An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

Synovial membrane immunohistology in early untreated juvenile idiopathic arthritis: differences between clinical subgroups

Abstract: Objective Juvenile idiopathic arthritis (JIA) consists of a heterogeneous group of inflammatory disorders, within which there are a number of clinical subgroups. Diagnosis and assignment to a particular subgroup can be problematical and more concise methods of subgroup classification are required. This study of the synovial membrane characterises the immunohistochemical features in early untreated, newly diagnosed JIA and compares findings with disease subgroup at 2 years.

Methods: 42 patients with newly diagnosed untreated JIA underwent synovial biopsy before the administration of steroids or disease-modifying antirheumatic drugs. Patients were classified as either polyarticular, persistent oligoarticular or extended-to-be oligoarticular. The location and semiquantitative analysis of T-cell subsets, B cells, macrophages and blood vessels were determined using immunohistochemistry.

Results: Synovial hyperplasia varied significantly between the three groups
(p<0.0001). There was a significant difference in the CD3 T-cell population between the three groups (p=0.004) and between the extended-to-be and persistent group (p=0.032). CD4 expression was significantly higher in the poly and extended-to-be oligo groups (p=0.002), again the extended-to-be group had more CD4 T cells than the persistent group (p=0.008). B-cell infiltrates were more marked in the polyarticular group and were significantly higher in the extended-to-be group compared with the persistent group (p=0.005). Vascularisation was more pronounced in the polyarticular and extended-to-be oligoarticular groups, the extended-to-be group had significantly more vascularisation than the persistent group (p=0.0002).

Conclusions: There are significant differences in the histomorphometric features of synovial tissue between patient subgroups. Immunohistological examination of synovial membrane biopsies may provide further insight into early disease processes in JIA.

Gene expression profiling in MDS and AML: potential and future avenues

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients. Leukemia (2011) 25, 909-920; doi:10.1038/leu.2011.48; published online 29 March 2011

Comprehensive Inhibitor Profiling Of The Proteus Mirabilis Metalloprotease Virulence Factor Zapa (Mirabilysin)

In this study we report for the first time the comprehensive inhibitor profiling of the Proteus mirabilis metalloprotease virulence factor, ZapA (mirabilysin) using a 160 compound focused library of N-alpha mercaptoamide dipeptides, in order to map the S1´ and S2´ binding site preferences of this important enzyme. This study has revealed a preference for the aromatic residues tyrosine and tryptophan in P1´ and aliphatic residues in P2´. From this library, six compounds were identified which exhibited sub- to low micromolar Ki values. The most potent inactivator, SH-CO2-Y-V-NH2 was capable of preventing ZapA-mediated hydrolysis of heat denatured IgA, indicating these inhibitors may be capable of protecting host proteins against ZapA during colonisation and infection.

Composition profiling of seized ecstasy tablets by Raman spectroscopy

Raman spectroscopy with far-red excitation has been investigated as a simple and rapid technique for composition profiling of seized ecstasy (MDMA, N-methyl-3,4-methylenedioxyamphetamine) tablets. The spectra obtained are rich in vibrational bands and allow the active drug and excipient used to bulk the tablets to be identified. Relative band heights can be used to determine drug/excipient ratios and the degree of hydration of the drug while the fact that 50 tablets per hour can be analysed allows large numbers of spectra to be recorded. The ability of Raman spectroscopy to distinguish between ecstasy tablets on the basis of their chemical composition is illustrated here by a sample set of 400 tablets taken from a large seizure of > 50000 tablets that were found in eight large bags. The tablets are all similar in appearance and carry the same logo. Conventional analysis by GC-MS showed they contained MDMA. Initial Raman studies of samples from each of the eight bags showed that despite some tablet-to-tablet variation within each bag the contents could be classified on the basis of the excipients used. The tablets in five of the bags were sorbitol-based, two were cellulose-based and one bag contained tablets with a glucose excipient. More extensive analysis of 50 tablets from each of a representative series of sample bags gave distribution profiles that showed the contents of each bag were approximately normally distributed about a mean value, rather than being mixtures of several discrete types. Two of the sorbitol-containing sample sets were indistinguishable while a third was similar but not identical to these, in that it contained the same excipient and MDMA with the same degree of hydration but had a slightly different MDMA/sorbitol ratio. The cellulose-based samples were badly manufactured and showed considerable tablet-to-tablet variation in their drug/excipient ratio while the glucose-based tablets had a tight distribution in their drug/excipient ratios. The degree of hydration in the MDMA feedstocks used to manufacture the cellulose-, glucose- and sorbitol-based tablets were all different from each other. This study, because it centres on a single seizure of physically similar tablets with the same active drug, highlights the fact that simple physical descriptions coupled with active drug content do not in themselves fully characterize the nature of the seized materials. There is considerable variation in the composition of the tablets within this single seizure and the fact that this variation can be detected from Raman spectra demonstrates that the potential benefits of obtaining highly detailed spectra can indeed translate into information that is not readily available from other methods but would be useful for tracing of drug distribution networks.

Interaction of prothrombin with a phospholipid surface: evidence for a membrane-induced conformational change

Prothrombin interacts with phosphatidylserine containing platelet membranes via its N-terminal, gamma-carboxyglutamate (gla) residue-rich domain. Once bound it is cleaved to form the active protease, thrombin (factor IIa). Human prothrombin was cleaved with cathepsin G in the absence of calcium and magnesium ions. Under these conditions, the gla domain was removed. Phospholipid protected the protein from this proteolytic event, and this suggests that a conformational change may be induced by interaction with phospholipids. Binding of prothrombin to a surface containing 20% phosphatidylserine/80% phosphatidylcholine was detected by surface plasmon resonance, whereas no interaction with gla-domainless prothrombin was observed. Binding of intact prothrombin in the presence of calcium ions showed complex association kinetics, suggesting multiple modes of initial interaction with the surface. The kinetics of the dissociation phase could be fitted to a two-phase, exponential decay. This implies that there are at least two forms of the protein on the surface one of which dissociates tenfold more slowly than the other. Taken together, these data suggest that, on binding to a membrane surface, prothrombin undergoes a conformational change to a form which binds more tightly to the membrane.

Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments

Diabetes mellitus was induced in male beagles by a single injection of an alloxan and streptozotocin cocktail and fasting blood sugar levels maintained between 15 and 20 mmol/l. Five years after induction of diabetes, three diabetic animals were sacrificed, together with sex and age-matched controls, and the retinas fixed for either transmission electron microscopy (TEM) or trypsin digestion. In TEM specimens, capillaries in close proximity to the major vessels were designated as either AE (arterial environment) or VE (venous environment) and the thickness of their basement membranes (BMs) measured using an image analyser based two dimensional morphometric analysis system. Results show that the BMs of retinal capillaries from the diabetic dogs were significantly thicker than those from control dogs. Furthermore, within the diabetic group the AE capillaries had thicker BMs than VE capillaries (p less than or equal to 0.05). The controls, however, showed no significant difference in BM thickness between AE and VE capillaries. Although many of the capillaries designated as AE or VE would actually have been derived from the opposite side of the circulation, with respect to BM thickness, they conformed to values of their specific group. The conclusion is that diabetic capillaries are more vulnerable to BM thickening in an arterial environment than in a venous environment.