Crystal structure of the streptococcal superantigen SpeI and functional role of a novel loop domain in T cell activation by group V superantigens

Superantigens (SAgs) are potent microbial toxins that bind simultaneously to T cell receptors (TCRs) and class II major histocompatibility complex molecules, resulting in the activation and expansion of large T cell subsets and the onset of numerous human diseases. Within the bacterial SAg family, streptococcal pyrogenic exotoxin I (SpeI) has been classified as belonging to the group V SAg subclass, which are characterized by a unique, relatively conserved ∼15 amino acid extension (amino acid residues 154 to 170 in SpeI; herein referred to as the α3-β8 loop), absent in SAg groups I through IV. Here, we report the crystal structure of SpeI at 1.56 Å resolution. Although the α3-β8 loop in SpeI is several residues shorter than that of another group V SAg, staphylococcal enterotoxin serotype I, the C-terminal portions of these loops, which are located adjacent to the putative TCR binding site, are structurally similar. Mutagenesis and subsequent functional analysis of SpeI indicates that TCR β-chains are likely engaged in a similar general orientation as other characterized SAgs. We show, however, that the α3-β8 loop length, and the presence of key glycine residues, are necessary for optimal activation of T cells. Based on Vβ-skewing analysis of human T cells activated with SpeI and structural models, we propose that the α3-β8 loop is positioned to form productive intermolecular contacts with the TCR β-chain, likely in framework region 3, and that these contacts are required for optimal TCR recognition by SpeI, and likely all other group V SAgs.     

PCR Based Diagnosis of Begomoviruses Associated with Tomato Leaf Curl Disease in India

This paper reports development of a set of common primers and four sets of virus specific primers to detect ToLCNDV, ToLCBV, ToLCGV and ToLCKV, which are associated with tomato leaf curl disease (TomLCD) in India. The primer sets were first validated by amplification of desired genomic fragments from the cloned genomes of the four begomoviruses and then were tested for the detection of these viruses in field samples collected from different locations in India. Of the 26 TomLCD samples tested by PCR, 22 tested positive with the set of common primers and 16 tested positive for the target begomoviruses. ToLCNDV was detected in four samples, ToLCBV in six, ToLCKV in four, and ToLCGV in five samples. This limited survey shows that ToLCNDV and ToLCBV are distributed in northern and southern India, whereas ToLCKV and ToLCGV have a wider distribution. Mixed infection by two target viruses was observed in three TomLCD samples collected from Maharashtra, Punjab and Uttar Pradesh; two of these samples were infected with ToLCKV, which is prone to recombination. The results of the present study are indicative of association of uncharacterized variants or new begomoviruses with TomLCD in India, as (a) 27% of the samples found positive by the set of common primers did not amplify with species specific primers, and (b) 16% of the samples tested negative by PCR using common primers, although all the samples were collected from plants showing typical TomLCD symptoms. These plants might have been infected by some uncharacterized virus(es).     

Development and characterization of lipidic cochleate containing recombinant factor VIII

Hemophilia A, a life-threatening bleeding disorder, is caused by deficiency of factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII-containing cochleate cylinders, utilizing PS-Ca²⁺ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze-thawing and rate-limiting diffusion studies revealed that small cochleate cylinders with a particle size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications.     

Cutaneous manifestations in non-Hodgkin's lymphoma

OBJECTIVE: To examine and subtype cutaneous lymphoma specimens for diagnosis. STUDY DESIGN: Aspiration smears from skin lesions and lymph nodes diagnosed as non-Hodgkin's lymphoma (NHL) on cytology in 6 cases over a period of 1 year were reviewed. Two were follow-up cases of nodal lymphoma and were receiving chemotherapy, during which they developed skin lesions. In 4, the patients had cutaneous lesions as a presenting manifestation. Cytologic findings were correlated with histologic and hematologic findings and immunocytochemical markers for subtyping. RESULTS: Patients ranged from 14 to 50 years, with equal sex ratio. All presented with 0.5-5 cm multiple nodular, ulcerated and fungating skin lesions at various body sites. The aspirate was satisfactory in all cases. Cytologically, all cases were diagnosed as NHL. They were then immunocytochemistry subtyped as various lymphomas. CONCLUSION: Cutaneous lymphoma should always be considered in the presence of predominantly atypical lymphoid cells in smears from nodular and fungating skin lesions, even in the absence of a definitive clinical diagnosis.     

Ligand-Mediated Friction Determines Morphodynamics of Spreading T?Cells

Spreading of T cells on antigen presenting cells is a crucial initial step in immune response. Spreading occurs through rapid morphological changes concomitant with the reorganization of surface receptors and of the cytoskeleton. Ligand mobility and frictional coupling of receptors to the cytoskeleton were separately recognized as important factors but a systematic study to explore their biophysical role in spreading was hitherto missing. To explore the impact of ligand mobility, we prepared chemically identical substrates on which molecules of anti-CD3 (capable of binding and activating the T cell receptor complex), were either immobilized or able to diffuse. We quantified the T cell spreading area and cell edge dynamics using quantitative reflection interference contrast microscopy, and imaged the actin distribution. On mobile ligands, as compared to fixed ligands, the cells spread much less, the actin is centrally, rather than peripherally distributed and the edge dynamics is largely altered. Blocking myosin-II or adding molecules of ICAM1 on the substrate largely abrogates these differences. We explain these observations by building a model based on the balance of forces between activation-dependent actin polymerization and actomyosin-generated tension on one hand, and on the frictional coupling of the ligand-receptor complexes with the actin cytoskeleton, the membrane and the substrate, on the other hand. Introducing the measured edge velocities in the model, we estimate the coefficient of frictional coupling between T Cell receptors or LFA-1 and the actin cytoskeleton. Our results provide for the first time, to our knowledge, a quantitative framework bridging T cell-specific biology with concepts developed for integrin-based mechanisms of spreading.     

Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.     

Leukocyte Activity in Patients with ST-Segment Elevation Acute Myocardial Infarction Treated with Anakinra

Anakinra, the recombinant form of the human interleukin (IL)-1 receptor antagonist, blunts the acute systemic inflammatory response in patients with ST-segment elevation myocardial infarction (STEMI), by determining a fall in peripheral blood leukocyte and plasma C-reactive protein levels. The aim of the present study was to determine the effects of anakinra on the activity of leukocytes measured ex vivo. Blood was collected 72 h after admission in 17 patients enrolled in the Virginia Commonwealth University - Anakirna Remodeling Trial (2) (VCU-ART2) and randomly treated with anakinra (N = 7) or placebo (N = 10). Whole blood was cultured at 37°C for 24 h to measure spontaneous production of IL-6 or stimulated with Escherichia coli lipopolysaccharide (LPS) for toll-like receptor (TLR)-4 or heat-killed Staphylococcus epidermidis (SE) for TLR-2 activation. The cultures of anakinra-treated patients produced significantly less IL-6 spontaneously (71 pg/mL [27–114]) compared with placebo-treated patients (290 pg/mL [211–617], p = 0.005). LPS- or SE-induced IL-6 production, on the other hand, was not statistically different between anakinra-versus placebo-treated patients (344 pg/mL [94–560] versus 370 pg/mL [306–991], p = 0.32 for LPS, and 484 pg/mL [77–612] versus 615 pg/mL [413–871], p = 0.31 for SE, respectively). IL-1 blockade with anakinra in STEMI patients results in reduced spontaneous leukocyte activity ex vivo without impairing the responsiveness to bacterial stimuli.     

Photoinduced oxidation of a tris(2,2'‐bipyridyl)ruthenium(II)–peroxodisulfate chemiluminescence system for the analysis of mebeverine HCl pharmaceutical formulations and biological fluids using a two‐chip device

A new method for the analysis of mebeverine hydrochloride (MEB) has been developed using a two‐chip device. The method is highly selective, sensitive, rapid and consumes minute amount of reagents. The developed method is free of interference from the degradation products of MEB and from common ingredients present in pharmaceutical formulations. The limit of detection was 0.043 µg/mL, and the limit of quantification was 0.138 µg/mL. The short analysis time per sample (20 s) allowed a large number of analyses to be performed within a very short time. Various samples were analyzed, including two different pharmaceutical formulations and a uniformity of content analysis for 20 tablets from a known batch and two biological samples at different concentrations. In addition, the method was compared with a validated high‐performance liquid chromatography (HPLC) method and the results clearly indicated the suitability of the developed method for routine analyses. A new mechanism for the tris(2,2'‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐peroxodisulfate (S₂O₈^2?) chemiluminescence (CL) system has also been proposed. The mechanism is based on photoinduced oxidation of Ru(bpy)₃²⁺ to Ru(bpy)₃³⁺ via the formation of Ru(bpy)₃²⁺* upon irradiation with visible light. S₂O₈^2? then oxidizes Ru(bpy)₃²⁺* to Ru(bpy)₃³⁺ and the analyte subsequently reduces the resultant Ru(bpy)₃³⁺ to Ru(bpy)₃²⁺*, which then produces the CL signal. Copyright © 2013 John Wiley & Sons, Ltd.