A Combined Proteomics and Mendelian Randomization Approach to Investigate the Effects of Aspirin-Targeted Proteins on Colorectal Cancer.
Summary (3 min read)
1. Introduction
- Several reports indicate that females have a stronger immune response, partly due to differences in hormonal profile [1, 2].
- Recently, TTV has received attention as a possible endogenous biomarker for immune function, with immunocompetent individuals carrying lower levels of TTV in serum than immunocompromised, indicating a suppressing role of the immune system on the viral load [3].
- The high turnover-rate of virions indicates that changes in immune status can be followed in a short time frame.
- The differences in female and male immunity towards pathogens have implications for treatment and prevention of infectious diseases and may ultimately lead to a different approach depending on the sex of the patient.
2.1. Subjects
- 27 healthy individuals were included according to a protocol approved by the Central Ethical Review Board (Swedish Research Council, Stockholm, Dnr: €O 24–2009) and consisted of 17 premenopausal women, 6 men and 4 postmenopausal women (Table 1).
- The subjects were included and sampled during 6 months (between March and September, 2010).
- Informed consent was obtained from the participants.
- Premenopausal women aged 20–40 years with regular menstrual cycles, without hormonal contraceptives or other hormonal, anti-inflammatory (including ASA, systemic cortisone and NSAIDs) or any morphine treatment since>3 months, and parturition no later than 12 months before inclusion, also known as The inclusion criteria were.
- Men (aged 20–70) and postmenopausal women (no menstrual bleeding since >12 months) without the above stated treatment during the last 3 months.
2.2. Blood sampling and hormonal analyses
- From all individuals blood was drawn at four timepoints, and for the pre-MP women Ovustick® was used to identify the LH-peak.
- Simultaneously, at one or more timepoints PBMC was also sampled.
- The buffycoat was transferred to new tubes and slowly frozen in 20% dimethylsulphoxide (DMSO)-albumin, using isopropanol-loaded Mr. Frosty® freezing-container overnight, before long-term storage at -80 C.
- Analyses were made of WBC, differential count (including B-monocytes, Blymphocytes, B-neutrophils, B-eosinophils, B-basophils), S-TSH (thyroid stimulating hormone), S-T4, S-SHBG (sex hormone binding globulin), Sestradiol, S-testosterone, S-progesterone, S-FSH, S-LH and S-prolactin.
- The participants were assessed for hypo- or hyperthyroidism, and preMP women also whether they had a normal ovulation.
2.3. TTV DNA isolation and analysis
- This was performed according to QIAamp® DNA Mini and Blood Mini Handbook to increase DNA yield.
- This contained pre-prepared solutions of 5, 50, 500 and 5000 copies plasmid TTV DNA per μL, as well as a sensitivity control containing 1 copy/μL.
- The sample wells were run in triplicates using 10 μL of concentrated DNA solution.
- According to the standard curve obtained, this corresponded to CT of 37.09, 42.09 and 39.14 respectively on the included three TTV qPCR plates.
- A sample was considered positive if 2 of 3 triplicate samples were above the detection limit (i.e. below the CT-threshold mentioned above).
2.4. Statistical analyses
- The average hormone levels of TSH, estradiol, LH and testosterone were calculated for each of the 17 pre-MP women.
- As a logistic model with logit-link, the following was used: TTV ~ log (Mean_TSH) þ log (Mean_estradiol) þ log (Mean_LH) þ log (Mean_testosterone).
- The explanatory variables are treated as covariates.
3.2. TTV prevalence and TTV levels
- The detected levels of TTVwere highest among the TTV positive pre-MPwomen and lower in the post-MP women and in the men, both in terms of detected TT viral copies/mL and when adjusting for total amount of DNA in the sample (Table 2).
- The differences in TTV prevalence between pre-MP and postMP women as well as between pre-MP women and men were not statistically significant (Fisher's exact test, p > 0.999 and p ¼ 0.2786) .
- The raw data suggested higher prevalence in men than in preMP women, but significance testing could not rule out a chance yroid status, sex hormone levels.
- For pre-MP women, day of the menstrual cycle, rone and LH, is indicated.
3.3. Hormonal status in TTV-positive pre-MP women
- To determine whether sex hormones influence the risk of being TTVpositive (TTVþ) the authors compared the average sex hormone levels in TTVþ (n ¼ 3) and TTV (n¼ 14) individuals using a binomial regression including S-estradiol, S-testosterone, S-LH and S-TSH.
- When comparing average TSH from TTVþ and TTV individuals in a binominal regression, there was no significant difference (p-value¼ 0.337, Table 3).
- Samples from themid-luteal phase revealed (using Welch's t-test) significantly lower progesterone levels (p ¼ 0.002) in TTVþ compared to TTV pre-MP women .
- Dispersion parameter for binominal family eviance: 5.5798 on 13 Df. Akaike information criterion (AIC): 15.58.
3.4. Hormonal status in men and post-MP women
- All three TTV men, and two out of three TTV post-MP women hade traces of TTV, but below cut-off (Table 4B).
- All men and post-MP women (TTVþ and TTV ) had normal levels of TSH.
- Due to the low number of participants, it was not possible to use regression models for analyzing hormone levels in relation to TTV status in men and post-MP women.
- TTV status, age and range of hormone levels are shown in Table 5.
4. Discussion
- The detection of TTV in 17.6% pre-MP females, 25.0% post-MP females, and 50.0%males suggests that TTV presence in the PBMC fraction of peripheral blood may be associated with sex.
- TTVþ samples from pre-MP women in their cohort were mostly found during the first half of the menstrual cycle, and pre-MP women who were positive for TTV had hormonal aberrances being either anovulatory, hypothyroid or both.
- Interactions of sex hormones with the immune system are established on multiple levels (reviewed in e.g. [10, 15, 16]).
- In a study by Maggi et al. (2001), TTV-levels were considerably lower in PBMC than in plasma from the same individuals [31].
- Only 2.4% of the plasma samples in the study from Fern andez-Ruíz et al. [45], were below lower limit of detection, whereas in their study on PBMC, the majority of individuals (74.1%) were below cut-off.
Author contribution statement
- Conceived and designed the experiments; Performed the experiments;, also known as P. Brundin.
- Analyzed and interpreted the data; Wrote the paper.
- B-M. Landgren, P. Fj€allstr€om and A. Johansson: Analyzed and interpreted the data.
- Conceived and designed the experiments;, also known as I. Nalvarte.
- Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.
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Frequently Asked Questions (17)
Q2. What is the role of TTV in the immune system?
TTV has received attention as a possible endogenous biomarker for immune function, with immunocompetent individuals carrying lower levels of TTV in serum than immunocompromised, indicating a suppressing role of the immune system on the viral load [3].
Q3. What were the inclusion criteria for premenopausal women?
The inclusion criteria were: Premenopausal women aged 20–40 years with regular menstrual cycles, without hormonal contraceptives or other hormonal, anti-inflammatory (including ASA, systemic cortisone and NSAIDs) or any morphine treatment since>3 months, and parturition no later than 12 months before inclusion.
Q4. What is the effect of estrogen on the immune system?
An interesting finding is that estrogen seems to stimulate anti-apoptotic properties in B-cells and increase somatic hypermutations and the class switch of immunoglobulins [38].
Q5. How many individuals were positive for TTV in PBMC?
Of 27 included individuals (6 men, 17 pre-MP women and 4 post-MP women), in total 7 were positive for TTV in PBMC; 3 men (50.0%), 3 preMP women (17.6%), and 1 post-MP woman (25.0%).
Q6. Why do the levels of estrogen change during the menstrual cycle?
This is probably due to a biphasic effect of estrogen, where low levels of estrogen stimulate a Th1 response (cell-mediated immunity) and high levels of estrogen stimulate a Th2 response (humoral immunity) [21].
Q7. What was used for amplification of TTV?
For TTV amplification, Argene TTV R-gene® (bioM erieux S.A., Marcy l’Etoile, France) kit (described in detail by [25]) was used on an Applied Biosystems 7500 Real-time PCR system.
Q8. What is the sex association of the TTV?
The detection of TTV in 17.6% pre-MP females, 25.0% post-MP females, and 50.0%males suggests that TTV presence in the PBMC fraction of peripheral blood may be associated with sex.
Q9. How many species of TTV are in the blood?
Most healthy humans (up to 90%) carry several species of TTV in their blood, and the levels normally range between 2-8 log10 copies/mL [3, 25, 26].
Q10. What is the relationship between TTV and sex hormones?
Although their study was performed on a limited number of subjects, the data suggests that TTV in PBMC is associated with an anovulatory menstrual cycle with low progesterone levels, and possibly with male sex.
Q11. What are the reasons why the TTV women are more susceptible to infections?
Social and behavioral factors may certainly contribute to sex differences in mortality from infectious diseases, however, even when controlling for exposure to infection in animal models, females seems to mount a stronger response towards pathogens [37].
Q12. Why was it not possible to use regression models for analyzing hormone levels in relation to TTV?
Due to the low number of participants, it was not possible to use regression models for analyzing hormone levels in relation to TTV status in men and post-MP women.
Q13. What should be done to determine the effect of ovulation on TTV?
future studies including sex and hormonal status (e.g. pre- or post-menopause, contraceptives, hormonal replacement therapies and ovulation, as well as signs of hypothyroidism), should be performed to obtain more information on the impact of the menstrual cycle on TTV load and immune response.
Q14. What is the effect of anovulation on TTV?
In line with the mentioned reports ([49, 50, 51]) and the findings from Benki et al. [48] the authors suggest that anovulation and the absence of a normal LH-peak in their data contributes to a favorable environment for TTV replication.
Q15. What is the relationship between TTV and progesterone?
TTVþ individuals were related to low progesterone in the present study, but most literature states that high progesterone levels increase the risk of infections.
Q16. How many copies of plasmidTTV DNA were used?
This contained pre-prepared solutions of 5, 50, 500 and 5000 copies plasmidTTV DNA per μL, as well as a sensitivity control containing 1 copy/μL.
Q17. What is the hypothesis that the hormonal deviation modifies the immune response?
the authors hypothesize that the hormonal deviation modifies the immune response, which would make the host more vulnerable to TTV replication.