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Summary Background Smoking is associated with cancer and cardio-respiratory mortality. Reducing smoking prevalence will lead to fewer deaths and more life-years. Here, we estimate the impact of hypothetical introduction of reduced-risk products (heat-not-burn products and e-cigarettes) in Germany from 1995 to 2015 on mortality from lung cancer, chronic obstructive pulmonary disease, ischaemic heart disease, and stroke in men and women aged 30–79 years. Methods We used a previously described population health impact model, with individuals with a defined baseline cigarette smoking distribution followed under a “Null Scenario”, with reduced-risk products never introduced, and various “Alternative Scenarios” where they are. Transition probabilities allow product use to change annually, with the individual product histories allowing estimation of risks, relative to never users, which are then used to estimate reductions in deaths and life-years lost for each Alternative Scenario. Results In the Null Scenario, we estimated 852,000 deaths from cigarette smoking (42,600 per year), with 8.61 million life-years lost. Had everyone ceased smoking in 1995, and with no use of reduced-risk products, these numbers would reduce by 217,000 and 2.88 million. Compared to the Null Scenario, the estimated reductions would be 159,000 and 2.06 million with an immediate complete switch to heat-not-burn products and 179,000 and 2.34 million with 50% of smokers immediately switching to heat-not-burn products and 50% to e-cigarettes. In four Scenarios with a more gradual switch, the estimated decreases were 39,800–81,000 deaths and 0.50–1.05 million life-years, representing 17.5%–37.5% of the effect of immediate cessation in 1995. These estimates assume that switching to heat-not-burn products and e-cigarettes involves risk decreases of 80% and 95% of those from quitting, respectively. The reductions in mortality would be greater with more diseases and a wider age range considered or with a longer follow-up period, as the decreases increased markedly with time. Various limitations are discussed, none affecting the conclusion that introducing these new products into Germany in 1995 could have substantially reduced deaths and life-years lost. Conclusions Deaths from cigarette smoking could be substantially reduced not only by cessation but additionally by switching to reduced-risk products. Respective public health campaigns might increase such switching.

Summary Background We previously estimated the impact of introducing heat-not-burn products and e-cigarettes in Germany on smoking-related disease mortality in men and women aged 30–79 years between 1995 and 2015. Here, we estimate the impact by socioeconomic group. Methods Individuals with a defined baseline cigarette smoking distribution were followed under a “Null Scenario” (no reduced-risk products) and “Alternative Scenarios” (reduced-risk products introduced). Transition probabilities allowed estimation of annual product use changes, with individual product histories used to estimate reductions in deaths and life-years lost. Here, however, individuals were classified into two socioeconomic groups defined by income and education, with allowance for variation by group in initial smoking prevalence and the probability of changing product use, or of changing socioeconomic group. Results With no allowance for socioeconomic group, deaths would have reduced by 217,000 (from 852,000 for continued smoking) had everyone immediately ceased smoking in 1995 and by 40,000 to 179,000 had one or two types of reduced-risk products – the heat-not-burn product and the e-cigarette – been adopted by smokers to varying extents. With such allowance, we estimate substantial drops in each socioeconomic group. Where all cigarette smokers switched immediately, half of them to heat-not-burn products, half to e-cigarettes, the estimated drops in deaths were 60,000 in group A (higher socioeconomic group) and 122,000 in group B (lower), about 82% of the drops associated with immediate cessation (73,000 in A and 148,000 in B). With more gradual conversion, the drops were 26,648 in A and 53,000 in B, about 35% of those from cessation. The drops in deaths and life-years saved were about 2 and 1.5 times higher in group B, respectively, associated with its greater numbers, older age, and higher smoking prevalence. The estimated reductions would increase upon considering more diseases, a wider age range, or longer follow-up. Methodological limitations would not affect the conclusion that introducing these products in 1995 in Germany could have substantially reduced deaths and life-years lost in both groups, more so in B. Conclusions Although cessation is optimal for reducing mortality, switching to reduced-risk products also provides substantial health gains. A public health approach encouraging lower socioeconomic group smokers to switch to reduced-risk products could diminish smoking-related health inequalities relative to continued smoking.

Background Smoking is associated with cancer and cardiorespiratory disease mortality. Reducing smoking prevalence reduces deaths and life-years lost. Here, we estimate the impact of introducing heat-not-burn products and e-cigarettes in Germany from 1995 to 2015 on mortality from lung cancer, chronic obstructive pulmonary disease, ischaemic heart disease, and stroke in men and women aged 30–79 years. Methods We used the previously described population health impact model. Modelling starts with individuals of a given sex and age range with a defined cigarette smoking distribution. They are then followed under a “Null Scenario”, where reduced-risk products are never introduced, and one of seven “Alternative Scenarios”, where they are. Transition probabilities allow tobacco product use to change annually, and the individual product histories then allow estimation of risks, relative to never users, for each year and Scenario, which are then used to estimate reductions in deaths and life-years lost for each Alternative Scenario. Results In the Null Scenario, we estimated 852,357 deaths from cigarette smoking, with 8.61 million life-years lost. Had everyone quit in 1995, with no further use of the three products, these numbers would reduce by 216,650 and 2.88 million. The reductions would be 159,278 and 2.06 million with an immediate complete switch from cigarettes to heat-not-burn products, and 179,470 and 2.34 million with an immediate switch to 50% heat-not-burn products and 50% e-cigarettes. In four Scenarios with a more gradual switch, estimated decreases were 39,818–81,293 deaths and 0.50–1.05 million life-years, representing 17.5%-37.5% of the effect of cessation. These estimates assume switching to heat-not-burn products and e-cigarettes involves risk decreases of 80% and 95% of those from quitting. The decreases would have increased, had more diseases and a wider age range been considered, and also with a longer follow-up period, the decreases increasing markedly with time. Conclusions Various limitations are discussed, none affecting our conclusion that introducing these new products into Germany in 1995 could have substantially reduced mortality. Our results imply deaths from cigarette smoking could be substantially reduced, both by cessation and switching to reduced-risk products. Risk-proportionate public health campaigns and regulation might increase such switching.

Background Previously, we estimated the impact of introducing heat-not-burn products and e-cigarettes in Germany between 1995 and 2015 on mortality from the four main smoking-related diseases in men and women aged 30–79 years. Deaths would have reduced by 216,650 (from 852,357 for continued smoking) had everyone quit smoking in 1995 and by 39,818 − 179,470 had one or both of heat-not burn products and e-cigarettes been introduced and adopted to varying extents. Here, we report substantial mortality reductions separately in two socioeconomic groups (A = higher, B = lower) defined by income and education. Methods Modelling is essentially as before, with individuals of a given sex, age range and cigarette smoking distribution followed over time under a “Null Scenario” (reduced-risk products never introduced) and various “Alternative Scenarios” (reduced-risk products introduced), the individual product histories generated then allowing estimation of reductions in mortality for each Alternative Scenario compared to the Null Scenario. Here, however, individuals are subdivided into two socioeconomic groups with transition probabilities between product use groups varying by group as well as by sex, age and length of follow-up. The possibility of transitioning between socioeconomic groups is also allowed for. Results Where all cigarette smokers switch immediately, half to each new product, the drops in deaths were estimated as 60,081 in group A and 122,343 in group B, about 82% of the drops associated with immediate cessation (72,725 A, 147,684 B). Where the conversion was more gradual, the drops were about 35% of those from cessation (25,648 A, 52,652 B). Drops were about two times higher in B, because of their greater numbers, older age, and higher frequency of current smokers. Years of life saved were also higher (about 1.5 times) in B than A. Conclusions Methodological limitations would not have affected our conclusion that introducing these products in 1995 in Germany could have substantially reduced mortality in each group, with greater gains in B. While cessation is the best choice to reduce mortality, switching to reduced-risk products also provides substantial health gains. Encouraging lower socioeconomic group smokers who would otherwise continue smoking to fully switch to reduced-risk products could diminish smoking-related health inequalities.

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8+ T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8+ T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8+ T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8+ T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP396) > gp33 > gp276 > NP205) was well maintained. We observed a slight reduction in the NP205-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8+ T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8+ T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.

We present a predictive method that can simulate an essential step in the antigen presentation in higher vertebrates, namely the step involving the proteasomal degradation of polypeptides into fragments which have the potential to bind to MHC Class I molecules. Proteasomal cleavage prediction algorithms published so far were trained on data from in vitro digestion experiments with constitutive proteasomes. As a result, they did not take into account the characteristics of the structurally modified proteasomes--often called immunoproteasomes--found in cells stimulated by gamma-interferon under physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC Class I ligands, i.e. the specificity of the immunoproteasome is different than the constitutive proteasome. The tools developed in this study in combination with a predictor of MHC and TAP binding capacity should give a more complete prediction of the generation and presentation of peptides on MHC Class I molecules. Here we demonstrate that such an approach produces an accurate prediction of the CTL the epitopes in HIV Nef. The method is available at www.cbs.dtu.dk/services/NetChop/.

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

The effectiveness of protection conferred by CD8+ memory T cells is determined by both their quality and their quantity, which suggests that vaccine efficacy might be improved if it were possible to increase the size of the memory pool. Approximately 90% of virus-specific CD8+ T cells die during the contraction phase and, herein, we have attempted to increase the memory pool by reducing CD8+ T cell death. CD8+ T cell contraction has been attributed to apoptosis, or programmed cell death (PCD), which, classically, is dependent on caspases. Caspase-dependent PCD can be prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD), and here we evaluate the effect of this compound on virus-specific T cell responses in mice. zVAD prevented caspase-dependent PCD of freshly isolated virus-specific T cells in tissue culture, and a fluorescent analog, FITC-VAD, entered CD8+ T cells following in vivo injection. However, despite using 11 different regimens of zVAD administration in vivo, no significant effects on CD8+ or CD4+ memory T cell numbers were observed. Furthermore, the CD8+ memory T cell responses to secondary virus infection were indistinguishable, both qualitatively and quantitatively, in zVAD-treated and normal mice. The absence of effect cannot be attributed to a technical flaw, because identical doses of zVAD were able to rescue mice from hepatocyte apoptosis and lethal intrahepatic hemorrhage, induced by inoculation of anti-Fas Ab. We conclude that the contraction phase of the virus-specific T cell response is unlikely to require caspase-dependent PCD. We propose that contraction can be mediated by an alternative, caspase-independent pathway(s).

In an attempt to define HCMV IE1-derived, HLA-A(*)0201-restricted epitopes, an advanced computer-based epitope prediction combining HLA binding and proteasomal cleavages in silico was performed.This prediction algorithm clearly confirmed VLEETSVML to be the most likely CTL epitope. By tetramer staining, HCMV pp65 NLVPMVATV-specific CD8(+) T cells were detectable in 18/24 HCMV seropositive HLA-A(*)0201-expressing individuals (median frequency 0.58%; range 0.1%-4.7%), and IE1 VLEETSVML-specific CD8(+) T cells in 5/24 (median frequency 2.1%; range 0.1%-4.3%), respectively (p0.01). Also in recipients of an allogeneic SCT, VLEETSVML- and NLVPMVATV-specific CD8(+) T cells were detectable in comparable frequencies, but again the number of patients with detectable pp65-specific CD8(+) T cells was higher (p=0.014). In 4/15 individuals, all demonstrating IE1 VLEETSVML-specific CD8(+) T cells prior to peptide stimulation, VLEETSVML-specific T cell lines (purity of 42.6%-98.6% of all CD3(+)/CD8(+) T cells) were successfully generated after 2-4 weeks of culture using the IFN-gamma secretion assay.In conclusion, this novel prediction strategy efficiently predicted an immunodominant viral T-cell epitope.

The proteasome system represents a major source of HLA class I- presented peptides exposed to CTLs. Stimulation of cells with IFN-gamma instantly induces the expression of the proteasome immunosubunits as well as the proteasome activator PA28. These proteins have been shown to optimize class I antigen presentation of several viral CTL epitopes; however, their contribution to tumor antigen processing remains poorly understood. Here, we analyzed the generation of an HLA-A*0201-presented epitope derived from the melanoma antigen tyrosinase-related protein 2 (TRP2). Melanoma cells that lacked the IFN-gamma-inducible proteasome activator PA28 and immunoproteasomes did not display the TRP2(360-368) epitope to specific CTLs. Our experiments demonstrate that epitope presentation correlated with the presence of PA28 and could be completely rescued by restoration of PA28 expression. In vitro digestion of TRP2 polypeptides with 20S proteasomes confirmed that PA28 is essential for epitope liberation. Thus, our experiments indicate that PA28 provides the threshold for CTL recognition of this epitope. Importantly, processing of a second TRP2-derived epitope, TRP2(288-296), was diminished in IFN-gamma-treated cells, even in the absence of immunoproteasome up-regulation. Therefore, the reported IFN-gamma-induced self-regulation of epitopes may not necessarily be a consequence of immunoproteasomes as suggested previously.

Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon γ–inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex–like (MECL)-1 leads to the formation of immunoproteasomes which have been associated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously.We now report that cells expressing immunoproteasomes display a different peptide repertoire changing the overall cytotoxic T cell–specificity as indicated by the observation that LMP-7−/− mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.

The 436-amino acid protein enolase 1 from yeast was degradedin vitroby purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active β subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.

Interactions and cleavage reactions of β-amino acids and β-oligopeptides (up to nine residues, carrying the side chains of Ala, Val, Leu, Ile, Phe, Ser, Lys, and Hop) with biological systems, such as the most potent peptidases (pronase, proteinase K, 20S proteasome), microorganisms (Pseudomonas aeruginosa and Pseudomonas putida), and mammalian blood (intravenous application to rats) have been investigated and compared with ?-peptides. The results are: i) the three peptidases do not cleave β-peptides at all (within 24 h), and they are not inhibited by a β-peptide; ii) except for certain 3-aminobutanoic-acid (β-HAla) derivatives, neither free, nor N-acetyl-β-amino acids, nor β-peptides (offered as sole N and C source) lead to growth of the two bacteria tested; iii) two water-soluble β-heptapeptides (with Lys side chains) were shown to have elimination half-lives t1/2(β) of 3 and 10 h at 100- and 30-ng/ml levels, respectively, in the rodent blood – much larger than those of α-peptides. Thus, the preliminary results described here confirm the much greater stability of β-peptides, as compared to α-peptides, towards metabolization processes, but they also suggest that there may be interactions (by hitherto unknown mechanisms) between the worlds of α- and β-peptides.