increasing doses of acetylcholine (0.072, 0.72, and 7.2 microg per minute) were assessed. The mean peak flow velocity was measured by Doppler velocimetry, and the diameter of epicardial coronary vessels was measured by quantitative coronary angiography. RESULTS: In the initial study, the two groups had similar vasoconstrictive responses to acetylcholine. After four weeks of exercise training, coronary-artery constriction in response to acetylcholine at a dose of 7.2 microg per minute was reduced by 54 percent (from a mean [+/-SE] decrease in the luminal diameter of 0.41+/-0.05 mm in the initial study to a decrease of 0.19+/-0.07 mm at four weeks; P<0.05 for the comparison with the change in the control group). In the exercise-training group, the increases in mean peak flow velocity in response to 0.072, 0.72, and 7.2 microg of acetylcholine per minute were 12+/-7, 36+/-11, and 78+/-16 percent, respectively, in the initial study. After four weeks of exercise, the increases in response to acetylcholine were 27+/-7, 73+/-19, and 142+/-28 percent (P<0.01 for the comparison with the control group). Coronary blood-flow reserve (the ratio of the mean peak flow velocity after adenosine infusion to the resting velocity) increased by 29 percent after four weeks of exercise (from 2.8+/-0.2 in the initial study to 3.6+/-0.2 after four weeks; P<0.01 for the comparison with the control group). CONCLUSIONS: Exercise training improves endothelium-dependent vasodilatation both in epicardial coronary vessels and in resistance vessels in patients with coronary artery disease.     

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.     

Immune response against the T-independent antigen alpha (1 leads to 3) dextran. I. Demonstration of an unexpected IgG response of athymic and germ-free-raised euthymic BALB/c mice

The primary antibody response in BALB/c mice to the T-independent bacterial antigen dextran B1355S [alpha(1 leads to 3)dextran] (Dex) was studied by means of isoelectric focusing, hemagglutination and immunodiffusion techniques. In response to a single immunization with 10 micrograms Dex all mice produce specific IgM antibodies. In addition, about 30% of conventionally raised BALB/c and BALB/c nu/ + mice, but 95% of germ-free (GF)-raised normal BALB/c and 100% of athymic BALB/c nu/nu mice produce specific IgG class anti-Dex antibodies. These antibodies include all IgG subclasses, carry predominantly the lambda light chain and the cross-reactive J558 idiotype and are specific for the alpha(1 leads to 3)glucosidic linkage. As compared to athymic and GF-raised mice, conventionally raised mice exhibit only a weak IgG response. The pronounced IgG production of GF-raised mice was not altered when adult mice were removed from their GF environment and housed under conventional conditions for several weeks prior to immunization with Dex. Reconstitution with isolated splenic T cells from conventionally raised, unprimed BALB/c mice reduces the remarkable capacity of BALB/c nu/nu mice to produce IgG anti-Dex antibodies. These findings suggest that the reduced capacity of conventionally raised BALB/c mice to mount an IgG response to the T-independent antigen Dex is due to a T cell-mediated suppressive mechanism which is neonatally induced by contact with environmental, i.e. bacterial, antigens.     

Molecular genetics of PKU in Eastern Europe: A nonsense mutation associated with haplotype 4 of the phenylalanine hydroxylase gene

Phenylketonuria (PKU) is a genetic disorder secondary to a deficiency of hepatic phenyalanine hydroxylase (PAH). Several mutations in thePAH gene have recently been reported, and linkage disequilibrium was observed between RFLP haplotypes and specific mutations. A new molecular lesion has been identified in exon 7 of thePAH gene in a Hungarian PKU patient by direct sequencing of PCR-amplified DNA. The C-to-T transition causes the substitution of Arg²⁴³ to a termination codon, and the mutant allele is associated with haplotype 4 of thePAH gene. The mutation is present in two of nine mutant haplotype 4 alleles among Eastern Europeans and is not present among Western Europeans and Asians. The rarity of this mutant allele and its restricted geographic distribution suggest that the mutational event occurred recently on a normal haplotype 4 background in Eastern Europe.     

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