Microbiota Modulation to Reduce Cardiovascular Risk in HIV Patients: A Systematic Review


 Background: human immunodeficiency virus (HIV) infection is associated with premature and accelerated aging linked to the development of several co-morbidities, such as cardiovascular events, even after controlling for traditional cardiovascular risk factors. Since both traditional and “novel” factors are involved in the increased cardiovascular risk (CVR) observed in HIV-infected patients, new biomarkers could be needed to diagnose, evaluate and prevent the evolution of these events. Furthermore, since microbiota disturbance is involved in HIV-associated co-morbidities, all strategies focused on reversing or modulating gut microbiota (GM) composition and/or functionality could be of interest. In this context, a solid scientific evidence based on well-designed clinical trials is needed to support the use of prebiotics, probiotics, symbiotics and fecal transplantation (FT) to modify GM in HIV-infected patients and, therefore, to reduce the incidence of cardiovascular disease (CVD) in this population.
Methods: a search in PubMed has been carried out covering all clinical trials (Clinical Trial filter) including adult humans (19+ years) and whose results have been published in the last 10 years. Besides, the results obtained from ClinicalTrials.gob have been included and described in the present review Thus, we have reviewed the main results obtained from 3 clinical trials concerning the effects of prebiotics, 25 concerning probiotics, 6 concerning symbiotics and 4 concerning FT.
Results: none of the trials included in this review investigated if these compounds were able to reduce cardiovascular events in HIV patients.
Conclusions: the huge variability observed in the type of compound used, the dose and the length of administration, makes difficult to analyse the results as a proper meta-3 analysis and, therefore, to adopt general recommendations. Thus, there is an urgent need to investigate in this direction through robust and well-designed clinical trials.


METHODS
The objectives of this review were, i) to summarize the link between HIV infection and CVDs with special emphasis on GM involvement; ii) to describe "novel" biomarkers for cardiovascular risk in HIV infected patients; iii) to investigate if there is a solid scientific evidence (based on well-designed clinical trials) that supports the use of prebiotics, probiotics, symbiotics and FT to modify GM in HIV-infected patients. To achieve the last objective a search in PubMed has been carried out covering all clinical trials (Clinical Trial filter) including adult humans (19+ years) and whose results have been published in the last 10 years. We have limited the search to clinical trials because they are in the top of the "pyramid of scientific evidence", just below meta-analysis and systematic reviews (41). Meta-analyses and systematic reviews have also been consulted to confirm the clinical trials chosen for this review. A ten-years period has been chosen because from the 688 results obtained in PubMed with the keywords "microbiota HIV", 657 were published in the last 10 years, which means a huge and representative proportion (95.49%) of the total investigation developed in this field. Besides, a search in ClinicalTrials.gob has also been carried out and these studies have also been included and described in the present review (42).

CARDIOVASCULAR DISEASE IN HIV-INFECTED PATIENTS: FOCUS ON ATHEROSCLEROSIS
In this review, we have focused on atherosclerosis due to the fact that it constitutes a risk "per se" of developing other CVDs such as heart attack or stroke both in the general population as well as in HIV-infected patients. There are few differences in the histology of the atheromatous plaques of necropsy studies between HIV patients and uninfected patients, which suggests that the physiopathology of the development of the atheromatous plaque is quite similar (43) and consists on the well-known 3 phases: i) leukocyte infiltration into the subendothelial space and endothelial dysfunction, ii) formation of the lipid core of the atheromatous plaque, and iii) destabilization of the atheromatous plaque (44). Thus, we assume that the same CVR factors that contribute to de development of atherosclerosis affecting general population will also affect HIV-infected patients. The role of these traditional CVR factors in the development of cardiovascular events in HIVinfected people have been brilliantly reviewed by others (45).
In addition to the "classical" CVR factors, some authors consider that HIV infection acts as a CVR factor "per se". At vascular level, during leukocyte (mainly lymphocytes and macrophages) infiltration into the subendothelial space, leukocytes from HIV-infected patients have greater penetration capacity (46), possibly due to the fact that viral protein TAT increase the permeability of the vascular endothelium (47). Once installed in the subendothelial space, the lymphocytes get in contact with the endothelial cells, and these induce a significant increase of the viral replication inside the lymphocytes (through major histocompatibility complex (MHC) class II -T-cell receptors (TCRs) interaction) and, therefore, of the expression of viral proteins (48). The endothelial cells in contact with the viral protein TAT show more expression of macrophage attracting cytokines such as monocyte chemoattractant protein 1 (MCP-1/CCL2) (49) and adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1) (50), which attracts macrophages towards the arterial wall and facilitates their penetration into the subendothelial space through fibronectin (51). Moreover, viral proteins TAT and GP120 induce a rise in the production of endothelin-1 in the vascular endothelium, the most powerful vasoconstrictor known (52,53). Once macrophages have been installed in the subendothelial space, they express the "scavenger" receptor CD36, which will recognize the oxidized lipoproteins (4). At this point, the macrophages start to phagocytize oxidized lipoproteins accumulating cholesterol esters inside and becoming a foam cell. These cells will die by various mechanisms and their cholesterol stores will merge forming the lipid core of the plaque (44). The reverse cholesterol transport mediated by HDL must be able to counteract this situation, but their receptor ABCA1 is inhibited by the viral protein NEF (54). Then, the atheromatous plaque can be eroded resulting in a desquamation of the endothelial cells, which uncovers the matrix and origins the thrombus formation (44). In this point, HIV "per se" may induce the expression of metalloproteases that promote the endothelial desquamation (55,56). The contact of the plaque core with the arterial space induces the platelet aggregation. When platelets are activated they liberate inflammatory cytokines such as RANTES (regulated on activation, normal T-cell expressed and secreted) that reactivate the process initiated by macrophages (44) (Fig2).
In addition, the immune activation and persistent chronic inflammation observed in HIV-infected patients despite ART, are two factors strongly predictive of atherosclerosis (57), and they are associated with the development of non-AIDS events, with an increase in morbidity and mortality and, therefore, with a substantial reduction of patients' quality of life (28,58-60). Besides, HIV infection induces GM dysbiosis and ART is not able to completely reverse this situation (27). Only preliminary studies carried out with a small number of patients have showed an improvement in GM composition after treatment with integrase inhibitors (27).

CARDIOVASCULAR DISEASE RISK MARKERS IN HIV-INFECTED PATIENTS
Assessment of soluble and cellular proteins together with other approaches such as metagenomic or metabolomic could be used as biomarkers of cardiovascular events in HIV-infected people (61)(62)(63)(64)(65)(66)(67). Thus, we have herein classified these potential biomarkers as: (i) biomarkers dependent or partially dependent on microbiota; (ii) biomarkers independent on microbiota.

Trimethylamine N-oxide
Choline (vitamin B), carnitine and betaine (trimethylglycine) present in diet (mainly in milk, red meat, fish, vegetables and mushrooms) are degraded by the action of trimethylamine lyases present in GM producing trimethylamine (TMA). TMA is subsequently processed by hepatic monooxygenases leading to trimethylamine N-oxide (TMAO) (Fig3). TMAO is normally measured in blood by ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), although there are also other methods developed to measure such metabolite (68). The relation between very high levels of TMAO and the development of future cardiovascular events such as atherosclerosis has been recently recognised in non-HIV patients (69)(70)(71)(72)(73)(74). In fact, elevated blood levels of TMAO are associated with worse outcomes in patients with CVD, such as coronary artery disease or heart failure, and with higher mortality (70,(74)(75)(76)(77)(78). In HIV patients, several studies have also demonstrated the relation between TMAO and cardiovascular events (14,15,79,80). In one of these studies, high levels of TMAO were significantly associated with an increased risk of plaque incidence in the carotid artery (80). Furthermore, levels of TMAO were associated with higher levels of sCD14 and sCD163, two clusters of differentiation also involved in CVDs, as will be described later (80). Another study demonstrated an independent association between higher TMAO concentrations and higher CVR and between higher TMAO concentrations and multimorbidity (presenting ≥3 comorbidities) in HIV-infected patients (81). In the study from Haismman JM et al. (2017) no differences in TMAO blood levels were observed between naïve and ART-treated HIV patients, although high levels of its precursors, carnitine and betaine, were increased in untreated HIV patients compared to treated ones (82). It will be necessary to ensure if the lack of differences observed in that study between naïve and ART-treated people could be explained by diet. If diet were similar among both groups, it could be suggested that GM is different among both groups in terms of the ability to metabolize TMAO's precursors. In addition, the lack of differences observed in this study did not discard a significant increased cardiovascular risk in HIVinfected patients and other biomarkers of CVD should also be analysed. In fact, a very recent study has failed to find a strong association between TMAO, gut dysbiosis and inflammation in HIV infection (83). Therefore, the role of this metabolite as a marker of CVD in HIV population deserves further investigation.

Kynurenine/tryptophan ratio
Tryptophan is an essential amino acid (obtained from turkey, chicken, milk, cheese, fish, eggs, tofu, soybeans, sesame and pumpkin seeds, nuts, peanuts and peanut butter) indispensable to promote the liberation of serotonin, involved in the regulation of sleep and desire. Kynurenine is a metabolite resulting from degradation of tryptophan through the action of GM (84). A study demonstrated that HIV patients (45-50 years old under protease inhibitors) presented a lipid profile that predispose to CVDs and also a higher kynurenine/tryptophan ratio (measured in plasma by UHPLC-MS/MS) than healthy controls (85). However, more studies are needed to validate this ratio as an easy plasma biomarker of CVR in HIV-infected patients.

sCD14 and sCD163
As mentioned before, sCD14 and sCD163 are two clusters of differentiation that act as monocyte activators and inflammation markers as well as indicators of BT. sCD14 (soluble CD14) is secreted by activated monocytes (by proteolytic processing) after the join of lipopolysaccharide (LPS) to the toll-like receptor 4/MD-2 (TLR-4/MD-2) complex mediated by lipopolysaccharide binding protein (LBP) and mCD14 (membrane CD14). sCD14 can join in turn to more LPS and transfer it to mCD14 triggering the activation cascade of a greater number of monocytes (86) (Fig4). On the other hand, CD163, expressed in monocytes, has also two forms, mCD163 (membrane CD163) and sCD163 (soluble CD163). mCD163 is responsible of the removal of plasma hemoglobin through endocytosis of the very high-affinity complex hemoglobin-haptoglobin, thus, preventing the oxidative stress triggered by free hemoglobin through the release of the free iron, bilirubin and carbon monoxide. Moreover, sCD163 acts in the immune system but it is not yet clearly defined how. It is elevated in pathologies such as diabetes, obesity, liver disease and atherosclerosis, all of them characterized by a low-grade inflammation (87).
Regarding HIV-infection, only one study has reported a statistically significant relationship between the levels of sCD14 and sCD163 and new atheromatous plaque formation in HIV patients (88). Obviously, more studies are needed in this regard.

Other biomarkers related to immune activation, inflammation and bacterial translocation
D-dimer is a soluble fibrin degradation product that results from ordered breakdown of thrombin by the fibrinolytic system (89), whereas interleukin-6 (IL-6) is a glycoprotein with pleiotropic effects on inflammation, immune response and hematopoiesis (90). High levels of IL-6 and D-dimer are associated with increased CVR in HIV patients (91,92). Besides, these biomarkers remain high even under ART, which suggest immune activation even with successful suppression of HIV replication (93).
On the other hand, interleukin-1 (IL-1) is an inflammatory cytokine involved in the pathogenesis of multitude inflammatory disorders, including atherosclerosis (94). One study revealed that high levels of IL-1 receptor antagonist (IL-1Ra) in HIV-infected patients were associated with an increased risk of myocardial infarction, supporting the hypothesis that activating IL-1 signaling pathway could be a useful way to reduce CVR in HIV patients. (95).
Finally, when platelets are activated, they secrete other inflammatory cytokines such as RANTES (also known as CCL5), which reactivates the processes initiated by macrophages attracting more leukocytes (44). It has been demonstrated that platelets of HIV patients liberate more RANTES than healthy controls (96,97), although it should be of interest to investigate if this increased RANTES production could be associated with the increased CVR observed in these patients. One study reported that people with coronary atherosclerosis had significantly higher levels of RANTES than controls (98), but no studies have been published in the context of HIV-infection. It is also interesting to point out that CCR5, the receptor of CCL5, acts as a co-receptor of some HIV-1 strains (99) and it has been linked to atherosclerosis (100). Besides, Maraviroc, a CCR5 antagonist used in some HIV-infected patients (those in which HIV-1 has tropism towards this receptor), has led to statistically significant improvements in several CVR markers (101,102) and inflammation (103).

Endothelin-1
Endothelin-1 is synthesized by the vascular endothelium and presents vasoconstrictive, proinflammatory, profibrosis and promitogenic actions. It has been demonstrated that viral proteins TAT and GP120 are able to induce in the vascular endothelium an increase in the production of this molecule which leads to a higher vasoconstriction (52,53). Specifically, TAT regulates endothelin-1 at the transcriptional level through NF-κβ responsive sites in the promoter (52). The mechanism by which GP120 induces the production of endothelin-1 has not been described yet, but it is also at transcriptional level (104).
Elevated blood levels of endothelin-1 are independently associated with HIVassociated pulmonary arterial hypertension (PAH) maybe through the impairment of pulmonary endothelial function (105). Besides, it has been reported that plasma endothelin-1 levels are higher in HIV patients with PAH than in a non-infected population and also that levels increase with the severity of the PAH (106). The influence of ART on endothelin-1 deserves be further dilucidated.

Markers of oxidative stress
Oxidative stress has been defined as a disturbance in the balance between the production of reactive oxygen species (free radicals) and antioxidant defences, which may lead to tissue injury (107). Besides, the relationship between oxidative stress, ART and mitochondrial dysfunction has been documented ant it is related to alterations in the Krebs cycle and increased levels of ROS (108,109). This suggests that HIV infection and ART may induce oxidative stress (110,111). It has been demonstrated a decrease in the activity of paraoxonase-1 (PON-1), an antioxidant enzyme responsible for inhibiting the lipid peroxidation (112), in HIV-infected patients, which corroborates that HIV infection is accompanied by an increased oxidative stress. However, further studies are required to dilucidated the relation between HIV, ART and oxidative stress (113).
The marker of endothelial dysfunction asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide synthase (NOS) and it has been proposed as a marker of CVR and oxidative stress in several pathologies. ADMA plasma levels are increased after HIV infection and they seem to be reduced during ART (114). It has been reported that HIVinfected patients with type 2 diabetes show higher ADMA plasma levels than HIVinfected patients without type 2 diabetes (115,116), and taking into account that type 2 diabetes is a CVR factor it is reasonable to assume that it could be a good CVR marker in HIV population.

Lipoprotein-associated phospholipase A2
Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as plateletactivating factor acetylhydrolase (PAF-AH), is an enzyme produced by inflammatory cells (mainly macrophages) and responsible for hydrolyzing the oxidated phospholipids present in LDLs (117). It has been suggested that this enzyme could adequately and independently predict the onset of cardiovascular events in the general population (118)(119)(120). HIV patients showed higher levels of Lp-PLA2 in comparison with a healthy noninfected population (121,122), and these levels where associated with an elevated CVR in this population (122).

CD4/CD8 ratio
A low CD4/CD8 ratio is considered a hallmark of immunosenescence and is an independent predictor of mortality in the general population (123-125). Specifically, in HIV-infected patients under ART, it has been found a negative correlation between this ratio and immune activation and immunosenescence (126,127). Besides, CD4/CD8 is independently associated with markers of aging and, specifically, to carotid intima-media thickness (cIMT) and vascular stiffness (128). In this context, the analysis of the ICONA cohort showed that CD4/CD8 ratio below 0.30 compared to one above 0.45 was associated to mortality and to have non-AIDS events (129). Finally, another study demonstrated that this ratio was negatively correlated with progression of cIMT (130).

Prebiotics
Prebiotics were firstly described in 1995 by Glenn Gibson and Marcel Ruberoid as "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health" (131).
Eight studies have been published in PubMed concerning prebiotics in HIVinfected patients in the last 10 years. Only 5 are derived from clinical trials and of these, 3 have been discarded because they are related to symbiotics and, therefore, they will be incorporated in the appropriate section of this review. Of the 2 remaining articles, only one (NCT01838915) is registered in ClinicalTrials.gov and, interestingly, one of its objectives was to evaluate the effectiveness of the treatment to modify markers of endothelial dysfunction. However, the results concerning this issue have not been presented yet. In addition, another clinical trial (NCT04058392) appears in this website and whose aim was to evaluate tolerance and changes in inflammation and microbe composition in stools of HIV-infected people and to assess changes of gut barrier by doing biopsies by colonoscopy. However, it is not completed yet. Table 2 shows these 2 clinical trials registered in ClinicalTrials.gov, however, we have discussed the results obtained from all 3 clinical trials focused on prebiotics and HIV infection independently of its registration in the website.
Concerning the results published, the administration of prebiotics in HIV patients has proven to be able to modify microbiota composition at several taxonomical levels.
Moreover, the administration of scGOS/lcFOS/pAOS for 12 weeks has also been associated with decreases in BT markers such as sCD14 and LPS in naïve patients (133).
However, using a similar mixture of prebiotics (scGOS/lcFOS/glutamine) for 6 weeks no statistically significant differences were observed in these BT markers (132). These discrepancies could be due to the different duration of the intervention (12 weeks vs 6 weeks). Thus, microbiota is rather stable in adulthood and it is necessary longer periods of administration in order to modify it.
It is also interesting to point out that although it has not been observed changes neither in the viral load nor in the number of lymphocytes T CD4 after prebiotic administration, a decrease in the activation of these lymphocytes has been observed after the administration for 12 weeks of scGOS/lcFOS/pAOS (from 0.52% to 0.27 % in naïve patients, p<0.01) (133) or after the intervention with scGOS/lcFOS/glutamine for 6 weeks (132). This could mean that, although the intervention is not able to restore the number of lymphocytes T CD4, it facilitates a reduction of the activation of the immune system, which could be beneficial for these patients, given the persistent chronic inflammation characteristic of HIV infection.
In these two studies, the administration of prebiotics turned out to be safe without being observed modifications in the hepatic or the renal function (132,133). Nevertheless, in terms of tolerability, one of the studies has reported complications (mainly diarrhea), which disappeared 4 weeks after start the intervention (133).
To sum up, the administration of prebiotics in HIV patients was able to modify microbiota composition and to reduce the activation of lymphocytes T CD4, while the effects on markers of inflammation and BT were not clear enough. Besides, there are not studies that analyze if these changes are able to reduce the incidence of co-morbidities in this population and, specifically, if they are able to reduce CVR. Moreover, the type of compound and the time of administration is a variable to consider when drawing conclusions and making general recommendations. Therefore, it would be of interest to carry out more clinical trials to evaluate the effects of these compounds on CVR factors in HIV-infected patients and, specially, to carry out a follow-up study that could corroborate a significant reduction in cardiovascular events in those subjects with improvements in inflammation and intestinal dysbiosis.

Probiotics
The internationally endorsed definition of probiotics is "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (134). It has been suggested that the use of probiotics, such as Lactobacillus, Bifidobacterium and Enterococcus that can benefit the intestinal and immune system may one has been discarded because it only contains results obtained from symbiotics and, therefore, it will be incorporate in the following section of the review. Another 3 publications have been dismissed because they do not include HIV-infected patients.
Besides, we have added two more clinical trials that did not appear in the search carried out in PubMed but meets the inclusion criteria (137,138) and from the unique metaanalysis and/or systematic review published (139), we have appended one more clinical trial that meets the selection criteria and have not been included previously (140). Of the 17 remaining articles, only 5 appear registered in ClinicalTrials.gov. In addition, in this website another 11 clinical trials different from the abovementioned appeared. Thus, Table 3 shows all clinical trials registered in ClinicalTrials.gov although the discussion of all 25 clinical trials focused on probiotics and HIV infection has been included in this section.
The administration of probiotics in HIV infected patients has proved to be able to modify microbiota composition both in naïve patients and in ART-treated patients.
Specifically, intervention of ART-treated patients with different combinations of probiotics was associated with an increase of the relative abundance of Actinobacteria  (141). In addition, the administration for 12 weeks of Saccharomyces boulardii has also been able to reduce IL-6 levels respect to the placebo group (137). These discrepancies could be due to the different combinations used and, therefore, it makes difficult to conclude the positive effects of probiotics on inflammation or at least on these parameters measured (D-dimer and/or IL-6). In relation to the effects on BT, different combinations of probiotics (capsules with 56.5 mg living Saccharomyces boulardii, packet A and Saccharomyces boulardii) have been able to decrease LBP levels with respect to the placebo group (137,143,146). However, no effects in sCD14 or sCD163 levels have been observed (141,143,145,146)  In all these studies, the administration of probiotics turned to be safe without side effects or modifications in hepatic or renal functions (138,140,146,(148)(149)(150)(151)(152).
In summary, to date, we have some scientific evidence showing that the administration of probiotics in HIV patients is able to modify microbiota composition, increase the number of lymphocytes T CD4, decrease their activation and reduce LBP levels. There are more studies than in the case of prebiotics but, in general terms, these studies do not address the potential effects of these probiotics on CVR in HIV population.
Moreover, it is necessary to assess if the changes observed in markers of inflammation and BT after the ingestion of probiotics could lead to improvements in the incidence of co-morbidities and, specifically, CVR, in this population. In addition, the huge variability in the type of compound used, the dose and the time of administration makes it impossible to obtain verified and confirmed conclusions. Therefore, the first step would be to make a good description using large cohorts of the bacterial species that are decreased in HIVinfected patients. Secondly, these depleted species would be good candidates to ingest as probiotics to restore GM, but they should pass the requirements imposed by FAO and WHO to be used with any doubt of their safety. And, finally, the effects of these probiotics should be assessed in HIV-infected patients taking into account markers of inflammation, BT but also CVR, among others.

Symbiotics
The term symbiotics was introduced by Gibson and Roberfroid in 1995 to describe a combination of synergistically acting prebiotics and probiotics (131).
Five studies have been published in PubMed concerning symbiotics in HIVinfected patients in the last 10 years. Only 4 were derived from clinical trials. Of the 4 remaining articles, 3 appear registered in ClinicalTrials.gov. In addition, 2 more clinical trials were included in this website. The first one (NCT03568812) is still recruiting patients and its objective was to assess changes in immunity and in inflammation and BT markers. The objective of the second trial (NCT03542786), also in a recruiting state, was to evaluate inflammation and premature aging. It will be interesting to analyze the results of these clinical trials as soon as they are concluded. Table 4 shows clinical trials concerning symbiotics registered in ClinicalTrials.gov. Again, the discussion of all 6 clinical trials has been included in this review.
The administration of symbiotics in HIV patients has proved to be able to modify GM composition. Specifically, the administration for 16 weeks of 10 g of agavins from Agave tequilana plus Lactobacillus rhamnosus HN001 and Bifidobacterium lactis Bi-07 In all these studies, the administration of symbiotics was safe without side effects (144,153,155). Besides, the administration of LACTOFOS© twice daily for 30 weeks in naïve patients was able to significantly reduce diarrhea (reduction of 21.8% in incidents), nausea and/or vomits (reduction of 28.8% in incidents), constipation (reduction of 13.2% in incidents) and dyspepsia (reduction of 24.5% in incidents) (155).
In general terms, the results showed that the administration of symbiotics in HIV patients was able to modify microbiota composition, while the effects on the number of

Fecal transplantation
FT is the transfer of stools from a healthy donor into a patient with a disease characterized by a significant altered microbiome. Its objective is to restore the microbiota and, therefore, to treat the disease (156). FT is not new, in fact, there are reports of its use in ancient China for various purposes (157). It was firstly described as a treatment for pseudomembranous colitis in the 1950s (158) and in the recent years it has gained acceptance as a safe and effective treatment for recurrent Clostridioides difficile infection (CDI) associated diarrhea (159)(160)(161)(162)(163).
It has been observed that HIV patients treated for CDI by FT do not show any adverse events (164). However, in the same study, 14% of the inflammatory bowel disease (IBD)-patients experienced a disease flare requiring hospitalization in some cases (164). Therefore, FT involves an uncontrollable risk, particularly in immunocompromised patients if donor samples are not checked (165)(166)(167). In this context, the use of fecal filtrates could be a great alternative (168). Thus, despite all the advances in this field, the optimal protocol for FT is unknown and more well-designed studies and clinical trials are required because controversy issues are arising nowadays (169).

To our knowledge, 4 clinical trials have been registered up to date in
ClinicalTrials.gov, while the results of these clinical trials have not been published yet nor uploaded to PubMed. Thus, it will be interesting to analyze the results of these clinical trials in the future. Table 5 shows clinical trials concerning FT registered in ClinicalTrials.gov.

DISCUSSION
Our review has revealed that among all the GM modulatory strategies analysed

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Availability of data and materials: all data generated or analysed during this study are included in this published article.
Competing interests: the authors declare that they have no competing interests.     Original. Made with Biorender.
Fig4. sCD14 is secreted by activated monocytes after the join of LPS to the TLR-4/MD-2 complex mediated by LBP and mCD14. sCD14 can join in turn to more LPS and transfer it tomCD14 triggering the cascade activation of a greater number of monocytes. sCD163 acts in the immune system but it is not yet clearly defined how. LPS (lipopolysaccharide); TLR-4 (toll-like receptor 4); LBP (lipopolysaccharide binding protein); sCD14 (soluble CD14); mCD14 (membrane CD14); sCD163 (soluble CD163).
Original. Made with PowerPoint.        sCD14 is secreted by activated monocytes after the join of LPS to the TLR-4/MD-2 complex mediated by LBP and mCD14. sCD14 can join in turn to more LPS and transfer it tomCD14 triggering the cascade activation of a greater number of monocytes. sCD163 acts in the immune system but it is not yet clearly de ned how. LPS (lipopolysaccharide); TLR-4 (toll-like receptor 4); LBP (lipopolysaccharide binding protein); sCD14 (soluble CD14); mCD14 (membrane CD14); sCD163 (soluble CD163). Original. Made with