Xueqing Lu

Comparison of polymeric samplers for accurately assessing PCBs in pore waters.

Assessing the hazard posed by sediments contaminated with hydrophobic organic compounds is difficult, because measuring the freely dissolved porewater concentrations of such low-solubility chemicals can be challenging, and estimating their sediment-water partition coefficients remains quite uncertain. We suggest that more accurate site assessments can be achieved by employing sampling devices in which polymers, with known polymer-water partition coefficients, are used to absorb the contaminants from the sediment. To demonstrate the current accuracy and limitations of this approach, we compared use of three polymers, polydimethylsiloxane, polyoxymethylene, and polyethylene, exposed to a single sediment in two modes, one in which they were exhaustively mixed (tumbled) with the sediment and the other in which they were simply inserted into a static bed (passive). Comparing porewater concentrations of specific polychlorinated biphenyl (PCB) congeners with results obtained using air bridges, we found the results for tumbled polymers agreed within 20%, and the passive sampling agreed within a factor of 2. In contrast, porewater estimates based on sediment concentrations normalized to f(OC)K(OC), the weight fraction of organic carbon times the organic-carbon normalized partition coefficient, averaged a factor of 7 too high. We also found good correlations of each polymers uptake of the PCBs with bioaccumulation by the polychaete, Neanthes arenaceodentata. Future improvements of the passive sampling mode will require devices that equilibrate faster and/or have some means such as performance reference compounds to estimate mass transfer limitations for individual deployments.

Enhanced CD4+ T-cell response in DR4-transgenic mice to a hybrid peptide linking the Ii-Key segment of the invariant chain to the melanoma gp100(48-58) MHC class II epitope.

Linking the Ii-Key functional group LRMK, through a simple polymethylene linker, to the melanoma gp100(48-58) MHC class II epitope significantly enhances the vaccine response to that epitope in DR4-IE transgenic mice. A homologous series of Ii-Key/gp100(46-58) hybrids was synthesized to test the influence of spacer length (between Ii-Key and the gp100(48-58) epitope) on in vivo enhancement of gp100(48-58)-specific CD4+ T-lymphocyte responses. As measured by IFN-γ and IL-4 ELISPOT cytokine assays, the most effective vaccine hybrid was the one with a shorter linker between Ii-Key and the epitope. Mechanistic reasons for this observation are considered. This structure-activity relationship was seen with bulk and CD4+ purified T cells, and both primary and secondary in vitro restimulation assays. CFA augmented the IFN-γ response and to a lesser extent the IL-4 response. CpG enhanced a strong IFN-γ response, with a negligible IL-4 response. The 3- to 5-times enhancement of the total ELISPOT responses (number of spots × mean spot area) observed after vaccination with peptides consisting of an MHC class II epitope engineered into an Ii-Key hybrid indicates a potent vaccine effect. Such constructs can be applied to many diagnostic and therapeutic uses.

Tumor immunotherapy by converting tumor cells to MHC class II–positive, Ii protein–negative phenotype

A potent antitumor CD4+ T-helper cell immune response is created by inducing tumor cells in vivo to a MHC class II+/Ii− phenotype. MHC class II and Ii molecules were induced in tumor cells in situ following tumor injection of a plasmid containing the gene for the MHC class II transactivator (CIITA). Ii protein was suppressed by the antisense effect of an Ii-reverse gene construct (Ii-RGC) in the same or another co-injected plasmid. The MHC class II+/Ii− phenotype of the tumor cells was confirmed by FACS analysis of cells transfected in vitro and by immunostaining of tumor nodules transfected by injections in vivo. Subcutaneous Renca tumors in BALB/c mice were treated by intratumoral injection with CIITA and Ii-RGC, in combination with a subtherapeutic dose of IL-2, to up-regulate the activation of T cells. Significant tumor shrinkage and decrease in rates of progression of established Renca tumors were seen in the groups injected with Ii-RGC, compared with groups in which only IL-2 plus empty plasmid controls were injected. Our method provides an effective immunotherapy warranting further development for human cancers.

Ii-Key/MHC class II epitope hybrid peptide vaccines for HIV

The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Hybrid peptides linking an immunoregulatory segment of the Ii protein (Ii-Key peptide) through a polymethylene bridge to MHC class II epitopes of HIV gp160 or gag are potent vaccines to elicit CD4(+) T cell responses. More potent responses to two MHC class II epitopes, HIV gp160(843-852) or HIV gag(279-292), occurred in mice immunized with Ii-Key hybrid peptides than with epitope-only peptides, as measured in IL-4 and IFN-gamma ELISPOT assays of splenic lymphocytes stimulated in vitro by epitope-only peptides. Both the number of responding cells and cytokine output per cell were increased. The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Such antigenic peptide constructs create opportunities to enhance greatly Th1 or Th2 responses to MHC class II epitopes for therapeutic purposes.

Ii-Key/MHC class II epitope hybrids: a strategy that enhances MHC class II epitope loading to create more potent peptide vaccines

Life-threatening diseases, such as cancer and pandemic influenza, demand new efforts towards effective vaccine design. Peptides represent a simple, safe and adaptable basis for vaccine development; however, the potency of peptide vaccines is insufficient in most cases for significant therapeutic efficacy. Several methods, such as Ligand Epitope Antigen Presentation System and ISCOMATRIX®, have been developed to enhance the potency of peptide vaccines. One way of increasing the loading of MHC class II peptides occurs through the use of Ii-Key technology. Ii-Key (LRMK), a portion of the MHC class II-associated invariant chain (Ii), facilitates the direct loading of epitopes to the MHC class II molecule groove. Linking the Ii-Key moiety via a simple polymethylene bridge to an MHC class II epitope, to generate an Ii-Key/MHC class II epitope hybrid, greatly enhances the vaccine potency of the tethered epitope. The combination of such Ii-Key/MHC class II epitope hybrids with MHC class I epitope-containing peptides might generate a potent peptide vaccine for malignancies and infectious diseases. The Ii-Key hybrid technology is compared with other methods that enhance the potency of a peptide vaccine.

Suppression of major histocompatibility complex class II-associated invariant chain enhances the potency of an HIV gp120 DNA vaccine.

One function of the major histocompatibility complex (MHC) class II‐associated invariant chain (Ii) is to prevent MHC class II molecules from binding endogenously generated antigenic epitopes. Ii inhibition leads to MHC class II presentation of endogenous antigens by APC without interrupting MHC class I presentation. We present data that in vivo immunization of BALB/c mice with HIV gp120 cDNA plus an Ii suppressive construct significantly enhances the activation of both gp120‐specific T helper (Th) cells and cytotoxic T lymphocytes (CTL). Our results support the concept that MHC class II‐positive/Ii‐negative (class II+/Ii–) antigen‐presenting cells (APC) present endogenously synthesized vaccine antigens simultaneously by MHC class II and class I molecules, activating both CD4+ and CD8+ T cells. Activated CD4+ T cells locally strengthen the response of CD8+ CTL, thus enhancing the potency of a DNA vaccine.

Radiation Improves Intratumoral Gene Therapy for Induction of Cancer Vaccine in Murine Prostate Carcinoma

Our goal was to convert murine RM-9 prostate carcinoma cells in vivo into antigen-presenting cells capable of presenting endogenous tumor antigens and triggering a potent T-helper cell-mediated immune response essential for the generation of a specific antitumor response. We showed that generating the major histocompatibility complex (MHC) class I+/class II+/Ii- phenotype, within an established subcutaneous RM-9 tumor nodule, led to an effective immune response limiting tumor growth. This phenotype was created by intratumoral injection of plasmid cDNAs coding for interferon gamma, MHC class II transactivator, and an antisense reverse gene construct (RGC) for a segment of the gene for Ii protein (-92,97). While this protocol led to significant suppression of tumor growth, there were no disease-free survivors. Nevertheless, irradiation of the tumor nodule on the day preceding initiation of gene therapy yielded 7 of 16 mice that were disease-free in a long-term follow up of 57 days compared to 1 of 7 mice receiving radiotherapy alone. Mice receiving radiotherapy and gene therapy rejected challenge with parental RM-9 cells and demonstrated specific cytotoxic T-cell activity in their splenocytes but not the mouse cured by radiation alone. These data were reproduced in additional experiments and confirmed that tumor irradiation prior to gene therapy resulted in complete tumor regression and specific tumor immunity in more than 50% of the mice. Increasing the number of plasmid injections after tumor irradiation induced tumor regression in 70% of the mice. Administering radiation before this novel gene therapy approach, that creates an in situ tumor vaccine, holds promise for the treatment of human prostate carcinoma.

Ii-Key/HPV16 E7 hybrid peptide immunotherapy for HPV16+ cancers

Activation of antigen-specific CD4+ T cells is critical for vaccine design. We have advanced a novel technology for enhancing activation of antigen-specific CD4+ T helper cells whereby a fragment of the MHC class II-associated invariant chain (Ii-Key) is linked to an MHC class II epitope. An HLA-DR4-restricted HPV16 E7 epitope, HPV16 E7(8-22), was used to create a homologous series of Ii-Key/HPV16 E7 hybrids testing the influence of spacer length on in vivo enhancement of HPV16 E7(8-22)-specific CD4+ T lymphocyte responses. HLA-DR4-tg mice were immunized with Ii-Key/HPV16 E7(8-22) hybrids or the epitope-only peptide HPV16 E7(8-22). As measured by IFN-gamma ELISPOT assay of splenocytes from immunized mice, one of the Ii-Key/HPV16 E7(8-22) hybrids enhanced epitope-specific CD4+ T cell activation 5-fold compared to the HPV16 E7(8-22) epitope-only peptide. We further demonstrated that enhanced CD4+ T cell activation augments the CTL activity of a H-2D(b)-restricted HPV16 E7(49-57) epitope in HLA-DR4+ mice using an in vivo CTL assay. Binding assays indicated that the Ii-Key/HPV16 hybrid has increased affinity to HLA-DR4+ cells relative to the epitope-only peptide, which may explain its increased potency. In summary, Ii-Key hybrid modification of the HLA-DR4-restricted HPV16 E7(8-22) MHC class II epitope generates a potent immunotherapeutic peptide vaccine that may have potential for treating HPV16+ cancers in HLA-DR4+ patients.