Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection

Because CD4(+) T cells play a key role in aiding cellular immune responses, we wanted to assess whether increasing numbers of gene-engineered antigen-restricted CD4(+) T cells could enhance an antitumor response mediated by similarly gene-engineered CD8(+) T cells. In this study, we have used retroviral transduction to generate erbB2-reactive mouse T-cell populations composed of various proportions of CD4(+) and CD8(+) cells and then determined the antitumor reactivity of these mixtures. Gene-modified CD4(+) and CD8(+) T cells were shown to specifically secrete Tc1 (T cytotoxic-1) or Tc2 cytokines, proliferate, and lyse erbB2(+) tumor targets following antigen ligation in vitro. In adoptive transfer experiments using severe combined immunodeficient (scid) mice, we demonstrated that injection of equivalent numbers of antigen-specific engineered CD8(+) and CD4(+) T cells led to significant improvement in survival of mice bearing established lung metastases compared with transfer of unfractionated (largely CD8(+)) engineered T cells. Transferred CD4(+) T cells had to be antigen-specific (not just activated) and secrete interferon gamma (IFN-gamma) to potentiate the antitumor effect. Importantly, antitumor responses in these mice correlated with localization and persistence of gene-engineered T cells at the tumor site. Strikingly, mice that survived primary tumor challenge could reject a subsequent re-challenge. Overall, this study has highlighted the therapeutic potential of using combined transfer of antigen-specific gene-modified CD8(+) and CD4(+) T cells to significantly enhance T-cell adoptive transfer strategies for cancer therapy.

Uptake and metabolism of ginsenoside Rh2 and its aglycon protopanaxadiol by Caco-2 cells

The uptake and metabolism profiles of ginsenoside Rh2 and its aglycon protopanaxadiol (ppd) were studied in the human epithelial Caco-2 cell line. High-performance liquid chromatography-mass spectrometry was applied to determine Rh2 and its aglycon ppd concentration in the cells at different pH, temperature, concentration levels and in the presence or absence of inhibitors. Rh2 uptake was time and concentration dependent, and its uptake rates were reduced by metabolic inhibitors and influenced by low temperature, thus indicating that the absorption process was energy-dependent. Drug uptake was maximal when the extracellular pH was 7.0 for Rh2 and 8.0 for ppd. Rh2 kinetic analysis showed that a non-saturable component (K-d 0.17 nmol (.) h(-1) (.) mg(-1) protein) and an active transport system with a K-m of 3.95 mumol (.) l(-1) and a V-max of 4.78 nmol(.)h(-1) (.)mg(-1) protein were responsible for the drug uptake. Kinetic analysis of ppd showed a non-saturable component (K-d 0.78 nmol (.) h(-1) (.) mg(-1) protein). It was suggested that active extrusion of P-glycoprotein and drug degradation in the intestine may influence Rh2 bioavailability.

Unidirectional inversion of ibuprofen in Caco-2 cells: Developing a suitable model for presystemic chiral inversion study

Intestinal chiral inversion of ibuprofen is still lacking direct evidence. In a preliminary experiment, ibuprofen was found to undergo inversion in Caco-2 cells. This investigation was thus conducted to determine the characteristics and influence of some biochemical factors on the chiral inversion of ibuprofen in Caco-2 cells. The effects of substrate concentration (2.5-40 mu g/ml), cell density (0.5-2 x 10(6) cells/ well), content of serum (0-20%), coexistence of S ibuprofen (corresponding doses), sodium azide (10mm), exogenous Coenzyme A (CoA) (0.1 - 0.4 mm),. and palmitic acid (5-25 mu m) on inversion were examined. A stereoselective HPLC method based on the Chromasil-CHI-TBB column was developed for quantitative analysis of the drug in cell culture medium. The inversion ratio (F-i) and elimination rate constant were calculated as the indexes of inversion extent. Inversion of ibuprofen in Caeo-2 cells was found to be both dose and cell density dependent, indicating saturable characteristics. Addition of serum significantly inhibited the inversion, to an extent of 2.7 fold decrease at 20% content. Preexistence of S enantiomer exerted a significant inhibitory effect (p < 0.01 for all tests). Sodium azide decreased the inversion ratio from 0.43 to 0.32 (p < 0.01). Exogenous CoA and palmitic acid significantly promoted the inversion at all tested doses (p < 0.01 for all tests). This research provided strong evidence to the capacity and capability of intestinal chiral inversion. Although long incubation times up to 120 h were required, Caco-2 cells should be a suitable model for chiral inversion research of 2-APAs considering the human-resourced and well-defined characteristics from the present study.

T1/ST2 - an IL-1 receptor-like modulator of immune responses

Th2-associated factors such as IL-4 are involved in both the development of Th2 responses (via modulating Th2 cell differentiation) and in the effector phase of Th2 responses (via modulating macrophage activation). The IL-1 receptor-like protein ST2 (T1, Fit-1, or DER4) is expressed as a membrane-bound (ST2L) or secreted form (sST2), and has been clearly implicated as a regulator of both the development and effector phases of Th2-type responses. Here we analyze the mechanisms and therapeutic implications of the unique ability of ST2 to promote development and function of type 2 helper T cells through a positive feedback loop, as well as to act as a negative feedback modulator of macrophage pro-inflammatory function. (C) 2004 Elsevier Ltd. All rights reserved.

Prediction of HLA-DQ3.2 beta ligands: evidence of multiple registers in class II binding peptides

Motivation: While processing of MHC class II antigens for presentation to helper T-cells is essential for normal immune response, it is also implicated in the pathogenesis of autoimmune disorders and hypersensitivity reactions. Sequence-based computational techniques for predicting HLA-DQ binding peptides have encountered limited success, with few prediction techniques developed using three-dimensional models. Methods: We describe a structure-based prediction model for modeling peptide-DQ3.2 beta complexes. We have developed a rapid and accurate protocol for docking candidate peptides into the DQ3.2 beta receptor and a scoring function to discriminate binders from the background. The scoring function was rigorously trained, tested and validated using experimentally verified DQ3.2 beta binding and non-binding peptides obtained from biochemical and functional studies. Results: Our model predicts DQ3.2 beta binding peptides with high accuracy [area under the receiver operating characteristic (ROC) curve A(ROC) > 0.90], compared with experimental data. We investigated the binding patterns of DQ3.2 beta peptides and illustrate that several registers exist within a candidate binding peptide. Further analysis reveals that peptides with multiple registers occur predominantly for high-affinity binders.

Vaccine-induced Th1-type responses are dominant over Th2-type responses in the short term whereas pre-existing Th2 responses are dominant in the longer term

The effect of adjuvant on induction of human papillomavirus type 16 E7 protein-specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG)(2a) antibody was studied in C57BL/6 J mice immunized with various adjuvants and E7 protein. Quil-A adjuvant, but not complete Freund's adjuvant (CFA) or Algammulin, induced a T-helper 1 (Th1)-type response to E7, which was characterized by CTL activity against a tumour cell line transfected with E7 protein and by E7-specific IgG(2a). All tested adjuvants elicited comparable levels of E7-specific IgG(1). The longest duration and greatest magnitude of CTL response was seen following two immunizations with the highest dose of E7 and Quil-A. Simultaneous immunization with a Th1 and a T helper 2 (Th2)-promoting adjuvant gave a Th1-type response. However, E7 and Quil-A were unable to induce a Th1-type response (as measured by the inability to generate anti-E7 IgG(2a) antibody) in animals with a pre-existing Th2-type response to E7. These results suggest that saponin adjuvants may be suitable for immunotherapy in humans where a Th1-type response is sought, provided that there is no pre existing Th2-type response to the antigen.

Ca2+-activated K+ channels in rat otic ganglion cells: Role of Ca2+ entry via Ca2+ channels and nicotinic receptors

1. Intracellular recordings were made from neurones in the rat otic ganglion in vitro in order to investigate their morphological, physiological and synaptic properties. We took advantage of the simple structure of these cells to test for a possible role of calcium influx via nicotinic acetylcholine receptors during synaptic transmission. 2. Cells filled with biocytin comprised a homogeneous population with ovoid somata and sparse dendritic trees. Neurones had resting membrane potentials of -53 +/- 0.7 mV (n = 69), input resistances of 112 + 7 M Omega, and membrane time constants of 14 +/- 0.9 ms (n = 60). Upon depolarization, all cells fired overshooting action potentials which mere followed by an apamin-sensitive after-hyperpolarization (AHP). In response to a prolonged current injection, all neurones fired tonically. 3. The repolarization phase of action potentials had a calcium component which was mediated by N-type calcium channels. Application of omega-conotoxin abolished both the repolarizing hump and the after-hgrperpolarization suggesting that calcium influx via N-type channels activates SK-type calcium-activated potassium channels which underlie the AHP. 4. The majority (70%) of neurones received innervation from a single preganglionic fibre which generated a suprathreshold excitatory postsynaptic potential mediated by nicotinic acetylcholine receptors. The other 30% of neurones also had one or more subthreshold nicotinic inputs. 5. Calcium influx via synaptic nicotinic receptors contributed to the AHP current, indicating that this calcium has access to the calcium-activated potassium channels and therefore plays a role in regulating cell excitability.

T cell regulation of the immune response to infection in periodontal diseases

Although T cells have been implicated in the pathogenesis and are considered to be central both in progression and control of the chronic inflammatory periodontal diseases, the precise contribution of T cells to the regulation of tissue destruction has not been fully elucidated. Current dogma suggests that immunity to infection is controlled by distinct T helper 1 (Th1) and T helper 2 (Th2) subsets of T cells classified on the basis of their cytokine profile. Further, a subset of T cells with immunosuppressive function and cytokine profile distinct from Th1 or Th2 has been described and designated as regulatory T cells. Although these regulatory T cells have been considered to maintain self-tolerance resulting in the suppression of auto-immune responses, recent data suggest that these cells may also play a role in preventing infection-induced immunopathology. In this review, the role of functional and regulatory T cells in chronic inflammatory periodontal diseases will be summarized. This should not only provide an insight into the relationship between the immune response to periodontopathic bacteria and disease but should also highlight areas of development for potentially new therapeutic modalities.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Lipid, sugar and liposaccharide based delivery systems

Although there are formidable barriers to the oral delivery of biologically active drugs, considerable progress in the field has been made, using both physical and chemical strategies of absorption enhancement. A possible method to enhance oral absorption is to exploit the phenomenon of lipophilic modification and mono and oligosaccharide conjugation. Depending on the uptake mechanism targeted, different modifications can be employed. To target passive diffusion, lipid modification has been used, whereas the targeting of sugar transport systems has been achieved through drugs conjugated with sugars. These drug delivery units can be specifically tailored to transport a wide variety of poorly absorbed drugs through the skin, and across the barriers that normally inhibit absorption from the gut or into the brain. The delivery system can be conjugated to the drug in such a way as to release the active compound after it has been absorbed (i.e. the drug becomes a prodrug), or to form a biologically stable and active molecule (i.e. the conjugate becomes a new drug moiety). Examples where lipid, sugar and lipid-sugar conjugates have resulted in enhanced drug delivery will be highlighted in this review.

Early pregnancy factor suppresses the infiltration of lymphocytes and macrophages in the spinal cord of rats during experimental autoimmune encephalomyelitis but has no effect on apoptosis

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties that has been shown to suppress acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP) in Lewis rats. EAE is associated with infiltration of the central nervous system (CNS) with inflammatory cells. Spontaneous recovery involves the loss of T lymphocytes from the CNS and the selective apoptosis of Vbeta8.2(+) cells. In the present study, T cell, macrophage (CD11b/c(+)) and B cell (CD45RA(+)) populations in spinal cord and popliteal lymph nodes (LN) of Lewis rats with EAE were quantitated and apoptosis was studied. Rats were treated with EPF or vehicle. Following treatment on day 14 after inoculation with MBP, neither 1 x 100 mug nor 2 x 100 mug doses of EPF affected the total number of cells infiltrating the spinal cord on day 15, although the higher dose caused a decrease in the number of CD5(+) and CD11b/c(+) cells. Treatment with 2 x 100 mug/day from days 10 to 14 decreased the total number of infiltrating cells, and the numbers of CD5(+), CD11b/c(+) and CD45RA(+) cells. Apoptosis was unaffected. No alteration on the number or type of inflammatory cells in the popliteal LN was observed after treatment on days 10-14. However, treatment with EPF from days 0 to 11 increased the total number of T and B cells and CD5(+) T cells found on day 12 in the LN. Similarly, there was an increase in the frequency of MBP-reactive cells in the LN as determined by limiting dilution analysis. These results suggest that EPF treatment reduces the numbers of lymphocytes and macrophages in the CNS, possibly through an effect on cell trafficking. (C) 2003 Elsevier B.V. All rights reserved.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.