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vitamin d yeast infection

J. H. J. L. and S. R. contributed equally to the manuscript.

Joan Hui Juan Lim, Sharada Ravikumar, Yan-Ming Wang, Thomas Paulraj Thamboo, Lizhen Ong, Jinmiao Chen, Jessamine Geraldine Goh, Sen Hee Tay, Lufei Chengchen, Mar Soe Win, Winnie Leong, Titus Lau, Roger Foo, Haris Mirza, Kevin Shyong Wei Tan, Sunil Sethi, Ai Leng Khoo, Wee Joo Chng, Motomi Osato, Mihai G. Netea, Yue Wang, Louis Yi Ann Chai, Bimodal Affect of Vitamin D in Host Response to Systemic Candida An infection—Vitamin D Dose Issues, The Journal of Infectious Illnesses, Quantity 212, Difficulty 4, 15 August 2015, Pages 635–644, https://doi.org/10.1093/infdis/jiv033

 

Summary

Past its classical function in calcium and bone metabolism, vitamin D3 is understood to exert a protean affect on the immune system [1, 2]. One such impact described is the propensity of vitamin D3 to skew host cytokine response away from a proinflammatory profile towards a T helper sort 2 (Th2) and regulatory T-cell response [3–5]. It additionally limits the antigen-presenting capability of macrophages and directs the differentiation of dendritic cells towards a extra tolerogenic phenotype, inducing heightened interleukin 10 (IL-10) ranges [6, 7]. With the innate and bridging adaptive immune system forming the crucial frontline protection defending the host in opposition to pathogens, it’s compelling to take a position whether or not vitamin D might affect host susceptibility to infections. Candidemia is the fourth commonest bloodstream an infection (BSI) in hospitals worldwide [8]. The choices of antifungal brokers obtainable are restricted, and sufferers with Candida BSI and invasive candidiasis have mortality charges of as much as 40% [9]. That is partly because of the host’s incapability to mount an efficient immune response in opposition to the invading fungi [10].

We had earlier investigated the function of 1,25(OH)2D3 on in vitro cytokine manufacturing induced by Candida stimulation of human peripheral blood mononuclear cells (PBMCs). We discovered that 1,25(OH)2D3 skewed cytokine response towards an antiinflammatory profile with downregulation of interleukin 6, tumor necrosis issue α (TNF-α), and interferon γ (IFN-γ) [11]. Nonetheless, the organic relevance of those results was not recognized. Therefore, we purpose to research and validate the sequelae of the immunomodulatory properties of vitamin D3 in vivo.

 

METHODS

Stimuli and Reagents

Candida albicans cells (SC5314) had been grown in a single day at 30°C in yeast extract–peptone–dextrose broth. Cells had been washed twice in phosphate-buffered saline (PBS) and diluted to five × 106 microorganisms/mL. Warmth-killed C. albicans blastoconidia had been used at a focus of 1 × 106 blastoconidia/mL. 1,25(OH)2D3 was bought from Fluka Biochemika, Sigma-Aldrich (St. Louis, Missouri), and dissolved in absolute ethanol. The pharmaceutical preparation of 1,25(OH)2D3, Calcijex (1 µg/mL ampoule), was from Abbott Laboratories (Quebec, Canada).

Mice

Eight weeks previous Balb/c mice weighing 20–24 g had been contaminated with 2.5 × 105 stay C. albicans (SC5314) by way of tail vein injection. 100 microliters of vitamin D3 (Calcijex) was administered intraperitoneally from day 2 after an infection at doses of 0.001 µg/kg, 0.01 µg/kg, 0.1 µg/kg, or 1 µg/kg for 3 days; 3-day administration of PBS served as a management. Weight and survival had been charted each day, and the fungal burden within the kidneys was assessed 6 days after an infection. The variety of viable Candida organisms was decided by plating serial dilutions on Sabouraud dextrose agar plates, and the colony forming models (CFUs) had been counted after incubation for twenty-four hours at 37°C. For the survival research, following Candida an infection (on day 0), the mice had been handled from day 2 onward with the stipulated each day doses of Calcijex for both 3 days or constantly.

PBMC Isolation

Separation and stimulation of PBMCs from wholesome volunteers had been carried out as beforehand described [12]. Blood specimens had been collected into sodium heparin tubes (BD Vacutainer, Franklin Lakes, New Jersey) after knowledgeable consent was obtained. PBMCs had been remoted by density centrifugation on Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden). Cells had been washed, counted, and adjusted to a focus of 5 × 106 cells/mL in tradition medium (Roswell Park Memorial Institute 1640 medium, Dutch modification, supplemented with gentamicin, l-glutamine, and sodium pyruvate). Stimulation assays had been carried out in 96-well round-bottomed plates, utilizing 100 µL of PBMCs with C. albicans and the respective fractions to attain a complete quantity of 200 µL/properly. After incubation for twenty-four hours or 48 hours in a humidified ambiance (5% CO2) at 37°C, the supernatants had been collected and saved at −20°C till additional assay.

Vitamin D Degree Measurement

Vitamin D ranges had been measured utilizing deidentified serum specimens from sufferers with Candida BSI in our hospital in opposition to anonymized serum specimens from hospitalized and wholesome topics archived in our hospital’s scientific laboratory. The Roche Elecsys vitamin D business assay was used to find out 25-hydroxyvitamin D ranges in human serum, and the assay revealed that >95% of the vitamin D (25-OHD) measured in serum was vitamin D3 [13]. This electrochemiluminescence-based immunoassay was learn on a Cobas e411 analyzer.

Recruitment and Phagocytosis Assay

Neutrophil and macrophage recruitment had been assessed following intraperitoneal injection of mice with 1 × 106 inactivated Candida microorganisms/mL at 4 hours and 72 hours, respectively. The peritoneal cells had been extracted by injecting 4 mL of ice-cold PBS-heparin into the peritoneal cavity. After washing, cytospin of the pattern extract, adopted by enumeration of the cell sorts, was carried out underneath microscopy. To determine phagocytosis and killing, peritoneal phagocytes as obtained had been washed and counted in a counting chamber. The processes of phagocytosis and intracellular killing had been studied in an adherent monolayer of phagocytes [14].

Histology

The mouse kidneys had been studied by mild microscopy with hematoxylin and eosin and Gomori methenamine silver staining after being mounted in 4% formalin, processed and embedded in paraffin, and sectioned at thicknesses of three µm. Sections had been assessed by way of mild microscopy for the presence of Candida and the areas within the kidney the place the organisms had been current (within the glomeruli, cortex, medulla, and pelvicalyceal areas).

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Stimulation and Cytokine Measurement

Spleen cells (5 × 105 cells/mL) obtained from mice on day 6 of an infection had been stimulated with heat-killed C. albicans (1 × 106 organisms/mL) in 96-well microtiter plates at 37°C. After 24 or 48 hours, supernatant was collected, and cytokine measurements had been made utilizing an enzyme-linked immunosorbent assay in response to the directions of the producer (eBioscience, San Diego, California). Detection limits had been 20 pg/mL (for interleukin 1b [IL-1b], TNF-α, and IFN-γ) and 10 pg/mL (for interleukin 17 [IL-17] and IL-10).

RNA Isolation and Quantitative Polymerase Chain Response (PCR) Evaluation

Splenocytes had been harvested from mice pretreated with 3 days of vitamin D3 (or PBS as management) whereas human PBMCs had been preincubated with vitamin D3 (or PBS as management) for half-hour. The consequences of each low-dose (0.01 µg/mL) and high-dose (1 µg/mL) 1,25(OH)2D3 had been studied. The cells had been then stimulated with heat-inactivated C. albicans for 4 hours. RNA was extracted from the splenocytes and PBMCs, utilizing 500 µL of TRIzol reagent (Sigma, St. Louis, Missouri). Subsequently, 200 μL of chloroform and 500 μL of 2-propanol had been used to separate the RNA from DNA and proteins. After washing with 75% ethanol, the dry RNA was dissolved in 50 μL of diethylpyrocarbonate water. To acquire complementary DNA, we reverse transcribed 1 μg of DNase-treated complete RNA with oligo(dT) primers (0.01 μg/mL) in a reverse transcription PCR combination with a complete quantity of 20 μL. Quantitative PCR was carried out utilizing the ABI Prism 7000 Thermocycler and SYBR Inexperienced. The primers we used are listed within the Supplementary Supplies. Quantification of the PCR indicators for every pattern was carried out by evaluating the cycle threshold (Ct) values, in duplicate, for the gene of curiosity with the Ct values for B2M as a housekeeping gene. Imply relative messenger RNA expression was calculated utilizing the comparative Ct methodology. Values are expressed as a ratio of fold improve to mRNA ranges of unprimed cells.

Chromatin Immunoprecipitation (CHIP)

To analyze vitamin D receptor (VDR) binding to the IFN-γ promoter, PBMCs from wholesome volunteers had been or weren’t handled with vitamin D3 for half-hour, adopted by stimulation with C. albicans for 4 hours. Nuclear proteins had been cross-linked to DNA with 1% formaldehyde and incubated at room temperature for 10 minutes on a rocking platform. Cross-linking was stopped by including glycine to a remaining focus of 0.2 M for five minutes. The cells had been collected by centrifugation and washed twice with ice-cold PBS. The cell pellets had been resuspended in 500 µL of sodium dodecyl sulfate lysis buffer, and the lysates had been sonicated to yield DNA fragments of 200–500 bp. Cell particles was eliminated by centrifugation. 5 % of the sonicated chromatin was put aside for enter management and the remaining was divided equally into 2 tubes, one for anti-VDR antibody and the opposite for the management immunoglobulin G (IgG). The lysates had been diluted 1:10 in ChIP dilution buffer. One microgram of anti-VDR antibody (catalog no. sc-1008; Santa Cruz, Dallas, Texas) was added to one of many tubes, and 1 μg of nonspecific IgG (catalog no. sc-2027; Santa Cruz) was added to the opposite, and the tubes had been incubated in a single day at 4°C on a rotating platform. The immunocomplexes had been collected utilizing Recombinant Protein A-Sepharose beads (Invitrogen, Frederick, Maryland). The beads had been washed sequentially for 10 minutes at 4°C with rotation, utilizing low-salt buffer, high-salt buffer, and LiCl wash buffer. The beads had been lastly washed twice with TE buffer, and the immune complexes had been eluted utilizing 300 µL of elution buffer at room temperature for 20 minutes with rotation. The immune complexes and the enter samples had been reverse cross-linked at 65°C in a single day within the presence of proteinase Ok (Invitrogen). DNA was extracted with phenol chloroform extraction and ethanol precipitation.

Actual-time PCR reactions had been carried out utilizing Sybr Inexperienced qPCR grasp combine (Utilized Biosystems, Warrington, United Kingdom) with genomic primers for the BED promoter areas [15] of the gene encoding IFN-γ (Supplementary Supplies), utilizing the ABI Prism 7000 equipment. The outcomes had been normalized with respect to the enter. Nonspecific IgG outcomes had been subtracted utilizing the components 2−ΔCt*100(VDR ab) – 2−ΔCt*100 (IgG), the place ΔCt is calculated as Ct(ChIP DNA) – Ct (Enter), and Ct is the cycle quantity.

Circulation Cytometry

Splenocytes had been harvested and washed twice with 1 × PBS supplemented with 0.2% (w/v) bovine serum albumin, stained with CD4-APC/CD25-PE fluorochromes, and stuck and permeabilized utilizing the FoxP3 staining equipment (catalog no. 320018; Biolegend, San Diego, California). The cells had been then intracellularly stained utilizing Foxp3-Alexa Fluor 488 or isotype management and had been analyzed on the BD LSRII Circulation Cytometer (BD Biosciences, San Jose, California). Information evaluation was carried out utilizing FlowJo software program (model 7.6.5; Tree Star, Ashland, Oregon).

Statistics

Outcomes had been pooled from at the very least 3 units of experiments (except in any other case acknowledged) and are offered as means ± commonplace error of the imply (SEM). Statistical evaluation was carried out utilizing the Mann–Whitney U take a look at or the paired Wilcoxon signed rank take a look at, when related. P values of <.05 had been thought-about statistically important. Ethics Assertion Institutional assessment board approval (from the Area Particular Evaluation Boards, Nationwide Healthcare Group, Singapore) was obtained to carry out the research involving human topics. Ethics approval for conduct of the animal research had been attained by the Organic Useful resource Centre Institutional Animal Care and Use Committee (Singapore).  

RESULTS

Candidemia Sufferers Have Decrease Vitamin D Ranges

To underline the function of vitamin D in candidemia within the scientific setting, we measured 25-OHD ranges in sera obtained from 28 hospitalized sufferers with Candida BSI and in contrast them to ranges in anonymized sera from 75 hospitalized sufferers in our hospital. As proven in Determine 1, sufferers with Candida BSI had considerably decrease 25-OHD ranges (imply [±SEM], 12.31 ± 1.69 ng/mL vs 17.04 ± 1.28 ng/mL; P = .045). The imply 25-OHD degree (±SEM) measured in a cohort of 30 wholesome volunteers was 20.88 ± 1.51 ng/mL, and this was considerably greater than in candidemic sufferers (P < .01) however was not considerably totally different from values for the anonymized hospitalized sufferers.

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1,25(OH)2D3 Reduces Candida Fungal Burden however Not at Excessive Dose Within the following experiments, we assessed whether or not vitamin D would possibly affect susceptibility to Candida an infection in vivo. Over a therapy vary of 0.001–1 μg/kg, Candida-infected mice that obtained 0.01 or 0.1 μg/kg of Calcijex had considerably decreased kidney fungal burden, in contrast with untreated mice (Determine 2A). The load loss traits mirrored that of fungal burden within the lower-dose vitamin D3–handled mice (Determine 2B). However, mice that obtained excessive dose of Calcijex (1 μg/kg) had elevated Candida development. Findings in histological sections of the mouse kidneys corresponded to the CFUs obtained by cultures. There have been fewer germinating yeast in mice handled with 0.01 or 0.1 µg/kg of Calcijex (Determine 2C). By way of general survival (Determine 2D), Candida-infected mice that obtained 3 each day doses of 1,25(OH)2D3 had elevated survival, in contrast with untreated mice (P = .006). The administration of each day doses of vitamin D3 (0.01 µg/kg of Calcijex intraperitoneally) didn't confer a further survival profit, in contrast with the 3-day Calcijex routine described above on the similar dose (P = .5). Not unexpectedly, mice given a excessive dose of Calcijex (1 μg/kg) confirmed poor survival, per the elevated fungal burden noticed. Recruitment Functionality and Phagocytic Operate To analyze whether or not vitamin D3 would possibly affect the inflow kinetics of inflammatory cells and phagocyte perform, cells had been harvested from the peritoneal cavity 3 and 72 hours after intraperitoneal injection with Candida in management mice and 1,25(OH)2D3-treated mice. There was no important distinction within the recruitment capability of neutrophils, monocytes, lymphocytes, and macrophages in mice handled with low-dose Calcijex (Determine 3A). Within the 1,25(OH)2D3-treated group, there was a pattern towards elevated phagocytosis of Candida, however general, there was no important enhancement of neutrophil perform. A marginal however important improve within the killing of Candida was noticed in macrophages extracted from mice handled with 1,25(OH)2D3 (Determine 3B). Nonetheless, the improved anti-Candida capabilities noticed had been modest and fewer more likely to be of related physiological significance. Vitamin D3 Dose-Associated Cytokine Response to Candida Utilizing splenocytes from contaminated management mice and mice primed with 3 each day doses of 1,25(OH)2D3, we studied cytokine induction by C. albicans. The splenocytes of mice that obtained the decrease Calcijex doses of 0.01 and 0.1 µg/kg confirmed a considerably elevated IFN-γ response (Determine 4B). On the greater dose of 1 µg/kg, ranges of cytokines had been largely suppressed. IL-17 ranges confirmed a dose-related pattern just like that of IFN-γ and a maximal response with 0.01 µg/kg Calcijex (Determine 4C). The induction of TNF-α and IL-10 by low-dose 1,25(OH)2D3 was restricted. Analysis of whether or not vitamin D3 has an affect on regulatory T cells revealed no important modifications in expression of CD4+CD25+FoxP3+ cells in splenocytes of 1,25(OH)2D3-treated mice (Determine 4E). Modulation of STAT and SOCS mRNA Transcription by Vitamin D3 In view of the unanticipated proinflammatory response induced by 1,25(OH)2D3 throughout Candida an infection, we studied the doable mechanisms by which this response could also be mediated. Particularly, the STAT and SOCS pathways are recognized to be central mediators of the host sort 1 T helper cell (Th1) response. Vitamin D3 exerted a differential dose-related impact on SOCS3 transcription: solely low-dose vitamin D3 inhibited SOCS3 mRNA transcription. Correspondingly, STAT-3 mRNA expression was enhanced (Determine 5A). However, there was lack of impact of vitamin D3 at greater doses. There was additionally no affect on the STAT1 and SOCS1 pathway (Determine 5B). These results had been seen in each the human PBMC system and in mouse splenocytes (Determine 5C and 5D). VDR Binding to the Promoter Area of the Gene Encoding IFN-γ The accentuated proinflammatory response, significantly the IFN-γ response, induced by vitamin D3 was not mediated to a major extent by the STAT1/SOCS1 pathway. Binding and activation of the VDR might induce its interplay with particular vitamin D response components current within the BED promoter of the gene encoding IFN-γ [15]. Utilizing ChIP-PCR, we confirmed that low-dose 1,25(OH)2D3 induced elevated VDR binding to the IFN-γ BED promoter throughout Candida an infection in PBMCs (Determine 6A). In distinction, high-dose vitamin D3 led to diminished binding. Cells handled with low-dose 1,25(OH)2D3 had elevated IFN-γ manufacturing downstream, validating the sequelae of the mechanistic modulation that we had elucidated above (Determine 6B).  

DISCUSSION

Within the current research, we confirmed that vitamin D3 exerts a bimodal impact on host immune response in opposition to Candida. At low doses, vitamin D3 conferred resistance in opposition to candidemia. However, supplementation of the host with high-dose vitamin D3 may very well be detrimental. The useful results noticed with low-dose vitamin D3 had been mediated by the enhancement of the host immune response. This was by potentiation of the proinflammatory cytokine response, amongst which IFN-γ, TNF-α, and IL-17 are recognized to have essential roles in antifungal host protection [16, 17]. Mechanistically, low doses of vitamin D3 suppressed SOCS3 on the degree of mRNA transcription and correspondingly elevated STAT3 exercise. Moreover, we confirmed that vitamin D3 additionally exerted an impact upstream by elevated binding to the IFN-γ promoter BED to induce IFN-γ manufacturing. The vitamin D nuclear receptor was concerned in mediating these responses.

These findings are each novel and unanticipated. The reported observations thus far typically describe the propensity of vitamin D3 to skew the immune response away from a Th1 sort response towards interleukin 4– and IL-10–pushed Th2 responses [3, 18]. This has been attributed to the capability of vitamin D3 to inhibit T-cell proliferation by suppression of interleukin 2 by the NFATp/AP-1 complicated [19, 20]. Equally, vitamin D3 inhibits IL-12 in dendritic cells [21]. This ends in suppression of not simply Th1, but in addition Th17 inflammatory responses. We and others have attributed this partly to vitamin D3’s capability to downregulate transcription and expression of floor pattern-recognition receptors [11, 22]. Nonetheless, a lot of these in vitro experiments, together with our personal earlier research on the immunomodulatory results of vitamin D3, had used greater doses of vitamin D3 that, looking back, is likely to be supraphysiological in vivo [23].

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On reassessment of our printed information in Candida-infected human PBMCs, we noticed a pattern towards elevated IFN-γ manufacturing with low-dose 1,25(OH)2D3 and cytokine suppression at greater doses [11]. As such, our present in vivo and ex vivo outcomes had been largely per our earlier remark. In addition to the bimodal cytokine traits elicited within the Candida-stimulated splenocytes, extra essential was our potential to exhibit the corresponding sequelae of such immunomodulation by vitamin D3 within the host, as evidenced by the fungal burden traits (each microbiologically and histologically). Whereas the recruitment of inflammatory cells throughout an infection and phagocytic perform didn’t appear considerably altered, there was a marginal improve in intracellular Candida-killing capability. We confirmed, in flip, that low-dose vitamin D3 enhanced host inflammatory response most clearly by IFN-γ within the anti-Candida Th1 effector arm and certain by induction of Th17 responses. This was mediated by the suppression of SOCS3 transcription. A resultant improve in IFN-γ may very well be accounted by the reported capability of SOCS3 to inhibit Th1 differentiation [24]. The corresponding upregulation of STAT3 expression is seen with the pattern towards elevated manufacturing of IL-17, as seen in our research.

The VDR performed a central function in mediating these responses. As a member of the nuclear receptor superfamily, the VDR could be activated by low concentrations of 1,25(OH)2D3 [25]. The VDR is a nuclear receptor that may act as a transcription issue to bind to particular vitamin D response components inside the regulatory areas of particular major goal genes [26]. To additional account for the elevated IFN-γ immunogenicity induced by vitamin D3, we demonstrated that low-dose vitamin D3 exerted a extra profound impact on IFN-γ manufacturing upstream. We confirmed that 1,25(OH)2D3 induced the elevated binding of VDR to the BED promoter area of the gene encoding IFN-γ and that this led to elevated manufacturing of IFN-γ.

This remark of a bimodal impact suggests the differential susceptibility to modulation of the varied immune targets and pathways by 1,25(OH)2D3. SOCS3 transcription, as an example, may very well be suppressed by low doses of 1,25(OH)2D3. Nonetheless, it is just with greater doses that we noticed common attenuation and even inactivation of different goal genes of curiosity. This moderately accounts for the variations between outcomes of our research and people of different research carried out earlier. Most of such earlier research on the immunomodulatory results of 1,25(OH)2D3 had been derived from in vitro research utilizing greater doses of 1,25(OH)2D3 that is likely to be supraphysiological. Thus, whereas the consensus remained in that the consequences of excessive concentrations of 1,25(OH)2D3 are largely antiinflammatory [27], the intrinsic greater susceptibility of some immune targets (eg, SOCS3) to low ranges of vitamin D3 and the elevated 1,25(OH)2D3-induced VDR binding to the IFN-γ BED promoter warrant highlighting.

The above observations are very related within the scientific context. Vitamin D3 deficiency has been properly described to be related to varied illness states, particularly susceptibility to infections [28–31], as we now have additionally demonstrated right here within the context of candidemia. Nonetheless, epidemiological information learning the affiliation between vitamin D ranges, supplementation, and particular infectious illnesses, similar to tuberculosis and viral infections, have been inconclusive [32–38]. This can be attributable to the variations within the supplemental doses used, as our findings recommend that the immunomodulatory impact of vitamin D3 is dose dependent. Nonetheless, the bimodal impact as demonstrated in our animal research raises the questions on what constitutes a low dose of vitamin D3 (with its doubtlessly useful results of supplementation) and what constitutes a better dose of vitamin D3 (with its maybe excessively immunosuppressive results) within the human host. Certainly, whereas a better vitamin D3 degree with its anticipated immunosuppressive affect could also be a requisite in opposition to inflammatory situations similar to a number of sclerosis or inflammatory illnesses, immunomodulation at such ranges might not be applicable within the context of infections. It stays to be decided as to what should be the optimum physiological degree of vitamin D3 within the physique, though a current suggestion had been to extend circulating 25(OH)D3 focus to >75 nmol/L [39]. In mild of this research, nonetheless, a extra prudent strategy and more-specific in vivo research must be carried out to reply this query.

In conclusion, we now have demonstrated that sufferers with candidemia have decrease ranges of vitamin D. In flip, restricted supplementation of vitamin D augments host immunity and enhances resistance in opposition to C. albicans an infection. However, extreme high-dose vitamin D suppresses the host immune response. On the background of the findings of this research and the continued debate over suggestions for vitamin D supplementation [40] in opposition to morbidity and mortality danger discount, the stability between vitamin D supplementation and potential warning in opposition to hypervitaminosis D must be highlighted.

 

Notes – “vitamin d yeast infection”

Monetary help. This work was supported by the Nationwide Medical Analysis Council, Singapore (new investigator grant, clinician scientist award, particular person analysis grant, bedside and bench and analysis coaching fellowship grants to L. C.); the Nationwide Kidney Basis, Singapore (to L. C.); and the European Analysis Council (consolidator grant 310372 to M. G. N.).

Potential conflicts of curiosity. L. C. has obtained grant help from and served as an advisor/marketing consultant for Pfizer, Gilead, Merck Sharp and Dohme. All different authors report no potential conflicts.

All authors have submitted the ICMJE Type for Disclosure of Potential Conflicts of Curiosity. Conflicts that the editors contemplate related to the content material of the manuscript have been disclosed.

 

References

 

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