Balancing Thymocyte Adhesion and Motility: A Functional Linkage Between β1 Lntegrins and The Motility Receptor RHAMM

Thymocyte differentiation involves several processes that occur in different anatomic sites within the thymus. Therefore, thymocytes must have the ability to respond to signals received from stromal cells and adopt either adhesive or motile behavior. We will discuss our data indicating human thymocytes use α4β1 integrin, α5β1 integrin and RHAMM to mediate these activities. Immature multinegative (MN; CD3–4–8–19-) thymocytes use α4β1 and α5β1 integrins to mediate weak and strong adhesion. This subset also uses α4β1 integrin to mediate motility. As thymocytes differentiate, they begin to express and use RHAMM to mediate motility in conjunction with α4β1 and α5β1 integrins. Motile thymocytes use β1 integrins to maintain weakly adhesive contacts with substrate to provide traction for locomoting cells, thus weak adhesion is a requirement of motile behavior. Hyaluronan (HA) is also required by thymocytes to mediate motility. HA binding to cell surface RHAMM redistributes intracellular RHAMM to the cell surface where it functions to mediate motility. We propose that the decision to maintain adhesive or motile behavior is based on the balance between low and high avidity binding conformations of β1 integrins on thymocytes and that RHAMM:HA interactions decrease high avidity binding conformations of integrins pushing the balance toward motile behavior

Multiple Myeloma Includes Phenotypically Defined Subsets of Clonotypic CD20+ B Cells that Persist During Treatment with Rituximab

Potential progenitor B cell compartments in multiple myeloma (MM) are clinically important. MM B cells and some circulating MM plasma cells express CD20, predicting their clearance by treatment with anti-CD20. Here we describe two types of clonotypic CD20+ B cell in peripheral blood of myeloma patients, identified by their expression of CD19 and CD20 epitopes, their expression of CD45RA and their light scatter properties. Thus, the circulating component of the MM clone includes at least two distinct CD19+ CD20+ B cell compartments, as well as CD138+ CD20+ plasma cells. To determine whether either or both B cell subsets and the CD20+ plasma cell subset were depleted by anti-CD20 therapy, they were evaluated before, during and after treatment of patients with rituximab (anti-CD20), followed by quantifying B cell subsets over a 5 month period during and after treatment. Overall, all three types of circulating B lineage cells persist despite treatment with rituximab. The inability of rituximab to prolong survival in MM may result from this failure to deplete CD20+ B and plasma cells in MM

Storing self-contained gel capillary cassettes for POC medical diagnostics

For effective clinical uptake of the lab on a chip/point of care technology (LOC-POC), in addition to cost advantages LOC-POC devices should offer multiple patient screening panels for related diseases as well as cold-chain transportation and storage abilities. We recently described a device that performs polymerase chain reaction (PCR) to simultaneously screen raw clinical samples from up to 16 patients for multiple infectious agents (Manage et al., Lab Chip, 2013, 13, 2576–2584). This cassette contains glass capillaries with desiccated semi-solid acrylamide gels that include all the reagents except for the sample, with integrated quality control. Here we report the development of protocols to store assembled PCR cassettes at room temperature, 4 uC or 220 uC as well as at +40 uC. We show that our cassettes are stable, with no loss of activity for at least 3 months at RT and at least 7 months at 4 uC and 220 uC. However, the activity of desiccated cassettes degrades when stored for more than 2 weeks at 40 uC, insufficient time for postmanufacture delivery and use of cassette PCR. To address this, we have evaluated two stage storage protocols. PCR cassettes can initially be stored at 4 uC and 220 uC for prolonged periods of time and removed for shorter term storage at RT, retaining activity for at least a month, which would facilitate transport to remote areas for testing. Effective use of cassette PCR in high temperature regions of the world, for experimental purposes defined here as 40 uC, appears to be feasible only after a first stage storage in the cold, followed by no more than 1 week at 40 uC. This should allow sufficient time for delivery by the manufacturer to a central area well served by power and refrigeration, for later ambient temperature transport and use in under-resourced areas that lack refrigeration

An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics

This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format can simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis. To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma Urealyticum (UU) and Mycoplasma Homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50'. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for most clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will permit on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices

Comparison of a Miniaturized Cassette PCR System with a Commercially Available Platform for Detecting Escherichia coli in Beef Carcass Swabs

Detection sensitivity of cassette PCR was compared with a commercial BAX® PCR system for detection of eae and stx genes in Escherichia coli from 806 beef carcass swabs. Cassette PCR detects multiple genetic markers on multiple samples using PCR and melt curve analysis. Conventional PCR served as a gold standard. Overall, for positive and negative concordance, cassette PCR was 98.6% concordant with conventional PCR, and BAX PCR was 65.4% concordant. Of 806 beef carcass swabs, 339 by cassette PCR and 84 by BAX PCR harbored eae + stx+E. coli. For BAX PCR reactions, 84% of eae+ swabs, 79% of stx+ swabs, and 86% of eae + stx+ swabs were also detected by cassette PCR. For cassette PCR reactions, 457 swabs were eae+ with only 117 scored as eae+ using BAX PCR for 26% positive concordance. For stx primers, cassette PCR scored 480 samples as stx+ but only 215 samples were stx+ by BAX PCR, giving 45% positive concordance. Importantly, cassette PCR scored 339 swabs as harboring eae + stx+ E. coli, but BAX PCR detected only 71 positives giving only 21% positive concordance, with many false negatives. Cassette PCR is a highly sensitive method for detection of STEC genes in E. coli found in carcass swabs