Antigen-Presenting Cell Characteristics of Human γδ T Lymphocytes in Chronic Myeloid Leukemia.

Human γδ T lymphocytes play a role in the immune system defense against cancer. Their broad anti-cancer activity against different types of cancers makes them outstanding candidates for cancer immunotherapy. An issue of recent interest is whether their antigen presentation features are similar to mature dendritic cells. The antigen-presenting cell (APC)-like phenotype and function of γδ T lymphocytes have been confirmed in many clinical trials. In this study, to support the strong role played by Vγ9Vδ2 T cells against cancer, we provide evidence that Vγ9Vδ2 T cells activated with chronic myeloid leukemia (CML) cell lysate antigens can efficiently express an APC phenotype and function. Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with tumor cell lysate, and the tumor-activated Vγ9Vδ2 T cells could recognize and kill CML through their cytotoxic activity. In conclusion, the Vγ9Vδ2 T cells activated by cancer cell lysate showed APC characteristics, and this may greatly increase interest in investigating their therapeutic potential in hematologic malignancies. Abbreviations: CML: chronic myeloid leukemia; APC: antigen-presenting cell; TCR: T cell receptor; MHC: major histocompatibility complex; N-BPs: nitrogen-containing bisphosphonates; IPP: isopentenyl pyrophosphate; PBMC: peripheral blood mononuclear cells; NKG2D: natural killer receptor group 2, member D; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand.

Solvent-sensitive nanoparticle-enhanced PCR assay for the detection of enterotoxigenic Escherichia coli.

Stimulus-responsive nanoparticles are among the most utilized nanoscale materials in biomedical applications. As these nanoparticles exhibit a manipulable response to a particular stimulus, such as pH, heat, and organic solvent, they are potential signalling units in diagnostic assays. This study aims to enhance the limit of detection and reduce the turnaround time of magnetic nanoparticle polymerase chain reaction (PCR) enzyme-linked gene assay (MELGA), an advanced PCR-based technique termed the solvent-sensitive nanoparticle (SSNP)-enhanced PCR assay. This technique was proposed to detect pathogenic enterotoxigenic Escherichia coli (ETEC) through applying stimulus-responsive nanoparticles. The SSNPs were elaborated with three main components, including mesoporous silica nanoparticles as a structural unit, organic dye (Nile red) as a payload, and the corresponding organic solvent-sensitive polymer shell as "gatekeeper" (poly(maleic anhydride-alt-methyl vinyl ether, PMAMVE). A suitable organic solvent capable of inducing polymer swelling and dye dissolution was investigated by considering a solubility parameter. Using ethanol, the encapsulated Nile red can diffuse out of the SSNPs faster than other solvents and reach a constant concentration within 15 min. For the PCR inhibition study, various SSNPs concentrations (10-30 µg/reaction) were mixed with the ETEC gene and PCR reagent. The results showed that the particles in this concentration range did not inhibit PCR. By comparing the efficacy of conventional PCR, MELGA, and SSNP-enhanced PCR assay, the proposed technique showed a better detection limit than that of PCR, whereas that of MELGA was the lowest. Moreover, compared to MELGA or conventional PCR, this technique provided remarkably faster results in the postamplification process.

Heat-enhancing aggregation of gold nanoparticles combined with loop-mediated isothermal amplification (HAG-LAMP) for Plasmodium falciparum detection.

Malaria infection represents a major public health and economic issue that leads to morbidity and mortality globally. A highly effective and uncomplicated detection tool is required for malaria control in geographical hotspots of transmission. We developed a simple and more sensitive novel approach for the detection of the 18S rRNA gene of Plasmodium falciparum based on loop-mediated isothermal amplification (LAMP) and visualization using colorimetric, streptavidin-functionalized gold nanoparticles (SA-GNPs). Two loop primers of LAMP were biotinylated to produce biotin-containing products during amplification. After the addition of SA-GNPs, clusters of avidin-biotin complexes were established in the LAMP structure. While the positive reactions remained wine red, the negative reactions became colorless with partial aggregations induced by hydrochloric acid (HCl) under heat enhancement (60 °C). All steps of the assay were completed within 50 min, its detection limit was 1 parasite/µL, and it was highly specific for P. falciparum. This effortless detection system with high sensitivity and specificity could provide an alternative choice for malaria diagnostics in resource-limited regions.

Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients.

Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 102 parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

Urinary PCA3 detection in prostate cancer by magnetic nanoparticles coupled with colorimetric enzyme-linked oligonucleotide assay.

PCA3 is one of the most prostate cancer-specific genes described to date. Of note, PCA3 expression is detectable at high level in the urine of prostate cancer (PCa) patients. Accordingly, PCA3 is an ideal biomarker for PCa diagnosis. Several techniques for the measurement of this biomarker in urine have been developed but there are still some drawbacks. In this study, magnetic nanoparticle-based PCR coupled with streptavidin-horseradish peroxidase and a substrate for colorimetric detection was established as a potential assay for urinary PCA3 detection. The method provided a high specificity for PCA3 gene in LNCaP prostate cancer cell line. Additionally, this technique could detect PCA3 at femtogram level which was approximately 1,000-fold more sensitive than the conventional RT-PCR followed by agarose gel electrophoresis. The effectiveness of the method was assessed by PCA3 detection in clinical specimens. The relative PCA3 expression of PCa patients determined by this assay was significantly greater than that of benign prostatic hyperplasia (BPH) patients and healthy controls. The results of our test were comparable with the results of qRT-PCR. The proposed method is promising to distinguish between cancerous and non-cancerous groups. Altogether, this simple assay is practicable and useful for prostate cancer diagnosis.

Enrichment of human Vγ9Vδ2 T lymphocytes by magnetic poly(divinylbenzene-co-glycidyl methacrylate) colloidal particles conjugated with specific antibody.

γδ T cells play a significant role in protection against cancer. Purification of γδ T cells is needed for insight when studying their anti-cancer functionality and their utilization in adoptive cell therapy. To improve the purification of γδ T cells, in this work, a composite material based on magnetic nanoparticles was developed for purification of Vγ9Vδ2 T cells, the predominant subset of γδ T lymphocytes in human peripheral blood. The epoxy-functionalized magnetic poly(divinylbenzene-co-glycidyl methacrylate) particles (mPDGs) were bio-conjugated with anti-human Vδ2 antibody to provide specific recognition sites for T cell receptors of Vγ9Vδ2 T cells. Using fluorescence-activated cell sorting (FACS) analysis, separation of Vγ9Vδ2 T cells from peripheral blood mononuclear cells of healthy donors was confirmed with high purity [89.77% (range 87.00-91.80, n = 3)]. More interestingly, the immobilized particles did not affect the viability of purified cells as high cell viability was indicated (>90%). By combining the properties of magnetic nanoparticles with specific antibodies, these immobilized particles were shown to be used as a cell-friendly purification tool of Vγ9Vδ2 T lymphocytes without any limits for the further use of cells. The purified Vγ9Vδ2 T cells using the antibody-immobilized epoxy-functionalized mPDGs could be used directly without a detachment step for further cultivation and expansion. This highlights the advantages of this method in allowing the study of cell function and further investigation of such rare T cell populations in immunotherapy.

Magnetic particles for in vitro molecular diagnosis: From sample preparation to integration into microsystems.

Colloidal magnetic particles (MPs) have been developed in association with molecular diagnosis for several decades. MPs have the great advantage of easy manipulation using a magnet. In nucleic acid detection, these particles can act as a capture support for rapid and simple biomolecule separation. The surfaces of MPs can be modified by coating with various polymer materials to provide functionalization for different applications. The use of MPs enhances the sensitivity and specificity of detection due to the specific activity on the surface of the particles. Practical applications of MPs demonstrate greater efficiency than conventional methods. Beyond traditional detection, MPs have been successfully adopted as a smart carrier in microfluidic and lab-on-a-chip biosensors. The versatility of MPs has enabled their integration into small single detection units. MPs-based biosensors can facilitate rapid and highly sensitive detection of very small amounts of a sample. In this review, the application of MPs to the detection of nucleic acids, from sample preparation to analytical readout systems, is described. State-of-the-art integrated microsystems containing microfluidic and lab-on-a-chip biosensors for the nucleic acid detection are also addressed.

Combination of PCR and dual nanoparticles for detection of Plasmodium falciparum.

Highly reactive particle-based DNA amplification was developed for the detection of the Pfg377 gene from P. falciparum gametocytes using functional magnetic latex particles (MLPs) and quantum dots encapsulated polymer particles (QDs-PPs). Firstly, MLPs were prepared from the precipitation of iron oxide, polymerization using initiator, and adsorption of aminodextran (AMD) so as to provide amino-functionalized MLPs. Furthermore, amino-containing polymer particles (PPs) were prepared by emulsifier-free polymerization and encapsulated with fluorescent quantum dots (QDs) for use as a signaling support. Subsequently, poly(maleic anhydride-alt-methyl vinyl ether) (PMAMVE) copolymer was effectively used for rapid and simple grafting of amino-modified DNA primers onto the surface of amino-functionalized particles thereby providing a promising method for particle immobilization. Herein, primer-grafted particles were applied in the amplification of the Pfg377 gene using the PCR approach. After amplification, PCR products containing PMAMVE-grafted MLPs and QDs-PPs were separated using a magnet and examined via a fluorescence microscope. PMAMVE-grafted particles were not found to inhibit the PCR reaction while facilitating efficient fluorescent detection of the PCR product. Results showed high sensitivity and specificity for the detection of amplified Pfg377 gene within only a few steps. This procedure represents a novel improvement to the post-amplification analysis.

Enhanced Sensitivity for Detection of Plasmodium falciparum gametocytes by magnetic nanoparticles combined with enzyme substrate system.

The highly sensitive and specific detection of Pfg377 gene of Plasmodium falciparum gametocyte using Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA) was successfully developed. The MELGA included amplification of the Pfg377 gene by polymerase chain reaction (PCR) using magnetic nanoparticles (MNPs)-conjugated forward primer and biotinylated reverse primer, followed by post-analytical process using horseradish peroxidase (HRP)-conjugated streptavidin (SA). The complexes of MELGA product were incubated with the peroxidase substrate and hydrogen peroxide to produce the signal for colorimetric measurement. Altogether, the MELGA technique provided a highly sensitive and specific detection at 1 P. falciparum gametocyte/µL, which was more efficient than that of microscopic examination and rapid diagnostic tests (RDTs). Additionally, the MELGA could detect target gene at femtogram level, which was greater sensitive than the conventional PCR, nested PCR and loop-mediated isothermal amplification (LAMP). The MELGA technique could become a novel and practical method that overcome limitation of traditional gametocyte detection.

Fluorescent chitosan functionalized magnetic polymeric nanoparticles: Cytotoxicity and in vitro evaluation of cellular uptake.

Nanoparticles possessing magnetic and fluorescent properties were fabricated by the covalent attachment of fluorescein isothiocyanate onto magnetic polymeric nanoparticles functionalized by chitosan. The synthesized magnetic polymeric nanoparticles-chitosan/fluorescein isothiocyanate were successfully used for labeling the living organ and blood-related cancer cells, i.e., HeLa, Hep G2, and K562 cells. The cytotoxicity test of nanoparticles at various incubation times indicated the high cell viability (>90%) without morphological change. The confocal microscopy revealed that they could pass through cell membrane within 2 h for K562 cells and 3 h for HeLa and Hep G2 cells and then confine inside cytoplasm of all types of tested cells for at least 24 h. Therefore, the synthesized magnetic polymeric nanoparticles-chitosan/fluorescein isothiocyanate would potentially be used as cell tracking in theranostic applications.

Enrichment of malaria parasites by antibody immobilized magnetic nanoparticles.

The simple and less expensive technique based on magnetic nanoparticles (MNPs) was developed for separation of malaria parasites containing specific antigens. The carboxylated MNPs were chemically bound with anti-P. falciparum IgG antibodies (Ab-MNPs) purified from the plasma of malaria patients and then used for removal of P. falciparum malaria-infected erythrocytes from other non-infected blood cells in malaria culture at a given percent parasitemia. The results from optical microscope showed that all blood stages parasites, i.e., ring, trophozoite and schizont, could be separated from other blood components with high purity (> or = 95%) and yield of 33.5% (the early stages of ring and trophozoite:the schizont stage were 1:1.34). Highly specific interaction between Ab-MNPs and the P. falciparum malaria infected erythrocytes was confirmed by scanning electron microscope. When compared to the centrifugation with Percoll gradient and depletion by sorbitol lysis which are specific to the mature and the ring stages, respectively, our technique would be more useful for production of high quality of parasites to use in malaria pathogenesis or immunological studies, and in detection techniques.