Immune escape of melanoma: first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway.

Sequence analyses of the transporter associated with antigen processing (TAP) in tumor cell lines with deficient MHC class I surface expression identified a bp deletion at position 1489 near the ATP-binding domain of Tap1, causing a frameshift in one melanoma cell line. The impaired TAP1 protein expression was associated with deficient TAP2 protein expression, peptide binding, translocation, and MHC class I surface expression. Stable TAP1 gene transfer reconstitutes the described defects, whereas lysis by HLA-A2-restricted CTLs was still abrogated. This was attributable to a 2-bp insertion at position 890 in the HLA-A2 gene and was corrected after HLA-A2 cotransfection. This study describes for the first time mutations in two distinct components of the MHC class I antigen processing pathway, suggesting an immune selection against CTLs recognizing both TAP-dependent and -independent T-cell epitopes.

Transporter (TAP)- and proteasome-independent presentation of a melanoma-associated tyrosinase epitope.

The melanosomal protein tyrosinase is considered as a target of specific immunotherapy against melanoma. Two tyrosinase-derived peptides are presented in association with HLA-A2.1 [Wölfel et al., Eur. J. Immunol., 24, 759-764 (1994)]. Peptide 1-9 (MLLAVLYCL) is generated from the putative signal sequence. The internal peptide 369-377 is posttranslationally converted at residue 371, and its presentation is dependent on functional TAP transporters and proteasomes [Mosse et al., J. exp. Med.187, 37-48 (1998)]. Herein, we report on the processing and transport requirements for the signal sequence-derived peptide 1-9 that were studied in parallel to those for peptide 369-377. After infection of TAP-deficient (T2) and TAP-positive (T1) cells with a Modified Vaccinia Ankara construct carrying the human tyrosinase gene (MVA-hTyr), we found that recognition by CTL against peptide 1-9 did not require TAP function as opposed to recognition by CTL against peptide 369-377. When target cells with intact processing and transport functions were infected with MVA-hTyr, lysis by CTL against peptide 1-9 was not impaired by lactacystin, a specific inhibitor for the proteasome, whereas lysis by CTL against peptide 369-377 was completely abrogated. Taken together, peptide 1-9 derived from the signal sequence of tyrosinase is presented in a TAP-independent fashion and does not require proteasomes for processing. Cellular immune responses against this hydrophobic peptide can be monitored with lymphokine spot assays as documented in the case of a patient with metastatic melanoma, in whom we observed a preferential T-cell response against tyrosinase peptide 1-9 subsequent to chemoimmunotherapy. Independence of cytosolic processing and transport pathways and potentially enhanced expression levels make signal sequence-derived peptides and their carrier proteins important candidates for specific immunotherapy.

Transient host range selection for genetic engineering of modified vaccinia virus Ankara.

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.

Recombinant Vaccinia Virus MVA for Generation and Analysis of T Cell Responses Against Tumor Associated Antigens.

Live attenuated viruses used as vaccines are known for their efficacy to elicit protective immunity against viral diseases. More recently, with an increasing number of tumor-associated antigens (TAA) being identified and molecularly cloned (1) the development of vaccines for cancer immunotherapy has gained considerable interest. In particular, live recombinant viral vectors seem to be appropriate delivery systems for efficient presentation of TAA to the immune system. The promise of viral vectors is likely to be founded on their capacity for high-level expression of target genes combined with their intrinsic property to activate immunological control systems mimicking an infection with a disease causing agent.

Modified vaccinia virus Ankara for delivery of human tyrosinase as melanoma-associated antigen: induction of tyrosinase- and melanoma-specific human leukocyte antigen A*0201-restricted cytotoxic T cells in vitro and in vivo.

Vaccination with tumor-associated antigens is a promising approach for cancer immunotherapy. Because the majority of these antigens are normal self antigens, they may require suitable delivery systems to promote their immunogenicity. A recombinant vector based on the modified vaccinia virus Ankara (MVA) was used for expression of human tyrosinase, a melanoma-specific differentiation antigen, and evaluated for its efficacy as an antitumor vaccine. Stable recombinant viruses (MVA-hTyr) were constructed that have deleted the selection marker lacZ and efficiently expressed human tyrosinase in primary human cells and cell lines. Tyrosinase-specific human CTLs were activated in vitro by MVA-hTyr-infected, HLA-A*0201-positive human dendritic cells. Importantly, an efficient tyrosinase- and melanoma-specific CTL response was induced in vitro using MVA-hTyr-infected autologous dendritic cells as activators for peripheral blood mononuclear cells derived from HLA-A*0201-positive melanoma patients despite prior vaccination against smallpox. Immunization of HLA-A*0201/Kb transgenic mice with MVA-hTyr induced A*0201-restricted CTLs specific for the human tyrosinase-derived peptide epitope 369-377. These in vivo primed CTLs were of sufficiently high avidity to recognize and lyse human melanoma cells, which present the endogenously processed tyrosinase peptide in the context of A*0201. Tyrosinase-specific CTL responses were significantly augmented by repeated vaccination with MVA-hTyr. These findings demonstrate that HLA-restricted CTLs specific for human tumor-associated antigens can be efficiently generated by immunization with recombinant MVA vaccines. The results are an essential basis for MVA-based vaccination trials in cancer patients.

Highly attenuated modified vaccinia virus Ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells.

Although desirable for safety reasons, the host range restrictions of modified vaccinia virus Ankara (MVA) make it less applicable for general use. Propagation in primary chicken embryo fibroblasts (CEF) requires particular cell culture experience and has no pre-established record of tissue culture reproducibility. We investigated a variety of established cell lines for productive virus growth and recombinant gene expression. Baby hamster kidney cells (BHK), a well-characterized, easily maintained cell line, supported MVA growth and as proficient expression of the E. coli lacZ reporter gene as the highly efficient CEF, whereas other cell lines were non-permissive or allowed only very limited MVA replication. Importantly, no virus production occurred in patient-derived infected primary human cells. These results emphasize the safety and now improved accessibility of MVA for the development of expression vectors and live recombinant vaccines.

Gene transfer into human dendritic antigen-presenting cells by vaccinia virus and adenovirus vectors.

In a search for means to deliver exogenous gene(s) into human dendritic cells (DCs) from the perspective of tumor-specific vaccination, we have evaluated two recombinant viruses, both of which carry a reporter gene which is namely a modified vaccinia virus Ankara (MVA) and an adenovirus, as possible expression vectors. The recombinant MVA-P11 LZ vector carries the Escherichia coli lacZ gene coding for the enzyme beta-galactosidase, and the recombinant Ad-MFG-AP vector carries a modified membrane-exposed alkaline phosphatase (AP) gene. DCs were generated ex vivo in the presence of tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, stem cell factor, and flk-2/flt-3 ligand taken from CD34+ hematopoietic progenitors that were mobilized into the peripheral blood of cancer patients treated with high-dose cyclophosphamide and filgrastim. The target cells used for gene delivery were either CD34+ cells that had been subsequently induced to differentiate into mature DCs or DCs transduced after ex vivo generation from CD34+ cells. The results showed that: (a) infection of CD34+ cell derived-DCs (mature DCs) with either viral vector resulted in the efficient synthesis of recombinant protein, and (b) CD34+ cells were permissive for the expression of the recombinant reporter gene after infection with Ad-MFG-AP but not after infection with MVA-P11 LZ. In conclusion, these results suggest that vaccinia and adenovirus vectors are candidate to act as vehicles in genetically engineering human DCs.

Comprehensive individualised nonpharmacological treatment programme for hypertension in physician-nurse clinics: two year follow-up.

A comprehensive programme of nonpharmacological control of hypertension (balanced nutrition, satisfactory weight, enhanced physical activity, relaxation technique, smoking cessation) by primary care physician-nurse (PN) teams who were instructed and routinely advised by a paramedical professional (PP) team (psychologist, nutritionist and physical activity instructor) was developed with the aim of increasing long-term compliance. To evaluate effectiveness, 52 mild and moderate hypertensives without target organ damage were randomly allocated to six weekly meetings of individual intensive instruction by PN teams alone, or direct group instructions by PP teams (24 and 28 patients, respectively). The respective results at 11 months and 24 months follow-up compared with baseline were: (1) 56.9% and 58.8% showed minimal satisfactory reduction of weight, (2) 49% and 58.8% showed minimal satisfactory increase in physical activity, (3) the reported increase in physical activity at 11 months follow-up was validated by significantly correlated improved performance in ergometry, (4) 75% and 40% of the patients performed relaxation vs. 2% at baseline and (5) 71% and 59% needed no medication or reduced dose to control BP, and these changes were significantly (P < 0.02) correlated with weight reduction and increased physical activity. As no differences were found between the two modes of treatment, we conclude that our programme can be successfully applied by the PN primary care teams to increase adherence to nonpharmacological measures in the control of hypertension.

Partial cost-benefit analysis of two different modes of nonpharmacological control of hypertension in the community.

In the Dan and Ashkelon areas of Israel, 28 male and 24 female mild to moderate hypertensives without target organ damage aged 35-65 years were randomly assigned to treatment programmes (based on nutritional management, exercise and stress management techniques) either on an individual basis administered by physician-nurse teams (PN) or on a group basis from a team of paramedical professionals (PP) consisting of a psychologist, nutritionist and physical activity instructor. At 11 and 24 months follow-up, there were similar significant improvements in both treatment modes for such risk factors as body mass index, caloric intake and physical activity levels. There was a significant decrease in drug use from $36.28 a month at baseline to $18.94 a month at 11 month follow-up (P = 0.01) and to an estimated $20.48 at 24 months. Mean BP remained unchanged, despite the reduction in drug use, indicating a reduction in the underlying BP. The net present value (NPV) of the reduction in drug utilisation totalled $740 per person over a five year time horizon and a 7.5% discount rate. The total extra costs of treatment, training, case-note writing, travelling and follow-up booster sessions, amounted to $95 per patient for the PN mode and $234 per patient for the PP mode, yielding benefit to cost ratios of 7.79/1 and 3.16/l, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The antihypertensive effect of atenolol and bopindolol in the elderly.

The antihypertensive efficacy and tolerability of two betablockers: atenolol and bopindolol, was compared in a group of 30 elderly subjects aged 64.8 +/- 4.6 years. The daily dose of the two agents was relatively low. Atenolol 50-100 mg and bopindolol 0.5-1.0 mg sufficed to cause reduction of DBP to the target of less than or equal to 95 mm Hg, when applied as monotherapy. This was achieved in 75% of cases with bopindolol and in 93% of cases with atenolol. Atenolol, 50-100 mg/dd, lowered blood pressure from 173.7 +/- 13.2/103.7 +/- 3.0 (weekly) to 155.5 +/- 16.5/86.5 +/- 8.2 mm Hg (week 12) (P less than 0.005) while bopindolol, 0.5-1.0 mg, lowered blood pressure from 171.6 +/- 11.3/104.1 +/- 3.6 to 158.7 +/- 20.9/86.1 +/- 6.0 mm Hg (P less than 0.005). Heart rate was reduced from 80.5 (week 4) to 66.7 +/- 7.3 (week 12) by atenolol (P less than 0.0001), and from 83.7 +/- 11.8 (week 4) to 71.1 +/- 7.5 (week 12) by bopindolol (P less than 0.0001). Between treatment differences: comparisons yielded P values which were not sufficiently low to reject the null hypothesis of no difference between the two treatments. Well-being and short-term memory were not affected by either agent and tolerability of both drugs was good. These findings demonstrate that both bopindolol and atenolol are useful agents for control of hypertension in the elderly.