The cytotoxic activity of mouse T lymphocytes against allogeneic cytotoxic T cells.

Using a 51Cr cytotoxicity assay, the sensitivity of murine cytotoxic T cells to T cell mediated cytotoxicity has been tested. Two experimental approaches have been used. First, cytotoxic T-blast-lymphocytes (CTBL), enriched by the velocity sedimentation at 1 g were used both as cytotoxic effector cells and as 51Cr-labelled target cells. It was found that murine CTBL are capable to lyse directly and specifically allogeneic CTBL within 200 minutes. The second approach used was to incubate CTBL together with CTBL, the cytotoxic activity of which was directed against the transplantation antigens of the added allogeneic CTBL population. After 10 hours, the residual cytotoxic activity was tested. Again it was found that CTBL were capable of functionally inactivating allogeneic CTBL. Therefore the results obtained are incompatible with the concept that target cell lysis by cytotoxic T lymphocytes is mediated by a non specific "lymphotoxin", secreted by activated T cells after the antigen-recognition phase in the confined space between T cells and target cells.     

Fronto-cingulate effective connectivity in major depression: a study with fMRI and dynamic causal modeling

Functional imaging studies are indicating disrupted error monitoring and executive control in a fronto-cingulate network in major depression. However, univariate statistical analyses allow only for a limited assessment of directed neuronal interactions. Therefore, the present study used dynamic causal modeling (DCM) of a fronto-cingulate network to re-analyze the data from a preceding fMRI study in 16 drug-free patients with major depression and 16 healthy controls using the Stroop Color-Word Test (Wagner et al., 2006). In both groups, a significant reciprocal interregional connectivity was found in a cognitive control network including prefrontal cortex (PFC) and dorsal anterior cingulate cortex (ACC). With regard to intrinsic connections we detected a significant difference for dorsal to rostral ACC connectivity between depressive patients and controls in terms of higher connectivity in patients. Additionally, a task by group interaction was observed for the bilinear interaction signaling enhanced task-related input from the dorsal to rostral ACC in subjects with depression. This could be related to the inability of patients to down-regulate rostral ACC activation as observed in the previous univariate analysis. The correlation between interference scores and intrinsic connections from dorsal ACC to dorsolateral PFC (DLPFC) was significant for both groups together, but no significant group differences in correlations could be detected. Thus, the observed relationship between control functions of the dorsal ACC exerted over DLPFC and interference scores appears to be valid in both patients with depression and controls. The findings are consistent with current models of a differential involvement of the fronto-cingulate system in the pathophysiology of major depression.     

The D category VI type 4 allele is prevalent in the Spanish population

BACKGROUND: The D category VI (DVI) is one of the clinically most important partial D. Three different molecular structures causing the DVI phenotype have been described. STUDY DESIGN AND METHODS: To determine the molecular basis of the DVI phenotype in the Spanish population, 20 DVI samples, previously detected in serologic screening, were examined by polymerase chain reaction with RHD exon-specific primers. Unexpected findings were further pursued by cDNA nucleotide sequencing. RESULTS: A novel pattern of RHD exon amplification was detected, which did not correspond to any of the previously described molecular structures. The cDNA sequence led to the identification of the new hybrid RHD-Ce(3-5)-D allele. The origin of exon 2 is undeterminable, because the 5' breakpoint was located within a region of RHD and RHCE identical sequence, which encompasses this exon. Sequencing of intron 5 allowed the 3' breakpoint to be mapped between the sixth and seventh polymorphic sites. Serologically, the hybrid protein has a D epitope expression pattern identical to the previously described DVI phenotypes and an antigen density slightly lower than DVI type 3. The new DVI variant is linked to the DCe haplotype and expresses the low-incidence BARC antigen. CONCLUSION: A novel structure causing the DVI phenotype, here named DVI type 4, has been characterized. This novel structure is the most frequent cause of DVI in Spain.     

An empirical evaluation of the performance of antibody identification tasks

Four empirical studies were conducted for better understanding of the nature of problem-solving activities by medical technologists and medical technology students when performing antibody identification tasks. The results indicated the importance of strategies that ensure the collection of converging evidence, as these strategies protect against the fallibility of commonly used heuristics and against errors due to simple slips. The results also indicate that not only do students make significant numbers of errors, but so do practicing technologists. In one of the studies covering a 1-year period, for instance, a group of 16 technologists made a total of 41 errors in 1057 cases. On the basis of these findings, several alternatives are proposed to reduce errors.     

Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes.

Surface-shielded DNA delivery systems have been synthesized with virus-like characteristics that target gene expression into distant tumor tissues. Polyethylenimine (PEI)/DNA complexes ('polyplexes') conjugated with the cell-binding ligand transferrin (Tf) or epidermal growth factor (EGF) were used to achieve receptor-mediated endocytosis. The surface charge of the complexes was masked by covalently linking PEI to polyethylene glycol (PEG). Three alternatives for generating these surface-shielded formulations were utilized, attaching ligand and PEG molecules to PEI either before or after DNA complex formation. The stabilized formulations could be ultra-concentrated, stored frozen, and applied systemically after thawing. Intravenous injection of Tf-PEG-coated polyplexes resulted in gene transfer to subcutaneous Neuro2a neuroblastoma tumors of syngeneic A/J mice; EGF-PEG-coated polyplexes were intravenously applied for targeting human hepatocellular carcinoma xenografts in SCID mice. In these models, luciferase marker gene expression levels in tumor tissues were 10- to 100-fold higher than in other organ tissues. Repeated systemic application of Tf-PEG-PEI/DNA complexes encoding tumor necrosis factor alpha (TNF-alpha) into tumor-bearing mice induced tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origin (Neuro2a, M-3 or B16 melanoma).     

T lymphocytes in implant-associated posttraumatic osteomyelitis: Identification of cytotoxic T effector cells at the site of infection

In implant-associated posttraumatic osteomyelitis, a massive infiltration of leukocytes into the infected site is seen. As described previously, the most infiltrated cells were highly activated polymorphonuclear neutrophils. In addition, a considerable T-cell infiltrate was noted. Whereas our previous work was mainly concerned with the phenotypical and functional characterization of the polymorphonuclear neutrophils, we now analyzed T lymphocytes of 32 patients with implant-associated posttraumatic osteomyelitis. We found evidence for an expansion of CD8 T cells in the peripheral blood of the patients and for an infiltration of these cells into the infected site. Further analysis of the surface-receptor pattern by three-color cytofluorometry revealed that the majority of these cells belonged to the cytotoxic-effector phenotype. Of note is that cytotoxic T cells are generally associated with virus infection. Thus, the detection of those cells in patients with bacterial infection was rather unexpected and points to a novel, not yet appreciated, role of CD8 T cells also in the defense of bacterial infections.     

Age-associated accumulation of CMV-specific CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1)

Large clonal expansions of peripheral CD8+ T cells carrying receptors for single epitopes of CMV and EBV are common in the elderly and may be associated with an immune risk phenotype predicting mortality. Here we show that the frequency of CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1), a marker of cells unable to undergo further clonal expansion, was markedly elevated in CD8+ T cells from old donors. Moreover, tetramer staining revealed that the elevated frequency of CMV-specific CD8+ T cells in the elderly was due to an accumulation of cells bearing this dominant negative receptor. The fraction of CMV-specific T cells able to secrete interferon-gamma after specific antigenic stimulation was significantly lower in the elderly than in the young, although the total number of functional cells was comparable. Therefore, the majority of the clonally expanded virus-specific CD8+ cells in the elderly was dysfunctional. Thus, T cell responses are altered in the aged by an accumulation of replicatively senescent dysfunctional T cells carrying receptors for persistent herpes viruses. The presence of clonal expansions of such virus-specific cells may shrink the available repertoire for other antigens and contribute to the increased incidence of infectious disease in the elderly.     

Fundamentals of pulmonary drug delivery

Aerosol administration of peptide-based drugs plays an important role in the treatment of pulmonary and systemic diseases and the unique cellular properties of airway epithelium offers a great potential to deliver new compounds. As the relative contributions from the large airways to the alveolar space are important to the local and systemic availability, the sites and mechanism of uptake and transport of different target compounds have to be characterized. Among the different respiratory cells, the ciliated epithelial cells of the larger and smaller airways and the type I and type II pneumocytes are the key players in pulmonary drug transport. With their diverse cellular characteristics, each of these cell types displays a unique uptake possibility. Next to the knowledge of these cellular aspects, the nature of aerosolized drugs, characteristics of delivery systems and the depositional and pulmonary clearance mechanisms display major targets to optimize pulmonary drug delivery. Based on the growing knowledge on pulmonary cell biology and pathophysiology due to modern methods of molecular biology, the future characterization of pulmonary drug transport pathways can lead to new strategies in aerosol drug therapy.