Small molecule- and amino acid-induced aggregation of gold nanoparticles.

To understand which organic molecules are capable of binding to gold nanoparticles and/or inducing nanoparticle aggregation, we investigate the interaction of gold nanoparticles with small molecules and amino acids at variable pH. Dynamic Light Scattering (DLS) and ultraviolet-visible (UV-vis) spectra were measured on mixtures of colloidal gold with small molecules to track the progression of the aggregation of gold nanoparticles. We introduce the 522 to 435 nm UV-vis absorbance ratio as a sensitive method for the detection of colloidal gold aggregation, whereby we delineate the ability of thiol, amine, and carboxylic acid functional groups to bind to the surfaces of gold nanoparticles and investigate how combinations of these functional groups affect colloidal stability. We present models for mechanisms of aggregation of colloidal gold, including surface charge reduction and bridging linkers. For all molecules whose addition leads to the aggregation of gold nanoparticles, the aggregation kinetics were accelerated at acidic pH values. Colloidal gold is maintained only in the presence of anionic carboxyl groups, which are neutralized by protonation at lower pH. The overall reduced charge on the stabilizing carboxyl groups accounts for the accelerated aggregation at lower pH values.

Tip preparation for near-field ablation at mid-infrared wavelengths.

A fabrication method for high-throughput, fiber-based tips for near-field scanning microscopy (NSOM) in the mid-infrared (λ ~ 3 µm) has been developed. Several fiber materials have been investigated and recipes for wet-chemical etching have been varied to produce tips that are physically robust and are capable of low-loss transmission of high-power pulses of mid-infrared light. Ultimately, wet-chemical etching techniques are used on glass fibers to produce tips capable of focusing mid-infrared light to ablate material from sub-micron-sized regions of organic films. The power throughput of the tips is significantly increased by using a novel material, previously unreported for NSOM applications: germanate fibers. The tips produced are mechanically strong and capable of transmitting high light fluence without sustaining physical damage. Here, the development of these tips and their performance are described.

Theoretical analysis of vertical colloidal deposition.

We have modeled the dynamics of a relatively new deposition technique, vertical colloidal deposition (VCD), for preparing nanoparticle thin films. In this process, the substrate is placed vertically in a nanoparticle suspension and is gradually exposed by evaporation or other slow solvent removal. During the film's formation, we observe that the colloidal particles are deposited only at the solid-liquid-gas interface. In contrast with the horizontal geometry, treated elsewhere, where the meniscus is pinned, we observe qualitatively different deposition behaviors. In particular, uniform films rather than rings or lines are produced. Thus, we are led to model a diffusion-driven rather than a convection-driven film growth kinetics, and we are able to predict, consistent with our experimental observations, that the film's areal density is inversely proportional to the descent speed of the suspension surface. Additionally, we find that for submonolayer films, the areal density is proportional to the square of the suspension concentration, converting to a linear dependence once monolayer coverage is attained.

Cavity resonators of metal-coated dielectric nanorods.

Metallic nanoparticles bridge the length scale between atoms and crystals, exhibiting mesoscopic properties unique to their size. Thus, they have generated much interest for their potential applications as chemical or biological sensors and particularly as waveguides for light in nanoscale structures. [Y. W. C. Cao, R. C. Jin, and C. A. Mirkin, Science 297, 1536 (2002); H. J. Lezec et al., Science 297, 820 (2002); S. A. Maier, P. G. Kik, and H. A. Atwater, Appl. Phys. Lett. 81, 1714 (2002); J. M. Oliva and S. K. Gray, Chem. Phys. Lett. 379, 325 (2003)]. One important direction of research into the properties of individual metal nanoparticles involves the controlled variation of their geometry, which can yield new and tunable optical properties that simple spherical configurations do not possess. [T. S. Ahmadi, Z. L. Wang, T. C. Green, A. Henglein, and M. A. Ei-Sayed, Science 272, 1924 (1996)]. A prime example of this is the core-shell nanostructure that has a central material surrounded by differing cladding layer.

Increased functional expression of transgene in primary human lymphocytes using retroviral vectors modified with IRES and splicing motifs.

Genetic modification of human lymphocytes is being employed in strategies to correct enzyme deficiencies, encode cytokines and to redirect lymphocytes to antigenic targets other than those encoded by their endogenous T cell receptor. However, expression of transgenes in primary lymphocytes is generally low. Reasoning that vector modification may lead to increased transgene expression and subsequent increases in function, we have performed two retroviral vector modifications and report their effect on the functional expression in primary lymphocytes. A chimeric receptor specific for the colon carcinoma-associated antigen, EGP40, was initially incorporated into the retroviral vector LXSN. In this vector, receptor expression is driven by the Moloney murine leukemia virus LTR, and neomycin phosphotransferase expression driven by the SV40 promoter. Replacement of SV40 with an internal ribosomal entry site (IRES) increased the transgene activity of a mouse T cell line and human PBL as judged by increased cytokine release in response to antigen positive target cells. A further increase in transgene function was generated by the additional incorporation of a splice acceptor motif into the construct. Human PBL transduced with vector incorporating both IRES and intron were consistently more effective at lysing antigen positive colorectal carcinoma cells.

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100.

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.

Genes and viruses in hepatobiliary neoplasia.

Hepatobiliary neoplasms comprise a significant portion of the worldwide cancer burden. Advances in basic science research have led to rapid progress in our understanding of the molecular events responsible for these dreaded diseases. The genetic changes associated with hepatocellular carcinoma (HCC) have received the most attention. Aflatoxin B1 exposure leads to mutations in the p53 tumor suppressor gene, most commonly a transversion in codon 249 that leads to a substitution of serine for arginine in the p53 protein. Numerous other tumor suppressor genes, oncogenes, and tumor gene pathways are altered in HCC. Hepatitis B virus (HBV) infection is strongly associated with HCC. HBV may cause HCC either directly via the HBV X protein, or indirectly by causing liver inflammation and cirrhosis. Hepatitis C virus (HCV) infection is also associated with HCC. Recent evidence suggests that the HCV core protein may play a role in hepatocarcinogenesis. Several inherited metabolic diseases are associated with HCC. It is likely that these diseases cause HCC indirectly by causing cirrhosis. The molecular pathogenesis of cholangiocarcinoma and gallbladder cancer has not been well defined. However, multiple tumor suppressor genes and oncogenes, including p53 and K-ras, are altered in these tumors. Further molecular characterization of hepatobiliary tumors may lead to earlier diagnosis, better staging, improved treatment planning, and the development of more effective therapies.

Melanoma. A multidisciplinary approach for the general surgeon.

Advances in the understanding of the biology and treatment of melanoma have moved the care of melanoma patients into an increasingly multidisciplinary environment. Surgeons must understand these advances because they will often be responsible for directing the overall care of these patients. Most patients with melanomas more than 1 mm in diameter and no evidence of metastatic disease should be offered SLNB to more accurately stage them and direct decisions about participation in postoperative adjuvant therapy trials. Until the results of the MSLT are known, the effect of SLNB and ELND on outcome remains unknown. SLNs should be analyzed with serial sectioning and immunohistochemistry to avoid missing micro-metastatic disease. Based on the results of the ECOG-1684 trial, the FDA approved IFN-alpha 2b for the adjuvant treatment of melanoma patients with thick primary tumors (> 4 mm) or resected nodal disease. IFN-alpha 2b treatment is expensive and potentially toxic. The data from ECOG-1684 do not support the use of IFN-alpha 2b in patients with node-negative disease. In light of the ECOG-1690 trial results, the role of high-dose IFN-alpha 2b in the management of patients with resected nodal disease is considerably less clear. Any recommendations for treatment with high-dose IFN-alpha 2b should be made only after weighing the costs, side effects, and potential benefits for individual patients. Numerous, less toxic, promising, adjuvant immunotherapeutic strategies have been developed and are being tested in multicenter, prospective, randomized trials. These strategies include GMK, PMCV, and Melacine. If the results of any of these trials show a survival advantage compared with placebo or equivalent survival compared with IFN-alpha 2b, these immunotherapeutic agents will become the adjuvant treatment of choice for patients with resected high-risk melanoma. RT-PCR detection of tyrosinase in SLNs can identify patients with submicroscopic nodal disease who may be at increased risk for recurrence or death from melanoma. An ongoing, prospective, randomized trial will determine whether patients with histologically negative but RT-PCR-positive SLNs will benefit from lymphadenectomy or adjuvant IFN-alpha 2b therapy. RT-PCR can also identify minimal residual disease in peripheral blood and bone marrow from patients with high-risk melanoma, but RT-PCR analysis of peripheral blood and bone marrow is still experimental, and procedural details need to be standardized and prospectively validated in large patient groups before its use can be considered the standard of care.

Dendritic cells retrovirally transduced with a model antigen gene are therapeutically effective against established pulmonary metastases.

Dendritic cells (DCs) are bone marrow-derived leukocytes that function as potent antigen presenting cells capable of initiating T cell-dependent responses from quiescent lymphocytes. DC pulsed with tumor-associated antigen (TAA) peptide or protein have recently been demonstrated to elicit antigen-specific protective antitumor immunity in a number of murine models. Transduction of DCs with TAA genes may allow stable, prolonged antigen expression as well as the potential for presentation of multiple, or unidentified, epitopes in association with major histocompatibility complex class I and/or class II molecules. To evaluate the potential efficacy of retrovirally transduced DCs, bone marrow cells harvested from BALB/c mice were transduced with either a model antigen gene encoding beta-galactosidase (beta-gal) or a control gene encoding rat HER-2/neu (Neu) by coculture with irradiated ecotropic retroviral producer lines. Bone marrow cells were differentiated into DC in vitro using granulocyte/macrophage colony-stimulating factor and interleukin-4. After 7 d in culture, cells were 45-78% double positive for DC phenotypic cell surface markers by FACS(R) analysis, and DC transduced with beta-gal were 41-72% positive for beta-gal expression by X-gal staining. In addition, coculture of beta-gal transduced DC with a beta-gal-specific T cell line (CTLx) resulted in the production of large amounts of interferon-gamma, demonstrating that transduced DCs could process and present endogenously expressed beta-gal. DC transduced with beta-gal and control rat HER-2/neu were then used to treat 3-d lung metastases in mice bearing an experimental murine tumor CT26.CL25, expressing the model antigen, beta-gal. Treatment with beta-gal-transduced DC significantly reduced the number of pulmonary metastatic nodules compared with treatment with Hank's balanced salt solution or DCs transduced with rat HER-2/neu. In addition, immunization with beta-gal-transduced DCs resulted in the generation of antigen-specific cytotoxic T lymphocytes (CTLs), which were significantly more reactive against relevant tumor targets than CTLs generated from mice immunized with DCs pulsed with the Ld-restricted beta-gal peptide. The results observed in this rapidly lethal tumor model suggest that DCs transduced with TAA may be a useful treatment modality in tumor immunotherapy.

Retroviral transduction of human dendritic cells with a tumor-associated antigen gene.

Dendritic cells (DCs) are potent antigen-presenting cells that can activate quiescent T lymphocytes. When pulsed with tumor-associated antigen (TAA) peptide or protein, murine DCs can provide antitumor immunity. We reasoned that DCs retrovirally transduced with TAA genes might have important advantages over peptide- or protein-pulsed DCs, including long-term TAA presentation in vivo, and presentation of important but undefined epitopes. Therefore, we attempted to retrovirally transduce human DCs with a melanoma TAA gene (MART-1) and determine whether these transduced DCs could raise a specific antitumor response from quiescent autologous T lymphocytes. After retroviral transduction, human CD34+ cells were differentiated into DCs in vitro using granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor. This method consistently yielded a population of DCs as analyzed by morphology, phenotype, and MLR. Flow cytometric analysis revealed that 22-28% of cells expressing the DC phenotype also expressed a transduced marker gene. When DCs were transduced with the gene encoding MART-1, they stimulated much higher levels of cytokine release by MART-1-specific tumor-infiltrating lymphocytes than control DCs transduced with an irrelevant gene. In vitro stimulation using MART-1-transduced DCs but not control-transduced DCs raised specific antitumor CTLs from autologous quiescent T cells. These results provide evidence that human DCs can be retrovirally transduced with a TAA gene and that these transduced cells can raise a specific antitumor immune response in vitro. Transduced DCs may be useful for in vivo immunization against TAA.

Improved gene transfer into human lymphocytes using retroviruses with the gibbon ape leukemia virus envelope.

Gene-modified lymphocytes have a potential role in the therapy of cancer, infectious diseases, and genetic disorders of the immune system. Current gene therapy protocols involving gene transfer into lymphocytes utilize retroviruses with amphotropic envelope proteins. However, transduction efficiencies in lymphocytes using these viruses are relatively low. A potential strategy to improve gene transfer efficiency is the utilization of alternative retroviral envelopes that target unique receptors on the cell surface. One such alternative retroviral envelope, the gibbon ape leukemia virus (GALV) envelope, targets a distinct surface receptor (GLVR-1) that is 60% homologous but not cross-reactive to the amphotropic receptor (GLVR-2/RAM-1). Understanding the relationship between receptor expression and transduction efficiency is important for designing new strategies to improve gene transfer. Therefore, we compared GLVR-1 and GLVR-2 mRNA levels in lymphocytes and found that GLVR-1 was expressed 8- to 19-fold higher than GLVR-2. We then analyzed whether this enhanced expression of GLVR-1 correlated with increased infectivity of lymphocytes by retroviral vectors that utilize the GALV envelope compared to those that use the amphotropic envelope. We evaluated retroviral vectors packaged with either PA317 or PG13, which express the amphotropic and GALV envelopes, respectively. Lymphocyte transduction with PG13-packaged vectors was 4- to 18-fold higher than that with PA317-packaged vectors. These findings suggest that receptor expression level is an important factor in retroviral-target interactions and that gene transfer into human T lymphocytes should be performed with retroviruses that use the GALV envelope as opposed to retroviruses that use the amphotropic envelope.

Biosorption of uranium by Pseudomonas aeruginosa strain CSU: characterization and comparison studies.

Pseudomonas aeruginosa strain CSU, a nongenetically engineered bacterial strain known to bind dissolved hexavalent uranium (as UO(2) (2+) and/or its cationic hydroxo complexes), was characterized with respect to its sorptive activity (equilibrium and dynamics). Living, heat-killed, permeabilized, and unreconstituted lyophilized cells were all capable of binding uranium. The uranium biosorption equilibrium could be described by the Langmuir isotherm. The rate of uranium adsorption increased following permeabilization of the outer and/or cytoplasmic membrane by organic solvents such as acetone. P. aeruginosa CSU biomass was significantly more sorptive toward uranium than certain novel, patented biosorbents derived from algal or fungal biomass sources. P. aeruginosa CSU biomass was also competitive with commercial cation-exchange resins, particularly in the presence of dissolved transition metals. Uranium binding by P. aeruginosa CSU was clearly pH dependent. Uranium loading capacity increased with increasing pH under acidic conditions, presumably as a function of uranium speciation and due to the H(+) competition at some binding sites. Nevertheless, preliminary evidence suggests that this microorganism is also capable of binding anionic hexavalent uranium complexes. Ferric iron was a strong inhibitor of uranium binding to P. aeruginosa CSU biomass, and the presence of uranium also decreased the Fe(3+) loading when the biomass was not saturated with Fe(3+), suggesting that Fe(3+) and uranium may share the same binding sites on biomass. Although the equilibrium loading capacity of uranium was greater than that of Fe(3+), this biomass showed preference of binding Fe(3+) over uranium. Thus, a two-stage process in which iron and uranium are removed in consecutive steps was proposed for efficient use of the biomass as a biosorbent in uranium removal from mine wastewater, especially acidic leachates.

Reductive microbial dechlorination of indigenous polychlorinated biphenyls in soil using a sediment-free inoculum.

In laboratory experiments, unagitated soil slurry bioreactors inoculated with micro-organisms extracted from polychlorinated biphenyl-contaminated (PCBs) sediments from the Hudson River were used to anaerobically dechlorinate PCBs. The onset of dechlorination activity was accelerated by the addition of certain organic acids (pyruvate and maleate) and single congeners (2,3,6-trichlorobiphenyl). Dechlorination was observed under several working conditions after 19 weeks of incubation with PCB-contaminated soil and nutrient solution. Best results showed a drop in average chlorine content from 4.3 to 3.6 chlorines per biphenyl due to a loss of m-chlorines. Soil used for these experiments was obtained from a PCB-contaminated (weathered Aroclor 1248) site at an electric power substation. Dechlorination was observed with no sediment particles or other matrix being added.

A metastatic tumor cell line has greatly reduced levels of a specific homotypic cell adhesion molecule activity.

The first step in tumor metastasis is the detachment of cells from the primary tumor. If metastatic tumor cells have decreased levels of active homotypic cell adhesion molecules (CAMs), this might aid their escape from the primary tumor. In order to determine whether homotypic CAM activity might be reduced in metastatic cells, a new direct CAM assay was developed. The assay gave a linear response with respect to the concentration of a CAM fragment preparation from Balb/3T3 cells and was able to follow partial purifications of the CAM activity. The yield of homotypic CAM activity was measured from metastatic cells, related tumorigenic but nonmetastatic cells, and parental cells. The parental Balb/3T3 and nonmetastatic Balb/3T3 MSV85 cells yielded 22.5 +/- 1.1 (SD) and 24.8 +/- 3.5 units of CAM activity per mg of protein, whereas the metastatic Balb/3T3 K234 cells yielded only 4.6 +/- 0.8 units/mg. The homotypic adhesiveness of each of the cell lines was closely correlated with the level of CAM activity. When the CAM activity from each of the three cell lines was serially fractionated with the same column, the parental Balb/3T3 and nonmetastatic Balb/3T3 MSV85 cells each had one major peak of CAM activity that eluted in the same place. However, the metastatic Balb/3T3 K234 cells were missing this peak of CAM activity. These results suggest that levels of homotypic CAM activity are greatly reduced in a metastatic cell line because the cells are missing a specific CAM activity. This would presumably allow the metastatic cells to escape more easily from the primary tumor and provide a molecular explanation for how they can complete the first step in metastasis.

Establishment of control parameters for in situ, automated screening of sustained hydrogen photoproduction by individual algal colonies.

An apparatus was constructed which allowed automated screening of individual microalgal colonies for sustained ability to photoevolve H(2) during anaerobic photosynthesis. The main components of this apparatus were a microcomputer, a He-Ne laser mounted on a computer-controlled X-Y translation stage, a flow-through chamber which contained an agar plate of colonies, and a H(2) detector which interfaced with the microcomputer for data collection. The system was capable of detecting a minimum production rate of 1 nanomole of H(2) per hour per colony and provided an efficient means of screening relatively large numbers of algal colonies. Examination of the effect of the spacing of colonies on the agar plate, light intensity, stability of colonies within a screening period, colony age, chlorophyll content, and colony size on H(2) yield indicated that, under optimum conditions, yields from genetically uniform colonies varied by no more than a factor of 2 in their H(2)-producing ability. Therefore, colonies of algae whose H(2) yields lie outside this intrinsic twofold variability can be identified and selected as natural variants or mutants. A description of the construction and of the apparatus is presented, and the experimental results used to establish the control parameters for Chlamydomonas reinhardtii colonies are discussed.