Flower-like Mn-Doped Magnetic Nanoparticles Functionalized with αvß3-Integrin-Ligand to Efficiently Induce Intracellular Heat after Alternating Magnetic Field Exposition, Triggering Glioma Cell Death.

Despite the potential of magnetic nanoparticles (NPs) to mediate intracellular hyperthermia when exposed to an alternating magnetic field (AMF), several studies indicate that the intracellular heating capacity of magnetic NPs depends on factors such as cytoplasm viscosity, nanoparticle aggregation within subcellular compartments, and dipolar interactions. In this work, we report the design and synthesis of monodispersed flowerlike superparamagnetic manganese iron oxide NPs with maximized SAR (specific absorption rate) and evaluate their efficacy as intracellular heaters in the human tumor-derived glioblastoma cell line U87MG. Three main strategies to tune the particle anisotropy of the core and the surface to reach the maximum heating efficiency were adopted: (1) varying the crystalline anisotropy by inserting a low amount of Mn2+ in the inverse spinel structure, (2) varying the NP shape to add an additional anisotropy source while keeping the superparamagnetic behavior, and (3) maximizing NP-cell affinity through conjugation with a biological targeting molecule to reach the NP concentration required to increase the temperature within the cell. We investigate possible effects produced by these improved NPs under the AMF (f = 96 kHz, H = 47 kA/m) exposure in the glioblastoma cell line U87MG by monitoring the expression of hsp70 gene and reactive oxygen species (ROS) production, as both effects have been described to be induced by increasing the intracellular temperature. The induced cell responses include cellular membrane permeabilization and rupture with concomitant high ROS appearance and hsp70 expression, followed by cell death. The responses were largely limited to cells that contained the NPs exposed to the AMF. Our results indicate that the developed strategies to optimize particle anisotropy in this work are a promising guidance to improve the heating efficiency of magnetic NPs in the human glioma cell line.

Short-chain PEG molecules strongly bound to magnetic nanoparticle for MRI long circulating agents.

This study developed an approach for the synthesis of magnetic nanoparticles coated with three different polyethylene glycol (PEG)-derived molecules. The influence of the coating on different properties of the nanoparticles was studied. Magnetite nanoparticles (7 and 12 nm in diameter) were obtained via thermal decomposition of a coordination complex as an iron precursor to ensure nanoparticle homogeneity in size and shape. Particles were first coated with meso-2,3-dimercaptosuccinic acid by a ligand exchange process to remove oleic acid, followed by modification with three distinct short-chain PEG polymers, which were covalently bound to the nanoparticle surface via 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride activation of the carboxylic acids. In all cases, colloidal suspensions had hydrodynamic sizes <100 nm and low surface charge, demonstrating the effect of PEG coating on the aggregation properties and steric stabilization of the magnetic nanoparticles. The internalization and biocompatibility of these materials in the HeLa human cervical carcinoma cell line were tested. Cells preincubated with PEG-coated iron nanoparticles were visualized outside the cells, and their biocompatibility at high Fe concentrations was demonstrated using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Finally, relaxivity parameters (r1 and r2) were used to evaluate the efficiency of suspensions as magnetic resonance imaging contrast agents; the r2 value was similar to that for Resovist and up to four times higher than that for Sinerem, probably due to the larger nanoparticle size. The time of residence in blood of the nanoparticles measured from the relaxivity values, and the Fe content in blood was doubled for rats and rabbits due to the PEG on the nanoparticle surface. The results suggest that this PEGylation strategy for large magnetic nanoparticles (>10nm) holds promise for biomedical applications.

Workload measurement in subspecialty dermatopathology.

AIM: To measure pathologist workload in subspecialty dermatopathology. METHODS: Three subspecialty dermatopathologists, working in a university-affiliated laboratory, participated in a time-motion study during which they reported 2891 consecutive skin cases received from community-based dermatologists. All pathology reports were retrospectively reviewed and workload measured using the Royal College of Pathologists (RCPath) guidelines and the level 4 equivalent (L4E) method. RESULTS: The majority of dermatopathology cases were scored as low (32%) or intermediate (52%) complexity using the RCPath matrix. Only 16% of cases were considered high or very high complexity. The mean RCPath score per case was 2.68 units. Using L4E complexity levels, 83% of specimens were level 3, 15% were level 4, and only 2% were higher complexity (levels 5 and 6). Mean values for specimens/case, blocks/case, and slides/case were 1.31, 1.52, and 2.92, respectively. Time-motion analysis demonstrated a mean workload per hour of 16.3 cases, 21.3 specimens, 45.1 slides, 43.0 RCPath units, and 12.2 L4E. All three dermatopathologists reported >35 RCPath units per hour. CONCLUSIONS: The RCPath histopathology workload guidelines underestimate the workload achievable by an experienced dermatopathologist, and thus are not directly applicable to subspecialty dermatopathology practice. Hourly work rates 3-4 times that recommended by the RCPath workload matrix are routinely achievable, but extrapolation to yearly workload estimates requires detailed knowledge of practice pattern and time required for non-clinical duties such as teaching, research and administration.

The biological activity of natural and mutant pTalpha alleles.

beta selection is a major checkpoint in early thymocyte differentiation, mediated by successful expression of the pre-T cell receptor (TCR) comprising the TCRbeta chain, CD3 proteins, and a surrogate TCRalpha chain, pTalpha. The mechanism of action of the pre-TCR is unresolved. In humans and mice, the pTalpha gene encodes two RNAs, pTalpha(a), and a substantially truncated form, pTalpha(b). This study shows that both are biologically active in their capacity to rescue multiple thymocyte defects in pTalpha(-/-) mice. Further active alleles of pTalpha include one that lacks both the major ectodomain and much of the long cytoplasmic tail (which is unique among antigen receptor chains), and another in which the cytoplasmic tail is substituted with the short tail of TCR Calpha. Thus, very little of the pTalpha chain is required for function. These data support a hypothesis that the primary role of pTalpha is to stabilize the pre-TCR, and that much of the conserved structure of pTalpha probably plays a critical regulatory role.

Inhibition of natural killer cell activation signals by killer cell immunoglobulin-like receptors (CD158).

The killer cell immunoglobulin-like receptor (KIR) family includes receptors that bind to HLA class I molecules on target cells and inhibit natural killer (NK)-cell cytotoxicity, and receptors such as KIR3DL7 with no known ligand and function. Inhibitory KIR recruit the tyrosine phosphatase SHP-1 to block signals transduced by any one of a number of activation receptors. Inhibition of overall protein tyrosine phosphorylation by SHP-1 during binding of KIR to MHC class I on target cells is selective, suggesting that a limited number of substrates are dephosphorylated by SHP-1. We have chosen to study KIR inhibition as it occurs during binding of KIR to MHC class I on target cells, despite the technical limitations inherent to studies of processes regulated by cell contact. KIR binding to MHC class I on target cells inhibits tyrosine phosphorylation of the activation receptor 2B4 (CD244) and disrupts adhesion of NK cells to target cells. Inhibition of proximal events in NK activation may increase the availability of NK cells by liberating them from non-productive interactions with resistant target cells. As the receptors and the signaling pathways that induce NK cytotoxicity are not fully characterized, elucidation of the inhibitory mechanism employed by KIR may provide insight into NK activation.

NF-kappa B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development.

Activation of the transcription factor NF-kappa B and pre-T cell receptor (pre-TCR) expression is tightly correlated during thymocyte development. Inhibition of NF-kappa B in isolated thymocytes in vitro results in spontaneous apoptosis of cells expressing the pre-TCR, whereas inhibition of NF-kappa B in transgenic mice through expression of a mutated, superrepressor form of I kappa B alpha leads to a loss of beta-selected thymocytes. In contrast, the forced activation of NF-kappa B through expression of a dominant-active I kappa B kinase allows differentiation to proceed to the CD4(+)CD8(+) stage in a Rag1(-/-) mouse that cannot assemble the pre-TCR. Therefore, signals emanating from the pre-TCR are mediated at least in part by NF-kappa B, which provides a selective survival signal for developing thymocytes with productive beta chain rearrangements.

Signals involved in gamma/delta T cell versus alpha/beta T cell lineage commitment.

In seeking an explanation for the complexity of tissue development, biologists are obliged to explain lineage commitment, the events that dictate whether or not a progenitor cell will differentiate into one cell type or another. Such explanations have been sought across a broad spectrum of biological systems, although in no case has a full under- standing been developed. For immunologists, attention has been focused on the lineage commitment of a T cell progenitor to becoming either a gammadelta T cell or an alphabeta T cell. In this review, we compare the signals that thymocytes may receive from the pre T cell receptor (preTCR) with signalling from TCRgammadelta. These signals may determine, co-determine, facilitate, or cement the alphabeta/gammadelta lineage decision, in concert with signals from additional molecules, such as Notch and cytokine receptors. Elucidating the pleiotropic signalling events, particularly those elicited by the preTCR, may in the near future contribute to a molecular definition of lineage commitment.

The expression in vivo of a second isoform of pT alpha: implications for the mechanism of pT alpha action.

A second isoform of pT alpha, "pT alpha(b)," is derived from the pT alpha locus by tissue-specific, alternative splicing. pT alpha(b) is coexpressed in the thymus with the previously characterized form of pT alpha (which we term pT alpha(a)) and is also expressed in peripheral cells without pT alpha(a). While pT alpha(a) acts to retain most TCR beta-chains intracellularly, pT alpha(b) permits higher levels of cell surface TCR beta expression and facilitates signaling from a CD3-TCR beta complex.

Primary gamma delta cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation.

The division of CD4+ alpha beta T cells into Th1 and Th2 subsets has become an established and important paradigm. The respective activities of these subsets appear to have profound effects on the course of infectious and autoimmune diseases. It is believed that specific programs of differentiation induce the commitment of an uncommitted Th0 precursor cell to Th1 or Th2. A component of these programs is hypothesized to be the nature of MHC-peptide antigen presentation to the alpha beta T cell. It has heretofore remained uncertain whether a Th1/Th2 classification likewise defines, at the clonal level, gamma delta T cells. Such cells do not, as a general rule, express either CD4 or CD8 alpha beta, and they do not commonly recognize peptide-MHC. In this report, gamma delta cell clones are described that conform strikingly to the Th1/Th2 classification, both by cytokine expression and by functional activities of the clones in vitro and in vivo. Provocatively, both the gamma delta cell clones and primary gamma delta cells in vivo showed a strong association of the Th2 phenotype with CD4 expression. These results are discussed with regard to the immunoregulatory role that is increasingly emerging for gamma delta cells.

T-cell receptor usage in alloreactivity against HLA-B*2703 reveals significant conservation of the antigenic structure of B*2705.

B*2703 is an exceptional HLA-B27 molecule in that it differs from the most common B*2705 subtype by a unique amino acid change (His59) altering N-terminal peptide anchorage. To assess how this unusual feature affects the antigenic structure of HLA-B27, TCR usage by alloreactive CTL raised against B*2703 from two individuals was analyzed. Only few CTL recognized B*2703 from two individuals was analyzed. Only few CTL recognized B*2703 but nor ot at a lower level B*2705. Limited heterogeneity of these CTL was revealed by: 1) identity of TCR in two pairs of such CTL clones, 2) identity of beta chains, paired to distinct alpha chains, in two clonotypes, and 3) almost identical fine specificity of these two clonotypes with site-specific HLA-B27 mutants. These results indicate that B*2703 "private" epitopes are rare. TCR usage among anti-B*2703 CTL was analogous as in anti-B*2705 responses in the predominant and donor-independent usage of V beta segments from homology subgroup 4, more moderate and donor-dependent V alpha skewing, N+D beta diversity limited by motifs shared among clonotypes, and restricted J alpha heterogeneity. Homology of N+D beta motifs and J alpha segments of anti-B*2703 with anti-B*2705 TCR suggested significant sharing of peptide-associated epitopes between both subtypes. The results indicate that allospecific TCR are recruited by B*2703 following similar rules as in the anti-B*2705 response, and suggest that the B*2703 change keeps unaltered much of the antigenic structure of the molecule relative to B*2705. Therefore, most of the peptides bound to B*2703 should be the same and keep a similar conformation as in B*2705.

T cell receptor diversity in alloreactive responses against HLA-B27 (B*2705) is limited by multiple-level restrictions in both alpha and beta chains.

The T cell receptors (TCR) in HLA-B27 (B*2705) alloreactivity were analyzed in cytotoxic T lymphocytes (CTL) from two individuals. Non-random usage was found in V beta, N+D beta, V alpha, and J alpha, but not in J beta segments or N alpha-regions. V beta segments from homology subgroup 4 were predominant and not associated to a particular donor or fine specificity, suggesting involvement in recognizing the HLA-B27 molecule. In contrast, preferential V alpha usage was associated with particular individuals and fine specificities, indicating distinct V beta and V alpha recruitment and contribution to allorecognition. Recurrent N+D beta motifs and J alpha segments, even from different donors, limited junctional diversity, suggesting that CDR3 usage was determined by the alloantigenic epitope independently of individuals. TCR were selected differently at various levels, as indicated by the following findings. Four clonotypes with similar fine specificity had identical beta and unrelated alpha chains. Similar alpha were associated with unrelated beta chains, and vice versa. CTL using V beta subgroup 4 did not globally show concomitant predominance of other TCR elements. V alpha 7, one of the preferred V alpha segments, was always associated with V beta subgroups other than 4. Sometimes, a TCR showed homology in elements of one chain to a second TCR or group of TCR, and to another in the other chain. These results are best explained by differential selection of TCR elements by different epitopes, providing a key to the inner structure of allospecific TCR repertoires.

Cross-reactive T cell clones from unrelated individuals reveal similarities in peptide presentation between HLA-B27 and HLA-DR2.

HLA-B27- responder cells were stimulated in vitro with B*2705+ lymphoblastoid cell lines and alloreactive CTL clones were obtained by limiting dilution. Of the CD3+ CD4-CD8+ HLA-B27-specific CTL clones obtained, two of them, possessing the same TCR, cross-reacted with HLA-DR2. The fine specificity of these CTL was established with HLA-B27 and HLA-DR2 subtypes. They recognized the B*2701 to B*2706 subtypes, but only DR2Dw2. Lysis of DR2+ target cells was specifically inhibited by anti-CD3, anti-class II, and anti-DR mAb, but not with an anti-CD8 antibody. The monoclonal nature of the cross-reaction was established by the mutual inhibition of HLA-B27 and DR2Dw2 cells in cold target competition experiments. The DR2 protein involved in the cross-reaction was the heterodimer carrying the B5*0101 product, as shown by using L cell transfectants expressing each of the two molecules encoded in the DR2Dw2 haplotype. A correlation between the fine specificity of these CTL clones and the amino acid sequences of HLA-B27 and HLA-DR2 subtypes revealed a shared structural motif between HLA-B27 and the DR2 B5*0101 chain, which could be related to the observed cross-reaction. This motif was contributed for by several residues located in adjacent beta strands, at the floor of the peptide-binding site. The contribution of two of these residues, as well as other beta-pleated sheet residues to HLA-B27 allorecognition by the cross-reactive CTL clones was directly demonstrated with site-directed mutants. These results suggest that the dual reactivity pattern reflects presentation of identical or structurally related peptide by HLA-B27 and HLA-DR2Dw2. As T cell cross-reactivity between HLA-B27 and HLA-DR2 was previously found in cells from an unrelated individual the results reported here are likely to reflect an intrinsic property of HLA-B27, rather than the fortuitous finding of a rare clonal reaction pattern. We speculate on the potential implications of these results for the pathogeny of HLA-B27-associated spondyloarthropathies.