Adenosine receptor A2a blockade by caffeine increases IFN-gamma production in Th1 cells from patients with rheumatoid arthritis.

OBJECTIVE: Studies indicate that caffeine uptake may be a risk factor for rheumatoid arthritis (RA), but a definitive link between caffeine consumption and RA has not been established. This study aimed to investigate the interplay between caffeine, adenosine receptor A2a, and interferon-γ (IFN-γ) production in CD4+ T cells from RA patients. METHOD: Peripheral blood mononuclear cells were obtained from the peripheral blood of healthy individuals and patients with RA. CD4+ T cells were isolated using the magnetic activated cell sorting technique and cultured in vitro with caffeine or mock control. In addition, adenosine was used as a competitive inhibitor of caffeine. After 48 h, expression of IFN-γ and interleukin-17 (IL-17) was analysed by flow cytometry. Ex vivo expression levels of adenosine receptor A2a were also assessed. RESULTS: Caffeine promoted IFN-γ production in Th1 cells in vitro. Significantly higher concentrations of caffeine were required to increase IFN-γ levels in Th1 cells from healthy individuals compared to Th1 cells from patients with RA. Moreover, ex vivo levels of adenosine receptor A2a expression on CD4+ T cells were significantly higher in RA than in healthy individuals. Caffeine-driven IFN-γ production was completely reversed by adenosine, a competitive agonist of adenosine receptor A2a. In contrast to IFN-γ, production of IL-17 was not affected by caffeine. CONCLUSION: Caffeine promotes IFN-γ production in Th1 cells from RA patients in vitro by competitive inhibition of adenosine receptor A2a. Excessive coffee consumption could contribute to T-cell activation and inflammation in RA.

Redirecting human CD4+CD25+ regulatory T cells from the peripheral blood with pre-defined target specificity.

Recent insight into the balance of self-tolerance and auto-aggression has raised interest in using human regulatory T (Treg) cells for adoptive immunotherapy of unlimited autoimmune diseases including type-1 diabetes, rhematoid arthritis and multiple sclerosis. The therapeutic use of Treg cells, however, is so far hampered by the inefficiency of current protocols in making them accessible for genetic manipulations. We report here that TCR/CD3 stimulation that is accompanied by extensive CD28 costimulation makes human Treg cells susceptible to retroviral gene transfer ex vivo while preserving their properties in vitro and in vivo. To show the power of genetic manipulation of human Treg cells, we engineered 'designer Treg cells' by retroviral expression of a chimeric immunoreceptor with defined specificity, which activates Treg cells in a ligand-dependent manner to proliferate, to secrete high amounts of interleukin-10 and to repress an ongoing cytolytic T-cell response in vivo. The procedure in genetically modifying human Treg cells ex vivo will open a panel of applications for their use in the adoptive therapy of deregulated immune responses.

Targeting lipid metabolism by the lipoprotein lipase inhibitor orlistat results in apoptosis of B-cell chronic lymphocytic leukemia cells.

Constitutively activated pathways contribute to apoptosis resistance in chronic lymphocytic leukemia (CLL). Little is known about the metabolism of lipids and function of lipases in CLL cells. Performing gene expression profiling including B-cell receptor (BCR) stimulation of CLL cells in comparison to healthy donor CD5+ B cells, we found significant overexpression of lipases and phospholipases in CLL cells. In addition, we observed that the recently defined prognostic factor lipoprotein lipase (LPL) is induced by stimulation of BCR in CLL cells but not in CD5+ normal B cells. CLL cellular lysates exhibited significantly higher lipase activity compared to healthy donor controls. Incubation of primary CLL cells (n=26) with the lipase inhibitor orlistat resulted in induction of apoptosis, with a half-maximal dose (IC(50)) of 2.35 microM. In healthy B cells a significantly higher mean IC(50) of 148.5 microM of orlistat was observed, while no apoptosis was induced in healthy peripheral blood mononuclear cells (PBMCs; P<0.001). Orlistat-mediated cytotoxicity was decreased by BCR stimulation. Finally, the cytotoxic effects of orlistat on primary CLL cells were enhanced by the simultaneous incubation with fludarabine (P=0.003). In summary, alterations of lipid metabolism are involved in CLL pathogenesis and might represent a novel therapeutic target in CLL.

Current status of immunotherapy in B cell malignancies.

Conventional treatment of hematologic malignancies mainly consists of chemotherapeutic agents or a combination of both, chemotherapy and monoclonal antibodies. Despite recent advances, chemotherapeutic treatments often remain unsatisfying due to severe side effects and incomplete long-term remission. Therefore the evaluation of novel therapeutic options is of great interest. B cell malignancies, in particularly follicular lymphomas, chronic lymphocytic leukemia and multiple myeloma, represent the most immune-responsive types of all human cancer. Several immunotherapeutic strategies are presently employed to combat these B-cell malignancies. Active immunotherapies include vaccination strategies with dendritic cells (DCs) and genetically-modified tumor cell preparations as well as DNA and protein vaccination. Most of these vaccines target the tumor-specific immunoglobulin idiotype and have already demonstrated some anti-lymphoma activity in early phase clinical trials while their definitive impact is evaluated in ongoing phase III randomized trials. In contrast to these active immunizations, T cells transduced with chimeric antigen receptors and donor leukocyte infusions (DLI) represent adoptive (passive) immunotherapies. Recent advances of gene transduction technologies enabled improvement of immunotherapeutic strategies based on genetic modification of malignant cells or adoptive T cells. Current early phase clinical trials are investigating the potential of these innovative approaches. At the moment it remains unclear if the novel immunotherapeutic strategies will be able to play a similar role in the treatment of B cell malignancies than the already established antibody-based immunotherapy.

The potential of gene transfer into primary B-CLL cells using recombinant virus vectors.

Despite recent advances, chronic lymphocytic leukemia (CLL) as the most common leukemia remains a largely incurable disease. Modern treatment options include novel drugs like purine analogues, monoclonal antibodies and transplantation strategies. Moreover, gene transfer of immunostimulatory molecules is another, but still experimental approach that can be used to potentiate immune responses against leukemic cells. CD40 ligand (CD40L) was shown to be a promising molecule for immunotherapy of B-CLL playing a critical role in immune activation. However, CLL B cells are resistant to transduction with most currently available vector systems. Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy in this area. Using replication defective adenovirus encoding CD40L (Ad-CD40L), immunologic and clinical responses were seen in CLL patients after infusion of autologous Ad-CD40L-CLL cells in a recent phase I trial. Due to the immunogenic nature of adenovirus vectors, alternative vector systems are currently explored. Recombinant adeno-associated virus (rAAV) was shown to enable efficient transduction of primary B-CLL cells. By use of a library of AAV clones with randomly modified capsids, receptor-targeting mutants with a tropism for CLL cells can be selected. Furthermore, helper-virus free Epstein-Barr virus (EBV)-based gene transfer vectors hold promise for development of CLL-targeted vaccines after remaining safety issues will be resolved. Herpes simplex virus (HSV)-based vectors, especially HSV amplicons, have favorable features for B-CLL gene transfer including high transduction efficiency, ability to infect postmitotic cells and a large packaging capacity. The challenge for the future will be to transfer these alternative vector systems into clinic and allow the detection of a CLL-specific immune response by use of defined tumor antigens. This will make it possible to establish the potential clinical role of gene therapy for CLL patients.

Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors.

Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.

Vero cytotoxin-producing Escherichia coli O157 infections in Austria.

To determine the prevalence of Vero cytotoxin-producing Escherichia coli (VTEC) serotype O157 associated diarrhea in the Austrian patient population, we surveyed all stool specimens of liquid consistency submitted to the Federal Public Health Laboratory (FPHL) in Innsbruck for 2 years for this organism. This laboratory serves a population of approximately 1 Million people. Of 5,265 stool specimens, 7 yielded O157 VTEC. Five isolates of E. coli O157 phage type 32, VT2 were cultured from specimens received during a three day period from residents in the county of Schwaz. During the investigation of this "outbreak" E. coli O157 strains were also isolated from two household contacts. Only 1 out of 8 persons with E. coli O157 diarrhea had bloody stools, although 5 of 7 tested specimens (= 71%) also yielded Campylobacter jejuni. None of our patients received antimicrobial therapy directed against E. coli O157 (one child had josamycin). There were no fatalities and no cases of hemolytic uremic syndrome (follow up period: 6 months). Consumption of hamburger, roast beef, and unpasteurized milk was not confirmed in this study. In Austria, no O157 VTEC strain was isolated till June 1992, although at the FPHL in Innsbruck stool specimens of liquid consistency were cultured for this organism since January 1991.

Complement-mediated enhancement of HIV-1 infection of the monoblastoid cell line U937.

To assess the role of complement and complement receptors in HIV-1 infection of monocytes and macrophages, we studied the infectivity of HIV-1, isolated from the peripheral blood of a patient with subacute AIDS-related encephalopathy, on the human monoblastoid cell line U937. HIV-1 and HIV-1-infected cells were capable of activating the complement system via the classical and the alternative pathways, respectively. Low concentrations of HIV-1 were able to infect U937 cells more easily in the presence than in the absence of complement. At higher virus concentrations, infectivity was no longer facilitated by the presence of complement. Infection of U937 cells was reduced in the presence of any of the monoclonal antibodies (MAbs), OKT4a (anti-CD4), OKM1 (anti-CR3), or M522 (anti-CR3). A combination of all three of these MAbs reduced the infection by an even greater amount. These data indicate that complement receptors may be a port of entry for complement-coated HIV-1.

Application of the tyrosinase assay to normal melanocytes in culture.

Using a special selection technique, normal guinea-pig melanocytes were maintained in highly purified but sparse cultures (approximately 10(4) cells/25 cm2 culture vessel) which showed little proliferative activity. The applicability of the Pomerantz tyrosinase assay was tested in this in vitro model system using three different approaches, namely crude cell extracts and viable cell cultures either in situ or in suspension. The latter modifications both proved too insensitive, whereas crude cell extracts allowed accurate measurements of the basal tyrosinase activity and its stimulation by various agents. In unstimulated cultures basal tyrosinase activities ranged from 30% to 700% (mean 260%) above the blank values; intra-assay and inter-assay variability were 4.2% and 77.5%, respectively. Stimulation with alpha-MSH (10(-5) M, 10(-6) M), beta-MSH (10(-5) M), choleratoxin (10(-11) M) and cAMP (10(-4) M) plus theophylline (10(-4) M) resulted in an increase of tyrosinase activity 30-65% above basal values. Melanotropin potentiating factor (10(-8) M) enhanced the effects of alpha-MSH (10(-6) M) by 20%. This assay modification provides a sensitive tool for comparative studies of melanogenesis in normal melanocytes, malignant melanocytes and otherwise altered melanocytes.