Allogeneic bone marrow transplantation in a child with hemoglobinopathy olmsted

The authors describe the first successful bone marrow transplant for the treatment of hemoglobinopathy Olmsted in a boy who presented with severe transfusion-dependent hemolytic anemia and jaundice at age 4 months. He received bone marrow from an HLA-identical sibling with normal hemoglobin electrophoresis after conditioning with busulfan, cyclophosphamide, and antithymocyte globulin when he was 18 months old. The posttransplant course was uneventful. Two years after transplantation the patient has a normal hemoglobin level without evidence of hemolysis. DNA analysis shows 100% chimerism of donor cell origin, confirming full engraftment with normal hematopoietic cells.     

Regulatory T Cells Modulate Staphylococcal Enterotoxin B-Induced Effector T-Cell Activation and Acceleration of Colitis

Oral administration of bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) activates mucosal T cells but does not cause mucosal inflammation. We examined the effect of oral SEB on the development of mucosal inflammation in mice in the absence of regulatory T (Treg) cells. SCID mice were fed SEB 3 and 7 days after reconstitution with CD4⁺ CD45RB^high or CD4⁺ CD45RB^high plus CD4⁺ CD45RB^low T cells. Mice were sacrificed at different time points to examine changes in tissue damage and in T-cell phenotypes. Feeding SEB failed to produce any clinical effect on SCID mice reconstituted with CD4⁺ CD45RB^high and CD4⁺ CD45RB^low T cells, but feeding SEB accelerated the development of colitis in SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone. The latter was associated with an increase in the number of CD4⁺ Vβ8⁺ T cells expressing CD69 and a significantly lower number of CD4⁺ CD25⁺ Foxp3⁺ T cells. These changes were not observed in SCID mice reconstituted with both CD45RB^high and CD45RB^low T cells. In addition, SEB impaired the development of Treg cells in the SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone but had no direct effect on Treg cells. In the absence of Treg cells, feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation.     

p62 Is a Common Component of Cytoplasmic Inclusions in Protein Aggregation Diseases

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.     

Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8+ T cells in an epitope‐specific manner

Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus‐harboring hepatocytes influences de novo priming of CD8⁺ T cells. We showed that surface antigen‐expressing transfectants exclusively display a K^b/S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K^b/S208 epitope to CD8⁺ T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K^b/S190‐ and K^b/S208‐specific T cells in C57BL/6 mice by endogenous/DNA‐ or exogenous/protein‐based vaccines, respectively. Silencing the K^b/S190 epitope (K^b/S190_V194F) in antigen‐expressing vectors rescued the presentation of the K^b/S208 epitope in stable transfectants and significantly enhanced priming of K^b/S208‐specific T cells in C57BL/6 mice. A K^b/S190‐mediated immunodominance operating in surface antigen‐expressing cells, but not in rSP‐pulsed cells, led to an efficient suppression in the presentation of the K^b/S208 epitope and a consequent decrease in the priming of K^b/S208‐specific T cells. This K^b/S190‐mediated immunodominance also operated in 1.4HBV‐S^mut transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K^b/S208‐ but not K^b/S190‐specific T cells in 1.4HBV‐S^mut tg mice. However, IFN‐γ⁺ K^b/S208‐specific T cells could not inhibit HBV replication in the liver of 1.4HBV‐S^mut tg mice. These results have practical implications for the design of T‐cell‐stimulating therapeutic vaccines.     

The role of immune-related myeloid cells in angiogenesis

Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.     

Mosaic genes and mosaic chromosomes-genomic variation in Streptococcus pneumoniae

The genome sequences of two strains of Streptococcus pneumoniae, one of the major human pathogens, are currently available: that of the nonencapsulated laboratory strain R6, the origin of which dates back to the early 20th century, and of the serotype 4 TIGR strain isolated recently. The two genomes are not only different in size (2 versus 2.16 Mb) but differ also by approximately 10% of their genes, many of which being organized in large clusters. Their strain-specific genes and gene clusters are described here. The R6 genome contains 69 kb organized in six large regions that are absent from the TIGR strain, which in turn contains an extra 157kb in twelve clusters compared to R6. In addition, the TIGR strain contains 13 clusters of 4 kb and larger that are not shared by a variety of genetically different S. pneumoniae strains. Many regions bear signs of gene transfer events such as the presence of insertion sequences, transposable elements, and putative site-specific integrases/recombinases. Three strain-specific regions are devoted to genes encoding proteins with the cell wall anchor motif LPXTG which are important for the interaction with host cells and appear to be highly variable, similar to cell wall-associated choline-binding proteins.     

In vitro quality control of red blood cell concentrates outdated in clinical practice.

The properties of red blood cell (RBC) concentrates stored in different additive solutions have been previously examined under laboratory conditions at the end of shelf-life. However, whether these data are representative for RBC units used in clinical practice has not been shown. Therefore, we examined 164 RBC units from six manufacturers outdated after clinical usage in a hospital-based transfusion service for cellular content, hemolysis, adenosin triphosphate, 2,3-DPG, pH, oxygen saturation and levels of beta-thromboglobulin and proinflammatory cytokines interleukin (IL) 1beta (IL-1), IL-6, IL-8 and tumor necrosis factor alpha (TNFalpha). Results were correlated with the number of interruptions of recommended storage conditions and with different manufacturers. TNFalpha and IL-8 levels in the supernatant of RBC concentrates showed a weak correlation with the number of interruptions of recommended storage conditions (TNFalpha: r = 0.25, P < 0.01; IL-8: r = 0.20, P < 0.01) for the whole series. We detected no significant correlation between hemolysis and interruptions of recommended storage conditions or any of the remaining studied parameters. However, we found significant differences between RBC concentrates supplied by different manufacturers with respect to cellular content and most of the studied parameters. RBC concentrates containing SAG-M from one single manufacturer had higher in vitro hemolysis at the end of shelf-life compared to all other manufacturers (P < 0.05). We conclude from our data that interruptions of optimal conditions for storage of red cell components during cross-match testing and transport in our setting play a minor role for in vitro properties of RBC units at the end of shelf-life. The influence of processes of production, storage and/or transport until entry of RBC units into our blood component depot seems to be much more important for final product quality at the end of shelf-life than subsequent events.     

Laser microdissection and pressure catapulting (LMPC) in paraffin sections mounted on glass slides. A methodological report

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.