Nanostructured and Photochromic Material for Environmental Detection of Metal Ions.

Compared to conventional spectroscopy or chromatography analysis, chemical sensing based on colorimetric changes offers an alternative to monitor potential metal hazards in aqueous environment through rapid and low-cost colorimetric changes which can be easily interpreted. In this work poly(ethylene glycol) (PEG 2000) was modified with a carboxylic acid spiropyran (SPCOOH) derivate by Steglich esterification (PEGSP2). PEGSP2 was incorporated into a poly(-caprolactone) (PCL) polymer matrix by electrospinning technique to produce nanofibers with photochromic properties. Spectroscopic analysis, thermal gravimetric analysis (TGA), and differential scanning calorimetry (DSC) were used to characterize PEGSP2. Drop shape analysis (DSA) and scanning electronic microscopy (SEM) were used to characterize the electrospun (ES) nanofibers morphology. Several metal ions solutions relevant to environmental hazards were prepared to be spotted on the surface of ES nanofibers for photochromatic sensing. Among them, Mg2+, Ca2+, Zn2+, Cd2+, La3+, and Er3+ demonstrated orange fluorescence when exposed to UV light. ES nanofibers also presented higher wettability when compared to a pure PCL polymer matrix, which is critical for sensitivity. Eighteen metals ions could be detected on the electrospun material. Additionally, among all metal ions Fe3+ was the most sensitive one in solution, in a µmol L1 range.

Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.

Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening.

The detection of rhodamine 123 efflux at low levels of drug resistance.

Although many cell models of multidrug resistance (MDR) have been developed, most have been high-resistance models which generate up to 100-fold increases in drug resistance. However, the drug concentrations required to achieve these levels of resistance are much higher than those found in vivo. In this paper we describe the development of a cell model that reflects the resistance levels that are likely to be found clinically. We then investigated the methods used to detect MDR1 expression at these low levels of drug resistance. We demonstrated that the immunological and PCR-based methods are unable to detect increased MDR1 expression in cells with a < 5.2- and 6.5-fold increase in vinblastine resistance, respectively, in our drug-resistant sublines. The rhodamine 123 (Rh 123) efflux assay was able to discriminate the vinblastine-sensitive parent cells from all the vinblastine-resistant sublines, including cells with a 1.7-fold increase in resistance. However, this assay is non-specific to the MDR1 gene and may detect the activity of other drug efflux proteins such as the MDR-associated protein (MRP). Our results show that the Rh 123 efflux assay is able to detect the activity of drug efflux proteins such as the P-glycoprotein, MRP or other efflux systems at the low levels of drug resistance that are likely to be attained in vivo.

Streptavidin-biotin immunotoxins: a new approach to purging bone marrow.

Immunotoxins have been used both experimentally and clinically to purge bone marrow of tumor cells or T cells before transplantation. We describe the synthesis of a streptavidin-biotin-toxin conjugate using whole ricin. Streptavidin-biotin-ricin (SA-BR) conjugates were synthesized by biotinylation of whole ricin, which was then complexed with streptavidin. Hybrid molecules consisting of a single biotinylated ricin moiety linked to a streptavidin molecule were separated by gel filtration. This SA-BR conjugate was used in an indirect cytotoxicity assay. The assay involved sensitizing of target cells with biotinylated monoclonal antibody (B-MCAB) followed by treatment with dilutions of SA-BR conjugate. The assay demonstrated a specific antibody-directed cytotoxicity. The strength of this SA-BR system is that a single conjugate was able to be used in conjunction with a library of B-MCABs to selectively target phenotypically different cell types. The application of the SA-BR conjugate is thus only restricted by the availability of B-MCABs specific for the desired target cells. The high affinity of avidin for biotin (Kd approximately 10(-15)) and the ability of a single conjugate to target phenotypically different cells through utilization of a library of B-MCABs gives SA-BR conjugates great potential in the selective targeting of individual cell types.

Monoclonal antibody-ricin conjugate cytotoxic to cells expressing the common acute lymphoblastic leukemia antigen (CALLA).

The monoclonal antibody PHM-6, which is specific for the common acute lymphoblastic leukemia antigen (CALLA), was conjugated to the plant toxin ricin. Binding of the PHM-6-ricin conjugate to cells via the ricin molecule was blocked by the presence of 100 mM lactose. The IC50 (concentration resulting in 50% inhibition) of the PHM-6-ricin conjugate for the CALLA-positive KM-3 cell line was 280-fold greater than for bone marrow stem cells, indicating the potential of this conjugate for immunological purging of autologous remission marrow.

Hyperthermia potentiates the activity of immunotoxin conjugates against common acute lymphoblastic leukaemia cells in vitro.

We studied the in-vitro cytotoxic effect of hyperthermia at 42 degrees C, both alone and in combination with ricin-linked immunotoxins, reactive with the common acute lymphoblastic leukaemia cell lines Reh and KM-3. Assessment of cytotoxicity was by incorporation of 3H-leucine and limiting dilutions analysis. The effect of immunotoxins alone and in combination with hyperthermia on normal human marrow progenitor cells was assessed by conventional colony forming units-granulocyte macrophage (CFU-GM) assay. We found that incubation of either of the cell lines with a mixture of the two immunotoxins, RPH-7-ricin and PHM-6-ricin, at 42 degrees C for one hour (h) potentiated the cytotoxic activity of the immunotoxins at 37 degrees C. At a concentration of 10(-8) mol/L, a 2.2-log kill was seen with KM-3 leukaemic cells at 37 degrees C and a 3.3-log kill at 42 degrees C, an increase of approximately 10 fold in cytotoxic activity. Survival of CFU-GM following treatment at 42 degrees C for one h with a similar concentration of immunotoxins was 26.2% (+/- 13.7%) (equivalent to 0.6 log kill) and 76.0% (+/- 1.83%) (0.1 log kill) when normal marrow was incubated with immunotoxins at 37 degrees C. This suggests relative sparing of normal marrow cells compared with the leukaemic cells tested as indicated by the 2.1-log kill difference (approximately 100 fold) between normal and leukaemic cells at 37 degrees C and the 2.7-log kill (approximately 500-fold) difference at 42 degrees C. We conclude that hyperthermia may have a role in addition to immunotoxins in the purging of marrow ex vivo to remove leukaemic cells.