Due to its metabolic activity, the human microbiota is defined as a virtual organ that consists of more than 1013
cells, which are found lining the skin and mucosal surfaces such as the gastrointestinal tract. The relative bacteria:human cell ratio in the literature varies from 1:1 to 10:1, nevertheless, the biological importance of the microbiota is undeniable [1
]. The microbiota has also been shown to have protective and structural effects on the epithelium and is responsible for maturation of the gut mucosal immune system [4
]. On the other hand, alterations in microbial composition are associated with many diseases, such as inflammatory bowel disease [5
] and colorectal cancer (CRC) [6
]. Dysbiosis is defined as any change in commensal communities leading to an imbalance between harmless and harmful bacterial species; such imbalances may account for either the cause or consequence of disease [7
]. There is, however, a strong correlation between dysbiosis and gut inflammation, as evidenced by a decreased proportion of Firmicutes and increased numbers of Bacteroides fragilis
, B. vulgatus
and bacteria from the Fusobacterium
]. This dysbiosis can be redressed by probiotics, which in effect change the microbiota within the gastrointestinal tract into more favourable species [11
]. Among probiotics, lactic acid bacteria (LAB) are the most frequently used [12
]. Apart from their ability to contribute to the inhibition of pathogens [13
], they have been shown to have immunomodulatory effects [14
Probiotic strains exhibit potential as dietary supplements against neoplastic transformation, through extensive effects on the host immune system [16
]. Such an investigative approach is urgently required to identify immunomodulatory probiotic strains in the treatment of colon cancer, which in effect support anti-tumour responses in both innate and adaptive immune cells. Innate immune cells are highly represented in the complex ecosystem of a tumour, with macrophages being the most abundant [17
]. In addition, epithelial-associated macrophages of the gastrointestinal tract, represent the largest population of these phagocytes in the body [18
]. Generally, macrophages exist in two distinct stages of polarisation, M1, which exhibit a pro-inflammatory phenotype, and M2, which have an anti-inflammatory phenotype. M1s are characterised by high production of pro-inflammatory cytokines and their induction of Th
1 immune responses whereas M2s produce anti-inflammatory mediators and drive wound healing and induce Th
2 immune responses [19
]. The M2 subset has also been linked to tumour-associated macrophages (TAMs), especially because of their pro-tumour angiogenic properties [21
]. This polarisation of macrophage response between pro-inflammatory M1-like and anti-inflammatory M2-like is suggested to be plastic, the manipulation of which represents a targeted therapeutic regimen for controlling immunity to pathogens and neoplastic transformation and cancer. The best example of this plasticity phenomenon is a growing body of evidence that suggests that the ability of tumours to switch between macrophage phenotypes has a beneficial effect on their own proliferation over different stages of cancer development [22
]. Probiotics have been shown to have both anti- and pro-inflammatory effects on macrophages [24
], thus it is possible that they can manipulate the plastic switching between these phenotypes in a favourable way to tumour surveillance and protection. The role of macrophages in tumour development is complex with dynamic macrophage phenotypic plasticity determining tumour initiation, progression and metastasis. Generally, TAMs have a dual effect on tumour growth and progression, acting in either a pro- or anti-tumour manner [26
]. In early stage tumorigenesis, M1 macrophages may stimulate production of pro-inflammatory mediators and cytokines, which can favour tumour cell growth. On the other hand, M2 macrophages play a pro-tumorigenic role mainly in late stage of tumours or in hypoxic areas, where they can promote angiogenesis, inhibit anti-tumour responses and also influence tumour relapse after conventional anticancer therapies [27
]. This M1/M2 classification however, is an over-simplification of a functional spectrum acquired by macrophages in response to stimuli of the tumour microenvironment. Given these key roles in homeostasis and cancer, manipulation of the functional plasticity of colonic macrophages and their cytokine production represent an exciting therapeutic intervention. Many macrophage-focused anticancer approaches have been investigated, including the blockade of tumour-promotion and activation of anti-tumour effector functions [28
]. Macrophage reprogramming towards an M2 anti-inflammatory subset in chronic inflammation (early onset CRC) and an M1 tumoricidal subset post-onset CRC, represents an attractive therapeutic strategy against cancer.
LAB Lactobacillus plantarum
VD23, C28 and MS18 and Lactobacillus salivarius
MS3, MS6 and MS16 were used in the present study. These strains have been tested previously for their probiotic features (viability under conditions of the human gastrointestinal tract, aggregation with pathogens and lactic acid production) and showed a positive effect on animal health [29
]. These strains were patented as fermented foodstuff product [31
]. Thus, the aim of this study was to investigate the in vitro immunomodulatory potential of these strains via their ability to modulate macrophage subset-specific phagocytic potential and cytokine production profiles. CRC exhibits a specific pathological sequence progression from pro-inflammatory (M1-driven) to immunosuppressive (M2-driven) stages, thus the translatability of these probiotic strains was studied using a bacterial mixture, based on the fact that some probiotic bacteria exhibit synergistic or suppressive effects on each other [32
], to assess modulation of anti-tumour responses in an in vivo model of CRC. For the further use of potential probiotic products containing multiple strains, their benefits to the host must be confirmed for the specified combination and not extrapolated from the evidence for the separate probiotic strains.
2. Materials and Methods
2.1. Macrophage Culture
The human monocytic cell line, THP-1, was obtained from ECACC and used for this study between passages 7 and 35. THP-1 cells were cultured in R10 medium composed of RPMI-1640 medium supplemented with 10% (v
) fetal bovine serum (Gibco, Massachusetts, Grand Island, NY, USA) and 2 mM L-glutamine, in the absence of exogenously added antibiotics. Pro-inflammatory (M1-like) macrophages and anti-inflammatory (M2-like) macrophages were generated by differentiation of these monocytes in the presence of 25 ng/mL phorbol 12-myristate 13-acetate (PMA) for three days or 10 nM 1,25-(OH)2
(Sigma-Aldrich, Poole, UK), for seven days, respectively [33
]. M1-like macrophages were then washed and allowed to rest in fresh R10 medium without PMA for an additional 24 h [34
]. Macrophages were plated out to a final density of 5 × 105
cells/500 µL/well in R10 medium in 24 flat-bottomed well tissue culture plates.
2.2. Bacterial Culture
Lactobacilli tested in this study are shown in Table 1
and were kindly provided by Dr. Jane Beal from internal microbiology stocks at the University of Plymouth (UK). All bacterial species were cultured in De Man Rogosa Sharp (MRS) (Sigma-Aldrich, Poole, UK) broth at 37 °C, 5% CO2 for 18–20 h until the beginning of the stationary phase. Bacterial cells were harvested by centrifugation of 4000 g for 5 min at room temperature and resuspended in R10 medium to the desired bacterial stock density.
2.3. Modulatory Effect of Live Probiotic Bacteria on Cytokine Production
To investigate probiotic regulation of macrophage cytokine release, different live bacterial strains were added in culture to a final density of 1 × 107 CFU/mL for 18 h (determined as the optimal concentration and time period for secretion of the cytokines TNFα, IL-1β, IL-18 and IL-23) in a humidified environment at 37 °C, 5% CO2. Supernatants were harvested and stored at −20 °C until assayed. Release of the pro-inflammatory cytokines, TNF-α, IL-1β, IL-18 and IL-23, were analysed by sandwich ELISA using commercially available capture and detection antibodies for TNF-α (BD-Pharmingen, Oxford, UK), IL-1β and IL-23 (eBioScienceTM, Paisley, UK) and an IL-18 human ELISA kit (InvitrogenTM, Paisley, UK). Protocols were followed according to the manufacturer’s instructions and compared to standard curves, using the recognised international standards available from NIBSC (Potter’s Bar, UK) over the range of 7 to 5000 pg/mL for TNF-α, IL-1β, IL-18 or 16 to 2000 pg/mL for IL-23, respectively. Colorimetric development was measured by an OPTIMax tunable microplate reader at 450 nm and analysed by Softmax Pro software, version 2.4.1 (Molecular Devices Corp., Sunnyvale, CA, USA). Cytokine secretion results are reported as the fold of induction (FOI) compared to the unstimulated macrophage control, to which an FOI of 1 was attributed.
2.4. Phagocytosis Assay of Live Probiotic Bacteria
To test macrophage subset phagocytosis of different bacterial strains, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-loaded bacteria were used. Briefly, bacterial strains were labelled with CFSE (Molecular Probes, Leiden, The Netherlands) at a final concentration of 1.5 mM in PBS for 30 min in a humidified environment at 37 °C, 5% CO2 in the dark. Concurrently, macrophages were treated with an inhibitor of actin polymerisation, cytochalasin D (Sigma Aldrich, Dorset, UK), at a concentration of 1 µg/mL for 35 min at 37 °C, 5% CO2. After incubation, bacteria were washed extensively to remove unbound CFSE and were added to a final density of 1 × 107 CFU/mL (determined as the optimal concentration for phagocytosis assay) to both untreated and cytochalasin D-treated macrophages and co-cultured in a humidified environment at 37 °C, 5% CO2. Phagocytosis was performed for a period of six hours with four time points—one, two, four and six hours. Cells were then washed with PBS, detached with trypsin and processed through flow cytometry. A total of 5000 events were recorded on a FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analysed with BD FACS-Diva software (BD Biosciences, San Jose, CA, USA). Degree of phagocytosis was determined as an increase in the mean fluorescence index (MFI) ratio between untreated and cytochalasin D-treated macrophages. Phagocytosis of each probiotic strain was carried out in triplicate samples for at least n = 3 independent replicate experiments.
2.5. Animals and Diet
Male Wistar rats (n = 56) (Velaz, Ltd., Prague, Czech Republic) were obtained at four weeks of age (mean body weight 130.45; range 114–157g), quarantined and housed in groups of four per cage at room temperature 22 ± 2 °C, 55 ± 10% humidity and a 12 h light/dark cycle. They had free access to a standard laboratory diet (Altromin 1324, Lage, Germany) and water. Food consumption for each cage was monitored daily and body weights were recorded weekly. All experiments were approved by the National Animal Ethical Committee of the Slovak Republic (license number Ro-4058/16-221) and were conducted in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS 123).
2.6. In Vivo Investigation of Probiotic Effects on Colorectal Cancer
After one week of acclimatisation, rats were randomly divided into four groups (Scheme 1
): control cancer group (n
= 20) (DMH), probiotic-fed cancer group (n
= 20) (DMH+P), probiotic-fed healthy group (n
= 8) (P) and control healthy group (n
= 8) (INT). Probiotic-fed groups were given a mixture of LAB (LP VD23, C28 and MS18 and LS MS3, MS6 and MS16) from Monday to Friday until the end of experiment. The probiotic mixture was prepared according to standard procedure (see Section 2.2
on bacterial culture) and administered orally in a final concentration of 109
/50 μL/dose in sterile water once a day. The proportion of bacteria in the mixture was equal. The probiotic mixture was used as prophylactic for four weeks (prevention period) prior to 1,2-dimethylhydrazine (DMH) administration. After the prevention period, cancer groups received subcutaneous injection of DMH. CRC was induced by DMH (Fluka Chemie, Buchs, Switzerland) prepared according to the standard method [35
]. DMH was dissolved in 0.9% NaCl containing 1 mM EDTA and adjusted to pH 6.8 with 1 M NaOH and administered subcutaneously at 20 mg/kg once a week for 15 weeks. A fresh solution was prepared prior to each application. Blood samples were collected monthly from the tail vein in EDTA-containing tubes (Sarstedt, Nümbrecht, Germany) until the fifth month of the study and analysed for leukocyte count on BC-2008 VET automatic analyser (Mindray, Shenzhen, China). At the age of 35 weeks, the rats were humanely euthanised by decapitation. For clarity, the experimental design is shown in Scheme 1
2.7. Measurement of Probiotic Effects on Tumour Incidence, Colon Length and Tumour Cytokine Production
During autopsy, the colon was removed and its length was measured. It was opened along the longitudinal axis, flushed with saline and examined macroscopically for tumours, the number, size and distribution of which were recorded. Tumour size was measured by digital calipers and volume calculated by the following formula
(where V stands for volume, L for length, W for width and H height) [36
]. Tumours were snap frozen on dry ice and stored at −80 °C until analysis. Analysis of TNF-α, IL-1β and IL-18 was performed by Bio-Plex®
Multiplex Immunoassay (Bioclarma Sr.l., Turin, Italy). Briefly, tissue samples were lysed according to the manufacturer’s instructions. All samples were then dosed with DC Protein Assay and diluted to a working concentration of 0.9 μg/μL. Fifty μL of this solution were analysed in each well (each sample was tested in a technical duplicate). The standard curve was optimised for lysate analysis with eight points according to manufacturer’s instructions over the range of 10.61 to 11,327.7 pg/mL for TNF-α, 3.49 to 14,002.96 pg/mL for IL-1 β and 3.56 to 9,048.72 for IL-18.
2.8. Statistical Analysis
All statistical analyses were performed using Minitab version 16 software (Minitab Inc., 2013, Coventry, UK). All data were examined for normal distribution, and appropriate tests applied. Statistical differences between groups were analysed using ANOVA followed by the Tukey post hoc test or Kruskal–Wallis test followed by pairwise multiple comparison procedures (Mann–Whitney test). All differences were considered statistically significant when p < 0.05.
The purpose of this research was to investigate the beneficial effects of a novel panel and mixture of probiotic species with respect to their immunomodulatory capacity on macrophages and their potential to be adopted for prophylaxis of colorectal cancer, a pathology that is associated with macrophage subset-specific effector responses. This study resulted in several important findings, namely: (1) probiotic strains used in this study differentially induced macrophage pro-inflammatory cytokines in a subset- and strain-specific manner, (2) phagocytosis of probiotic was both macrophage subset- and bacterial strain-specific, (3) probiotic mix abrogates DMH-induced shortening of the colon and exhibits a beneficial effect on leukocyte count and colon tumour development, and (4) probiotic mix induces secretion of pro-inflammatory IL-18 by tumour tissue.
In agreement with similar studies, the probiotic bacteria tested in this study induced secretion of pro-inflammatory, hence potentially anti-tumour cytokines (TNF-α, IL-1β, IL-18, IL-23) in a strain-specific and cell-subset specific manner [14
]. M1-like macrophages generally secreted higher levels of TNF-α compared to M2-like (anti-inflammatory) macrophages. In the case of M1s, strains of genus LP were 10 to 20-fold less potent than strains of LS, whereas in M2s, the LP strains C28 and MS18 induced similar levels to the LS strains, indicative of a more pro-inflammatory effect of these bacteria on this subset of macrophage. LP VD23 was shown to have the least potent effect on secretion of TNF-α in M2s. TNF-α, originally described for its anti-tumour properties [39
], is a multifunctional cytokine, which, at high doses, can induce tumour cell apoptosis whereas it accelerates tumour progression via tumour invasion and metastasis with chronic low doses [40
]. It was also found, that TNF-α concentration increases with advanced stage of CRC [42
]. The same trend was exhibited for IL-1β production where LS strains induced 2.5 to 16-fold higher levels of this cytokine compared to LP strains in M1-like cells. LP VD23 showed poor ability to induce secretion of this cytokine in both macrophage subsets tested, however it must be noted that the fold induction was much higher in M2 than M1 macrophages. IL-1β can be considered as both pro- and anti-tumoral; that is, it promotes anti-tumour responses via its pro-inflammatory nature and its ability to promote Th17
differentiation and neutrophil activation and it is also able to drive pro-tumour responses via tumour invasiveness and immunosuppression [44
]. Higher IL-1β levels can induce myeloid-derived suppressor cells (MDSC), which exhibit phenotypic similarity to anti-inflammatory M2 macrophages capable of suppressing anti-tumour immunity, and resulting in tumour growth. MDSCs and M2 macrophages are present at appreciable levels in many different types of cancer including CRC [46
]. This would suggest that the probiotic strain-induced cytokine profile can effectively manipulate macrophage subset plasticity and the switching from pro-inflammatory to pro-tumour effector phenotypes. Similar results were observed for IL-18, an IL-1β family cytokine that promotes protective host immunity mediated by CD8+
cytotoxic T cells (Tc
), NK cells [47
] and Th1
-driven macrophage activation. NK cells exhibit an anti-tumour responses, therefore an increase in IL-18 secretion may be relevant to the anti-tumour activity of specific lactobacilli strains in rat colorectal cancer models [49
]. In addition, IL-18 is crucial for intestinal epithelial cells’ (IECs) homeostasis and mucosal repair, inducing IEC proliferation/differentiation, goblet cell mucus production, the expression of tight junction proteins and secretion of anti-bacterial peptides [51
] essential in preventing CRC development [53
]. LS species potently induced this cytokine in both cell subsets, whereas LP strains had little or no ability to stimulate IL-18 secretion compared to control. Interestingly, LS MS3 induced 2.5-fold higher production of IL-18 in M2-like cells compared to M1-like cells, which may be indicative of a subset plasticity switch towards a pro-inflammatory/anti-tumour M1 phenotype. IL-23 is a heterodimeric pro-inflammatory cytokine, which, in ulcerative colitis, activates and induces cytokine production of NK cells, intraepithelial lymphocytes (IEL), innate lymphoid cells (ILCs), and Th
17 (reviewed in [54
]), whereas it blocks activation of regulatory T cells (Treg
]. This pro-inflammatory nature however, is contradicted by several observations where serum IL-23 levels were increased in CRC patients and positively correlated with VEGF [56
] and high primary tumour tissue levels of IL-23 were predictive of increased CRC metastasis [57
]. LS strains stimulated both macrophage subtypes to secrete this cytokine, whereas strain MS3 potently induced secretion of this cytokine in M2 cells. LP strains, on the other hand, showed little or no induction of IL-23.
Differential cytokine production profiles is a key feature of polarised macrophages exhibiting differential effector functionality, such as phagocytic capability [58
]. Homeostatic mucosal macrophages resemble the M2 subset, and are characterised by high phagocytic activity, scavenger receptors (CD36, CD68, CD206) and low pro-inflammatory cytokine production, whereas during inflammation, they polarise towards an M1-like subset (Phagolo
, Scavenger Receptorlo
, Pro-inflammatory Cytokinehi
]. Macrophage phagocytic capability and the nature/health of target cells being recognised for phagocytosis have a profound effect on the macrophage cytokine profile, and hence the plasticity of the effector phenotype [61
]. Phagocytic activity was higher in M2- than M1-like macrophages immediately after the first hour of co-culture with all tested probiotic strains. M1s also exhibited phagocytic activity, but at delayed onset time points. In M2-like cells, the highest phagocytosis was observed after six hours of co-culture. In general, LS MS3 induced low phagocytic activity in both M1s and M2s, whereas the cytokine profile stimulated by this strain in both macrophage subsets was TNF-αhi
. This is indicative of the potential of MS3 to drive towards an M1 phenotype characterised by low phagocytic activity and high pro-inflammatory cytokine expression. Lactobacillus
MS6 also stimulated cytokine secretion to high levels in both macrophage subsets, but interestingly phagocytosis in M1-like cells was the highest for all tested strains. This might indicate that both pro- and anti-inflammatory properties are inducible by this bacterial strain and that macrophage polarisation may be intermediate between conventional M1 and M2 subsets. MS16 potently induced pro-inflammatory cytokines whilst inducing a low to medium level of phagocytosis, which demonstrates the pro-inflammatory potential in both M1s and M2s. Both LP strains MS18 and C28 had poor capability to induce cytokine release and generated medium phagocytic activity in both macrophage subsets, suggesting the more anti-inflammatory capacity of these strains. Finally, both M1s and M2s displayed a TNF-αlo
profile after stimulation by LP VD23 whereas M2s were Phagohi
and M1s Phagomed
. Of all the tested lactobacilli, this would appear to be the most anti-inflammatory strain with potentiation of an M2 phenotype. From these data, the tested probiotic bacteria showed pro-, anti- and mixed pro- and anti-inflammatory properties in vitro. It is however, essential to test production of other cytokines, expression of scavenger receptors, Arg-1 and iNOS to confirm the potential of these strains to polarise towards distinct macrophage subsets. Nevertheless, this study has demonstrated an association between probiotic strain LS MS3 and M1 macrophage phenotyping, whereas LP VD23 showed an association with M2 macrophage phenotype. Other strains showed an intermediate polarising capacity between these two distinct macrophage subsets. Consequently, these probiotic bacterial strains may differentially drive functional plasticity between M1 and M2 macrophage subsets, effectively reprogramming macrophage effector functions towards pro-inflammatory/anti-tumour responses or anti-inflammatory/immune suppressive responses.
As a consequence of the in vitro data, which demonstrated a range of probiotic-driven effects such as pro-, anti- and both pro- and anti-inflammatory, and taking into account the differential immune responses exhibited at different stages of tumour progression, the in vivo evaluation of anti-cancer effects of probiotics on a DMH model of colon carcinogenesis utilised a mixture of the six probiotic strains. Studies in experimental CRC models suggest that tumour infiltrating immune cells and their inflammatory cytokines either directly or indirectly stimulate the uncontrolled growth of cancer cells, from neoplastic transformation to tumour expansion and metastasis. The onset and the progress of CRC could be potentially modified using a panel of lactobacilli with different macrophage reprogramming capability. During carcinogenesis, the tested lactobacilli mix, especially the anti-inflammatory M2-programming VD23 strain, ameliorates the inflammatory conditions (in the early stages) and/or the pro-inflammatory M1-programming MS3 strain can boost an anti-tumour immune response with the down-stream effect of eliminating dysplastic and cancerous cells. With respect to long-term study of CRC, where cancer arises from chronic inflammation and leads to an immunosuppressive state with tumour presence, a mixture of probiotic bacteria with both anti- and pro-inflammatory (M2- and M1-programming) features was used, and this may represent a realistic approach to harnessing probiotic strains in the modulation of CRC.
While body weight gain over the experimental period did not differ, there was a significant difference in daily food intake between all experimental groups. Despite the increased food intake of the DMH group compared to the DMH+P group, the rats’ ability to convert food into body mass (expressed by FER) was not significantly affected. The probiotic-fed group was shown to have the highest FER, therefore it can be suggested that probiotic treatment can improve absorption and digestion of food [64
]. In general, the improved health status of probiotic-fed cancer animals was obvious. In addition, probiotic treatment in the DMH+P group significantly increased colon length, effectively rescuing the reduced colon length observed in the DMH group. Similar results were found by [65
], who reported that probiotic supplementation restored colon length.
Although the incidence and multiplicity of tumours were not significantly influenced by probiotic supplementation, a trend in decreasing tumour volume and numbers in the DMH+P group compared with cancer control was evident. The total number of tumours was 33% less for the DMH+P group, suggesting a positive effect of probiotic treatment. Cytokine profiling in tumour tissue is a good indicator of tumour immunity, tumour development and prognosis [66
]. Probiotic treatment had no significant effect on tumour secretion of the pro-inflammatory cytokines, TNF-α and IL-1β, whereas IL-18 was significantly increased in tumour tissue of DMH+P animals. TNF-α and IL-1β exhibit both pro- and anti-tumour activity in CRC, dependent on stage of progression; IL-18 however, is considered an anti-tumorigenic cytokine, that not only affects Th1
-cell-mediated immunity but also stimulates anti-tumour activity of NK cells, thus, a probiotic-mediated augmentation of IL-18 suggests it had a protective effect in this in vivo system. Better tumour surveillance of probiotic-fed DMH rats can be also linked to this probiotic potential to induce IL-18 secretion, as IL-18 is critically involved in protection against colorectal tumorigenesis [67
]. Additionally, IL-18 also induces the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells derived from cancer patients, suggesting that IL-18 conferred NK cells on an APC-like phenotype, which indicates increased recognition and responsiveness to tumour antigens [68
]. As with TNF-α and IL-1β, IL-18 may also exhibit both pro- and anti-tumour activity in CRC, depending on the stage of progression and immune effector cells responsive to IL-18. It was found, that in models of IBD, secreted IL-18 potentiates IL-18 responsiveness via augmentation of lamina propria
T cell IL-18 receptor expression and that IL-18 signalling restricts intestinal inflammation by limiting the Th
17 differentiation and promoting Treg
effector responses [69
Leukocyte count slightly decreases with age in rodents and humans [70
]. The same trend was also observed in the present study with a slight difference between experimental groups. Interestingly, there was a significant decline in leukocyte count after five months of experiment in the DMH group (p
< 0.05), while leukocyte count in the DMH+P group did not change compared to the INT group. More profound changes in peripheral leukocyte count (especially a decrease in monocytes and lymphocytes count) can be seen in patients where disease has been systematically spread [72
], therefore these findings confirm the hypothesis that probiotics can increase quality of life or delay the onset of the disease [73