Induction of donor specific tolerance (DST) by the introduction of donor cellsinto a recipient’s portal vein is one of the approaches used to solve theproblem of transplant engraftment. However, the mechanism of DST developmentremains unclear to this moment. In the present work, we first studied thechange in the content of immunoproteasomes and macrophages of the liver atearly stages of the development of allospecific portal tolerance in rats byWestern blotting and flow cytofluorimetry. On the basis of the data obtained,we can conclude that the induction of DST is an active process characterized bytwo phases during which the level of the proteasome immune subunits LMP2 andLMP7 in liver mononuclear cells, including Kupffer cells, and the number ofKupffer cells change. The first phase lasts up to 5 days after the beginning ofDST induction; the second phase – from 5 to 14 days. In both phases, thelevel of the subunits LMP2 and LMP7 in the total pool of mononuclear cells andKupffer cells increases, with maximum values on days 1 and 7. In addition, thetotal number of Kupffer cells increases in both phases with a shift in severaldays. The most noticeable changes take place in the second phase. The third dayis characterized by a lower content of mononuclear cells expressingimmunoproteasomes compared to the control value in native animals. Presumably,at this time point a “window of opportunity” appears for subsequentfilling of an empty niche with cells of different subpopulations and, dependingon this fact, the development of tolerance or rejection. The results obtainedraise the new tasks of finding ways to influence the cellular composition inthe liver and the expression of immunoproteasomes on the third day after thebeginning of DST induction to block the development of rejection. The problem of transplant engraftment remains one of the concerns intransplantology. Transplantation is used in terminal organ failures, when othermethods of treatment have proved ineffective. Allogenic transplantationactivates the immune response, which leads to transplant rejection. Modernimmunosuppressive protocols are not always able to prevent rejection.Therefore, it is necessary to search for other approaches to induce toleranceto transplants in recipients.The critical role of the liver in the development of transplant tolerance hasbeen known for a long time. Spontaneous liver allotransplant engraftment inrecipients mismatched from donors by the major histocompatibility complex (MHC)was revealed for outbred pigs , inbredAnother key factor involved in the induction of transplant tolerance is thepresence of immunocompe tent cells of donor origin within the liver. This issupported by studies showing that allograft tolerance is not induced afterdepletion of passenger leukocytes from the donor liver-9.The method of inducing donor-specific tolerance (DST) is based on adherence tothese two conditions. The induction is performed by transfusion of donor cells into the liver via the portalvein. This method leads to significant lifespan extension of allografts of theheart , kidneysMultiple forms of immunoproteasomes that contain the immune subunits LMP2,LMP10, and/or LMP7 with proteolytic activities have been regarded as potentialcandidates for the role of messengers of immune response, which can direct theimmune response either toward allograft acceptance or rejection.Immunoproteasomes participate in the formation of antigenic epitopes for theMHC molecules, the regulation of the expression of co-stimulatory molecules onantigen-presenting cells (APCs), and the differentiation of T-lymphocytesubpopulations-21.We have found previously that the proportion of LMP2 and LMP7 immunoproteasomesubunits changes in the liver and allografts of ovaries and thyroid after the induction of DST, 23.AllPrevious experimental and clinical studies have shown that induction of DST fails in some cases, 25.MorIt is evident that the immunological events occurring in the liver of arecipient immediately after the administration of donor cells and that arerelated to the recognition and presentation of antigen can determine thedevelopment of tolerance. Therefore, the study of the cascade of cell-mediatedreactions and the change in the pool of proteasomes at early stages after theadministration of a donor antigen is important for understanding the mechanismunderlying DST induction.The aim of this study was to evaluate the level of immunoproteasomes and thequantity of resident macrophages in a rat liver in the first two weeks afterthe beginning of DST induction.ReagentsDMEM medium, collagenase and DNAse I , percoll, saponin , rabbit pAb to subunit LMP7,mouse mAb to subunit LMP2 , phycoerythrin-conjugated mouse mAb to macrophages , mouse mAb to β-actin , Alexa 488-labeled anti-rabbit IgG ,phycoerythrin-conjugated anti-mouse IgG  were used in thisstudy.Animalsn = 12)– intact control; group 2 (n = 48) –false-operated animals ;and group 3 (n = 48) – animals with DST induction ; group 4 (n = 30)– animals with DST disruption  and an intraportal administration ofsplenocytes after 24 h).The experiments were performed using 5- to 6-month-old Wistar and August femalerats. The donors were Wistar rats, and the recipients were August rats. Allmanipulations with the animals were performed in compliance with the EuropeanConvention for the Protection of Vertebrate Animals used for Experimental andOther Scientific Purposes . For the experiments, thefollowing groups of animals were used: group 1  at 37°C for 10 min. Afterwards, the tissue wasgrinded with scissors and further incubated in collagenase buffer at 37°Cfor 30 min, disintegrated through pipetting, filtered through a nylon sieve,and centrifuged at 20 g at 4°C for 2 min. The supernatantwas collected and centrifuged at 400 g for 3 min. The pelletof cells was resuspended in 30% percoll and centrifuged at 400 gfor 30 min at 4°C. The cells were collected from the interfaceand washed two times with 0.1 M phosphate buffer saline at 4°C.The rat liver was perfused via the portal vein with a calcium-free buffer  for 5 min. The liver was thenextracted and perfused with 0.1 M phosphate buffer saline containing 0.4 mg/mlcollagenase, 3.7 M CaClPhenotypical analysis of the cells using flow cytofluorimetry6 cells and the corresponding antibodies . After washing, thecells were incubated with secondary antibodies: Alexa 488-labelled anti-rabbitIgG or phycoerythrin-conjugated antibodies to mouse IgG at a dilution of 1 :500 for 30 min at room temperature.In order to identify subunits of immunoproteasomes, the isolated hepaticmononuclear cells were fixated in 4% paraformaldehyde for 15 min andpermeabilized for 15 min in a 1% saponin solution prepared with 0.1 M phosphatebuffer saline. The cells were treated with rabbit pAb to subunit LMP7 and mousemAb to subunit LMP2 overnight at 4°C in a sample containing 1 ×106 cells wereresuspended in 0.25 ml of phosphate buffer saline containing 10% fetal bovineserum and incubated for 30 min with phycoerythrin-conjugated anti-mouse mAb.For the identification of Kupffer cells, 1 × 10The cells were analyzed using the BD FACSCalibur flow cytometer  and the CellQuestPro software.Western blottingThe relative contents of proteasome subunits and β-actin were evaluated inclarified liver homogenates using mouse mAb to subunit LMP7, subunit LMP2, andβ-actin as described previously .Statistical analysisStatistical analysis was performed using the Excel and Statistica 7.0 softwarepackages. The data are given as a median; the difference significance betweenthe samples was estimated using a nonparametric Manne– Whitney test witha significance level of 0.05. A Bonferroni correction was used for multiplecomparisons.Content of the immunoproteasome subunits LMP2 and LMP7 in cells of therat liver after intraportal infusion of splenocytesth day after the beginning of DST inductioncompared to the false-operated controls. It isevident that the mechanisms that facilitate the DST effect were impaired in oneanimal. In the false-operated animals there were no differences in the contentof immune subunits at all studied stages after the administration of thephysiological solution. No differences were also revealed in the content ofimmune subunits in the animals of the 4th group with gadolinium chlorideinfusion. Hepatic mononuclear cells were studied using flow cytofluorimetry inorder to establish whether these changes in the immunoproteasome pool areassociated with hepatic mononuclear cells.A Western-blot analysis revealed an elevated level of proteasome subunits inthe clarified liver homogenates of five of the six rats with induction of DST(group 3) on the 7Fig. 2Ashows histograms derived during the analysis of therat hepatic mononuclear cells stained with antibodies to the immune subunitsLMP2 and LMP7. The quantity of cells expressing the subunits LMP2 and LMP7after the beginning of DST induction was found to change.It was established that as early as onthe 1st day, the quantity of LMP7-positive cells increased 1.8 timesin the rat livers of both groups with intraportal injection of splenocytes(groups 3 and 4) and the content of LMP2-postivie increased by 3 timescompared to the false-operated controls (group 2).Gadolinium chloride is a widely used specific inhibitor of the antigenpresenting the function of Kupffer cells. As showst day from the quantity of cells in the animals of the3rd group (without injection of GdCl3), but it was elevated comparedto the false-operated animals (group 2).We established that the quantity of cells containing the subunits LMP2 and LMP7in the animals treated with GdCl3 (group 4) did not differ significantly on the1rd and 4th groups: group 4should contain fewer of these cells than group 3. The absence of significantdifferences indicates that the increase in the quantity of cells expressingLMP2 and LMP7 on the 1st day was mostly due to a transfer of donorsplenocytes containing immune proteasomes into the liver. Macrophages, even ifthey contribute to the total number of mononuclear cells enriched inimmunoproteasomes in this period, do so in minimal fashion and the contributiondoes not influence the outcome.Taking into account the rich composition of hepatic APCs, which, in addition toKupffer cells and a liver sinusoidal endothelial cell (LSEC), includesdendritic and stellate cells , it islrd group decreased on the 3rd daycompared to the 1st day.This could be a result of donor splenocytes leaving the liver of a recipientand migrating to regional lymph nodes [+ T-lymphocytes[The quantity of mononuclear cells containing immunoproteasomes in the liver ofthe animals of the 3ph nodes .It is aphocytes.rd day not only compared to the 1st dayof DST induction, but also relative to their basal level in the control animalsof the 1st group. Taking into account the fact thatimmunoproteasomes are expressed mainly in APCs and immunocompetent cells, thisfact indirectly points to a decrease in their quantity in the liver on the3rd day after the beginning of induction. This can be associatedwith the apoptosis of activated T-lymphocytes observed in the liver during theinitiation and maintenance of the tolerance status [Interestingly, the quantity of mononuclear cells containing immune proteasomesdecreases on the 3e status .Howeverth day after the infusion of splenocytes, the maximum risein the content of mononuclear cells expressing immunoproteasomes was observedin the liver of animals of the 3rd group, which exceeded the valuesfor the control group almost 100 times for LMP2, and 200 times for LMP7.This excellent response could be rooted in both thetransfer of immunocompetent cells into the liver in response to the infusion ofdonor splenocytes and the activation of the resident APC pool in the liveritself, which is accompanied by an increase in the content of immune subunits[On the 7subunits, 34.In th day after DST induction. In addition, the discovery ofthis effect not in all the animals supports the hypothesis of the differentpossibilities of niche filling after the 3rd day that is criticalfor the development of tolerance or rejection.On the whole, the results of flow cytofluorimetry are consistent with Westernblot findings that indicate a burst in immunoproteasome expression in the liveron the 7th day exceeded the controlvalues only four times. The difference between groups 3 and 4 indirectlyindicates that the inhibition of Kupffer cells influences the processesdependent on the immunoproteasomes that occur in the early stages of DSTinduction.In animals treated with GdCl3, there was no similar noticeable increase in theamount of cells enriched in immunoproteasomes. The quantity of LMP7-positivecells did not differ from their quantity in false-operated animals, and thenumber of LMP2-positive cells on the 7Relationship between the content of Kupffer cells and the change in theexpression of immunoproteasomes during DST inductionOur results led to a need for a direct assessment of the Kupffer cell contentin different periods after splenocyte infusion. We used monoclonal antibodiesthat recognize ED2-like antigens on the membranes of rat resident macrophages,including Kupffer cells .Fig. 4),with the first peak occurring with a two-day shiftand the second peak with a three-day shift later compared to the peaks in thecontent of the total mononuclear cell pool expressing immunoproteasomes.This shift can be accounted for by the fact that, at first, APCs present a foreign alloantigenwith the participation of immunoproteasomes. This process is accompanied by a release ofmediators of the immune response, which serve as a signal for the proliferation of Kupffercells [The profile of the dynamics of ED2-positive cells also had two maximums was associated with the transfer of donor immune system cellsinto the liver; the second (7–10 days) – with the activation of aresponse in the liver of a recipient involving Kupffer cells.Does the expression profile of the inducible subunits LMP2 and LMP7 change inKupffer cells after intraportal alloantigen infusion?Fig. 5).First, two peaks of an increase in the quantity ofthe subunits LMP2 and LMP7 – on the 1st and 7thdays – were revealed. Second, it was revealed that the proportion of LMP2and LMP7 subunit expression in ED2-positive cells changes with time after DSTinduction. During the first 5 days, the quantity of LMP2 increased morenoticeably than that of LMP7; the level of both immune subunits was similarlyhigh on the 7th day.In order to answer this question, we studied the changes that occurred in theproteasome pool of ED2- positive cells in rat liver with DST induction (group3) in different periods after the administration of donor splenocytes.In the second phase, allthese events occurred in the same sequence: a peak in the expression ofimmunoproteasome subunits in ED2-positive cells was observed on the7th day, similar to that in the total mononuclear cell pool,followed by an increase in the quantity of ED2- positive cells on the10th day.The time changes in the content of ED2-positive cells and immunoproteasomeswere characterized by two phases. In the first phase, the quantity of LMP2 andLMP7 increased on the 1de novo LMP2 and LMP7 subunitsynthesis in mononuclear cells of a recipient’s liver, including inKupffer cells. This synthesis is induced in the first phase, resulting from theencounter of splenocytes with hepatic APCs. LSEC are known to cross-present aforeign antigen immediately to CD8+ T-lymphocytes[de novo immunoproteasome synthesis in the secondphase is also supported by the fact that the expression of immunoproteasome subunitsin response to cytokines reaches a peak only in 5–7 days[de novo synthesis.It is likely that differences exist in the mechanisms underlying the increasein the expression of immunoproteasome subunits in the total mononuclear cellpool in the first and second phases. The first peak of expression of LMP2 andLMP7 subunits reflects mostly the transfer of splenocytes enriched inimmunoproteasomes. At the same time, to a greater or a lesser extent the secondpeak can be associated with phocytes, and thephocytes and is aphocytes, 41thatphocytes, 42.The5–7 days, 44.TheThe proportion of proteasome immune subunits influences the activation ofmacrophages and their polarization into either an ED1- or ED2-phenotype. Therefoth and 6th daysafter adoptive transfer of CD8+ T-lymphocytes in the liver of arecipient [+ T-lymphocyte subpopulation on the 2md dayafter their intraportal infusion, followed by a gradual decline by the4th day, was also revealed[md day, followed by response extinction by the 6th day[th day and the dynamics ofthe rest subpopulations was biphasic, with peaks on the 1st and4th days.The established dynamics of immunoproteasome expression in hepatic mononuclearcells reflects the changes in the reactivity of their subpopulations inresponse to the introduction of foreign antigens. Previous data showed theappearance of peaks reflecting cell activation within the liver after peptideantigen administration or adoptive transfer of lymphocytes. For example, theproliferation of donor cells occurred on the 2.5ecipient . An eighrevealed.Stimula 6th day. It is iFig. 6).In the first phase, LSEC and Kupffer cells encounter donor cells, which, due tothe specific ability of the liver to retain activated CD8+ T-lymphocytes[Thus, a strong immunologic basis underlies the biphasic pattern of immunereactivity of the liver in response to intraportal infusion of a donor antigen(phocytes, remain phocytes, 51.Thi+ T-lymphocytes with Kupffer cellsleads to their apoptosis due to the absence of adequate proinflammatorystimulation [The interaction of activated CD8mulation . In addimulation , 53.A r+ T-lymphocytes via apoptosis, in theabsence of positive costimulation [The ability of immunocompetent hepatic cell subpopulations of primary antigenpresentation , which rmulation ,presentThe second phase is associated with the clonal expansion and transfer of theactivated T-lymphocytes and macrophages of a recipient into the liver. Thephenotypical profile of the cells that fill the immunological niche of theliver in this phase likely contributes to either tolerance or rejectionreactions. Activation of immune defense mechanisms in the liver directedtowards eliminating/alleviating inflammation can shift the balance in the sideof tolerance. These mechanisms include the apoptosis of the activatedT-lymphocytes of a recipient ,ED1-polThe repertoire of immune subunits influences the hierarchy of the presentedantigenic epitopes on APCs. At least four forms of immune proteasomes have beendescribed. One form contains all three proteolytic immune subunits: LMP7, LMP2,and LMP10. Two forms contain two immune subunits and one proteolyticconstitutive subunit: β5-LMP2-LMP10 and LMP7- LMP2-β2. One formcontains the immune subunit LMP7 and two constitutive subunits β1 andβ2 -60. The rd day after the beginning of DSTinduction for blocking rejection.This paper has studied for the first time the changes that occur in theimmunoproteasome pool of mononuclear cells within the liver during theinduction of allospecific portal tolerance. Based on the findings, it isconcluded that DST induction is an active process characterized by two phaseswherein the proportion of the immunoproteasome subunits LMP2 and LMP7 and thequantity of hepatic APCs, including Kupffer cells, change. Apparently, thebalance of these parameters is important for the development of tolerance totransplanted tissues. The third day after the beginning of DST induction is thekey point when a kind of “window of opportunity” opens for asubsequent filling of an empty niche with cells of different subpopulationsand, depending on this factor, the development of either tolerance orrejection. The results present new tasks related to the search for ways toinfluence the cellular composition of the liver and the expression ofimmunoproteasomes on the 3


Errors were introduced in the preparation of this article for publication. The last line of values in Table 2, group of EBV & H. pylori neg, is not complete. The complete Table 2 can be viewed here:


This paper presents the results of studies on the influence of oxygen partial pressure (vacuum level in the chamber) on the properties of FeAl intermetallics. One of the problems in the application of classical methods of prepared Fe-Al intermetallic is the occurrence of oxides. Applying a vacuum during sintering should reduce this effect. In order to analyze the effect of oxygen partial pressure on sample properties, five samples were processed (by a pressure-assisted induction sintering—PAIS method) under the following pressures: 3, 8, 30, 80, and 300 mbar . The chemical and phase composition, hardness, density, and microstructure observations indicate that applying a vacuum significantly impacts intermetallic samples. The compact sintered at pressure 3 mbar is characterized by the most homogeneous microstructure, the highest density, high hardness, and nearly homogeneous chemical composition. Alloys based on the Fe-Al intermetallic phases can be prepared by conventional methods, such as melting, casting, or powder metallurgy; however, the preparation of such materials by conventional methods leads to technological problems due to the large difference between the melting points of iron 1535 °C) and aluminum (660 °C), cross-reactivity, and strong affinity for oxygen 535 °C an,3. One oAnother problem in applying classical methods is the occurrence of oxides. Powders of iron and aluminum that are stored in air are covered with a layer of oxide . During the formation of FeAl, iron oxides are reduced according to the scheme :(1)3FeO2O3 precipitates in the finished sample. Simultaneously, on the surface of aluminum powder, there are reduction-resistant, continuous Al2O3 films. Their presence is an important barrier that hinders the interdiffusion of iron and aluminum, which significantly delays or prevents the complete reconstruction of the structure of orderly-sintered FeAl. To overcome this, Durejko and Bojar [2O3, located at the border of fine grains. Oxidation of the surface is a negative phenomenon due to the deterioration of the mechanical properties of the produced samples [Increasing the proportion of the Alnd Bojar ,8 used a samples . The sol samples ,11.Sintering in a vacuum facilitates removal of the adsorbed gases from the surface of the powder; a sufficiently high vapor pressure reduces the concentration of oxygen, and the thermodynamic conditions protect against oxidation and enable the thermal dissociation of some oxides, nitrides, and hydrides. Applying a vacuum during sintering has also important technological advantages: Vacuum, as an excellent thermal insulator, considerably reduces the heat necessary to heat the chamber, so the process is efficient and saves energy.A disadvantage of carrying out the sintering process under vacuum is the considerably higher cost of equipment, difficulties in process automation, and the long time needed to pump out a chamber, which increases with increasing vacuum level. The question is: What is the minimum vacuum level necessary to provide adequate purity of sintered FeAl? In addition, the influence of the level of vacuum to changes in the phase composition is unknown.ca. 60 μm. The iron powder particles were bigger and their average particle size was ca. 100 μm.The morphology and particle size distribution of the iron and aluminum powders is shown on The hardness of the samples  follows 3Al phase. As the pressure in the chamber was reduced, the samples homogenized. The peak Fe3Al phase—Angle 30.9° disappeared, as did the phase FeAl2—Angles 31.2 and 31.4, and the phase of Fe2Al5 (50.2°). However, most important is the reduction of the intensity of the iron peak (52.4°). If the pressure in the chamber was greater than 30 mbar, there was a significantly higher share of iron in the sample, suggesting that higher pressure inhibits the reaction between iron and aluminum, which results in a more non-equilibrium structure of the resulting sample. However, the mechanism of this phenomenon still requires further analysis. The compacts sintered at 3 and 8 mbar showed only minor proportions of iron.X-ray diffraction (XRD) experiments  showed t2 were confirmed in all the samples. Diffraction studies showed the presence of only the FeAl2 phase in the sample obtained at a pressure of 80 mbar. To confirm the presence of this phase, additional tests of phase composition into a micro-area, using electron backscatter diffraction (EBSD), were performed. EBSD studies showed its presence in all the samples. 2 phase is too small to detect by XRD.All obtained samples were re-studied to determine the oxygen content (EDS and XRF method) and the presence of trace impurities (XRF). Re-examinations of the trace contaminants were performed. i.e., increasing the homogeneity of the samples. The reasons for this influence of the pressure level on the homogeneity of samples are not clear. Below, there is proposed mechanism to explain this phenomenon, but further research is required to confirm it empirically. It is well-known that plasticization of aluminum takes place during the SHS reaction. Simultaneously, pressured vapor is locked in the pores and tries to get out towards the vacuum. The motion of the air molecules may force a more uniform distribution of the aluminum and thereby provide a better homogeneity of the sample after the reaction.Microstructure observation a confirmDuring analysis, the binary images showed that with increasing pressure, the dark areas are linked together to form long chains. This confirms the average aspect ratio shown in During the analysis of the microstructure in all samples, oxide particles with a characteristic bright border were observed. Typical examples of the oxide particles are shown in Starting materials were commercial powders of iron and aluminum, stored in the air. Iron powder was under the trade name NC 100.24  and aluminum powder was under the trade name AG 90/99.7 . The data regarding the powders are summarized in Sintering is carried out by a PAIS method. 3 [Density and porosity of samples were tested by the Archimedes method in water . A VickThe chemical composition was investigated by two methods. To determine the content of the oxides and other impurities in the powders and samples, XRF was used via a Rigaku Primus II and by EDS, using a Scanning Electron Microscope Quanta 3D FEG.Research on phase composition was carried out using XRD, using an X-ray diffractometer Ultima IV  using a cobalt lamp. Diffraction patterns were collected using Co radiation in parallel beam optics mode. Continuous scanning, 1 deg/min, was performed with sampling of each 0.02 deg. Linear detector was used . Before starting the analysis, all the obtained diffraction patterns were normalized. Normalization consisted of dividing all obtained measurement points by the highest intensity in the pattern. Studies of phase composition in a micro-area were performed using the EBSD method.From the analysis described above, the following conclusions can be drawn: Applying a vacuum has a significant impact on the microstructure and properties of the obtained sintered Fe40Al; sintering at a pressure of 3000 mbar reduces the oxygen content in the final material; and applying a vacuum enables the removal of trace impurities. Simultaneously, however, undesirable elements in the form of oxides and porosity formed elongated shaped regions that may reduce the strength properties; only carrying out sintering at lower pressures limits that unfavorable phenomenon.3 and Fe2Al5). It also limits the participation of the Fe3Al phase. The optimal pressure to consolidation was 3 mbar. The sample obtained at that pressure was characterized by the most homogeneous microstructure, the highest density, high hardness, and nearly homogeneous chemical composition. In order to remove insignificant quantities of Fe2Al phase and uniform distribution of iron in the phase Fe40Al, further annealing is necessary. Because iron is no longer present in samples obtained that way, the homogenizing process may be conducted in air. In addition, it has been shown that the SHS reaction between iron and aluminum may result in a reduction of primary iron oxides occurring in a green compact.Pressure reduction also causes substantial improvements of the microstructure and phase composition. Reducing pressure below 30 mbar eliminates unreacted iron and the unfavorable phases with high aluminum content (FeAl


The pathogenesis of pulmonary arterial hypertension remains undefined. Changes in the expression and effects mediated by a number of vasoactive factors have been implicated to play a role in the onset and progression of the disease. The source of many of these mediators, such as nitric oxide (NO), prostacyclin and endothelin-1 (ET-1), is the pulmonary endothelium. This article focus in the role of nitric oxide in PAH, reviewing the evidence for its involvement in regulation of pulmonary a vascular tone under physiological conditions, the mechanisms by which it can contribute to the pathological changes seen in PAH and strategies for the use of NO as a therapy for treatment of the disease. While the precise mechanism(s) that mediated the onset and progression of the disease remain undefined, several factors have been implicated in the pathology of PAH. These include endothelial dysfunction, oxidant stress, metabolic dysfunction, immune dysregulation and genetic factors disease .10,11. More recently, data has shown that depending on the presence of co-morbidities the survival 3 years after diagnosis is between 54.4% and 58.2%12. One year survival of PAH has been shown to be influenced by a range of prognostic indicators including renal insufficiency, PAH associated with connective tissue disease, functional class III heart failure, mean right atrial pressure, resting systolic blood pressure, heart rate, 6-minute walk distance, brain natriuretic peptide levels, percentage predicted carbon monoxide diffusion capacity and pericardial effusion on echocardiogram13. There is a predominance of the condition in women, which varies according to the aetiology of the disease14.PAH has an incidence of 15–50 people per million. Initially, median survival was calculated to be only 2.8 years15. Despite the wide range of causative factors, the lungs of patients with pulmonary hypertension exhibit a range of classical histological changes. These include remodelling of the pulmonary vessels, regions of neovascularisation, fibrotic changes in the vessel wall, thrombus formation and formation of plexiform lesions16. Plexiform lesions are composed of proliferating endothelial cells, matrix proteins and fibroblasts that obliterate the vascular lumen17. The reasons for their formation are poorly understood, however hypoxia, inflammation, shear stress, drugs, viral infections and genetic susceptibility have all been implicated18.The aetiology of PAH is varied, this is reflected in the World Health Organisation’s clinical classification of pulmonary hypertension 15. Despi1–5,19. At the cellular level dysfunction of the pulmonary endothelium seems to underpin many of the changes seen in PAH. Endothelial cells regulate vascular tone, vascular remodelling and inflammation via the release a range of vasoactive molecules that interact with blood elements and the underlying vascular smooth muscle. These mediators include nitric oxide (NO), prostacyclin and endothelin-1 (ET-1). The role of both ET-1 and prostacyclin has recently been reviewed in this journal2,3. The focus of the present article is on the role of NO in the onset and progression of PAH as well as the use of NO therapies for the alleviation of the clinical symptoms and improving the quality of life of patients with PAH.A number of factors and agents responsible for initiating and progressing the increases in pulmonary artery pressure have been suggested. Given the variety of different forms of the disease, it’s not surprising that so many different mediators and mechanisms are believed to be responsible , many of20.As with all other vascular beds, the production of NO by the pulmonary endothelium helps to regulate vascular tone. While a diverse range of endogenous chemical mediators have been identified to stimulate the release of NO from endothelial cells, the frictional force of the flow of blood over the surface of the endothelial cells (shear stress) is the principal physiological stimulus for NO release21. It was not until 7 years later that the chemical nature of EDRF as NO was identified and the metabolic pathway leading to its synthesis elucidated. Nitric oxide is formed by the enzymatic cleavage of the terminal amino group from the amino acid L-arginine22,23. The enzyme, termed nitric oxide synthase (NOS) was specific for the L isomer, but had no activity against the D isomer of arginine arginine . At apprG-monomethyl Arginine (L-NMMA), L-NG-Nitroarginine methyl ester (L-NAME) and NG-Methyl-L-arginine (L-NMA), allowed researchers to probe the role of NO in the vascular system, leading to the first demonstration that inhibition of NO increases blood pressure in animals and induces vasoconstriction in human blood vessels25–27. In addition, it was shown that the effects of NO are not confined to the vascular system alone. Besides the endothelial NOS two additional isoforms of the NOS enzyme, a neuronal isoform and an inducible isoform, have been identified. These three isoforms release NO for a variety of different biological functions28. All NOS enzymes bind calmodulin and contain haem and utilise L-arginine and molecular oxygen as substrates and requires the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-) 5,6,7,8-tetrahydrobiopterin (BH4). They differ in their distribution, dependence on calcium and the amount of NO that they produce , which leads to the activation myosin phosphate and a subsequent release of calcium from intracellular stores allowing smooth muscle cells to relax. cGKs may also activate transcription factors that may change gene expression in the cell and mediate other responses of the cell to NO or alter its response to other stimuli31,32. The bioavailability of NO is influenced by its inactivation by excessive amounts of oxygen-derived species such as superoxide (O2−) or changes in the protective effects of endogenous antioxidants.Most of the biological actions of NO depend on its ability to stimulate the enzyme soluble guanylate cyclase with the subsequent formation of cyclic 3′, 5′ guanosine monophosphate (cGMP)2− include NADPH oxidases, xanthine oxidase, cytochrome P450 enzymes and from complexes I and III of the electron transport chain in mitochondria. An additional source of O2− may come from uncoupling of NOS, which has been suggested to occur in several pathological conditions33–35. Unless inactivated by oxygen free radicals, the effects of NO are terminated by the breakdown cGMP by phosphodiesterase (PDE) enzymes. There are a number of different isoforms of PDEs that have specific distributions in different organs and vascular beds36.Sources of O37, however it is now considered more as an organ in its own right that regulates vascular homeostasis. In addition to NO, the endothelium also releases prostacyclin (PGI2) and ET-1, which also both have a direct effect on the vessel wall38–41. ET-1 is a powerful vasoconstrictor peptide that can be considered as a functional antagonist to the vasodilator effects of NO. ET-1 has also been implicated in the pathogenesis of PAH and inhibition of its effects is a pharmacological target for the treatment of the disease42,43. In contrast, PGI2 is a vasodilator and anti-aggregating substance. The effect of prostacyclin on platelet aggregation are enhanced by the presence of NO44. The effects of PGI2 are mediated via its action on cell surface receptors that in turn stimulate the accumulation of cAMP. Administration of PGI2, has been the most widely used, and to date most successful, therapeutic strategy for the treatment of patients with PAH. The labile nature of PGI2 has meant that such strategies have relied upon the development of stable analogues of PGI2 such as Iloprost, which unfortunately is not orally active and has to be inhaled or given as an I.V. infusion45. There have been some clinical trials that have combined PGI2 analogues or ET-1 receptor antagonists with compounds that promote the effect of NO43. The role of PGI2 in PAH, the therapeutic potential of PGI2 analogues and receptor agonists in the treatment of the disease and potential novel strategies to maximise drug delivery and effect have recently been reviewed in this Journal3.The endothelium was initially regarded simply as monolayer of cells that line the vasculature serving as a barrier between the blood and the vessel wall46. NO has also been shown to activate mitochondrial biogenesis via a cGMP-dependent mechanism47. In conditions associated with over-production of NO, such as septic shock, its been shown that low mitochondrial complex I activity is associated with raised nitrite/nitrate concentrations48. The irreversible nature of the inhibition of complex I suggest that it is due to the action of reactive nitrogen species, rather than an inhibitory effect of NO. The direct relevance to how NO regulation of mitochondrial function affects the pulmonary vasculature is currently been investigated. In the fetal lung, it has been shown that interactions between endothelial NOS and mitochondria reduce the levels of oxidative stress and facilitates vasodilation at birth49. The recent development of fluorescent molecular probes specific for the mitochondria and cytosol, that specifically and directly respond to NO have provided the opportunity to obtain a quantifiable and real-time readout of NO dynamics in different cellular compartments , which persist even under normoxic conditions58,59. HIF-1a can regulate mitochondrial fission, fusion and metabolism of pulmonary artery smooth muscle cells, which contribute to proliferation of smooth muscle cells and remodelling of the vessel wall60,61. The regulatory role of NO on mitochondria and the activation of these hypoxia-related pathways remain to be determined.Reductions in endothelial NOS have been shown to contribute to impaired mitochondrial biogenesis an ovine model of PAH56. ADMA is a methyl derivate of arginine, and is produced by the physiological degradation of methylated proteins proteins . Metabol72–74. However, the usefulness of inhaled NO as a treatment is limited due cost, technical difficulties and the fact that not all patients respond to the therapy. The ability of NO to oxidise haemoglobin to form methaemoglobin, which has been shown to be related the cumulative exposure to the gas, may also limit its effectiveness75. Rapid withdrawal of inhaled NO therapy can also have deleterious effects with levels of oxygenation and pulmonary hypertension returning to levels worse than those seen prior to the commencement of therapy76,77. However, developments in delivery technology have allowed optimisation of inhaled NO therapy by administering pulsed dosing of the gas 90. In addition to sildenafil, tadalafil and vardenafil, two newer PDE5 inhibitors, are currently being assessed for use in patients with PAH. Tadalafil, which is a once a day oral preparation has been shown to improve exercise capacity, pulmonary haemodynamics and time to clinical worsening, effects that last for up to 1 year in the PHIRST-1 trial 91. Similar results have been reported for vardenafil in the EVALUATION  trial, albeit in a much smaller cohort of patients92.The SUPER-1  trial showed that sildenafil improves the functional capacity and pulmonary haemodynamics in patients with PAH, with only mild adverse effects93,94. However, in larger clinical trials this effect of sildenafil was not seen95. It has recently been suggested that this may be due to the expression of PDE9 in myocytes. While PDE5 is found in contractile filaments, where is degrades cGMP produced by the action of NO on the soluble guanylyl cyclase (sGC) receptor, PDE9 is located near the T-tubular’ invaginations of the plasma membrane96. PDE9 in believed to be responsible for the degradation of cGMP produced by atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) hormones acting on the guanylyl cyclase type A (GC-A) receptor that is located the cardiomyocyte plasma membrane.PDE5 is also expressed in the heart and some small studies have suggested that sildenafil is capable of preventing the remodeling of the myocardium seen in heart failure97. Results of the PATENT-1  trial showed an improvement in exercise capacity, pulmonary haemodynamics, N-terminal pro-brain natriuretic peptide levels and time to clinical worsening. While adverse effects were similar to PDE5 inhibitors there was a greater incidence of systemic hypotension and haemoptysis.As an alternative to inhibition of the breakdown of cGMP, a new class of drugs are now being developed that directly activates soluble guanylate cyclase, thereby mimicking the effects of NO by increasing levels of cGMP. One such drug is riociguat, which has been assessed for efficacy in the treatment of PAH43. The results of some of the trials of combination therapy do show some promise, however some results are contradictory. For example the COMPASS-2  trial was able to show an improvement in the 6-minute walk test after 16 weeks of treatment but failed to delay the time to the first morbidity/mortality event98. However, meta-analysis of data from studies that involve combination therapy do indicate that this approach is effective in the treatment of PAH9.The mainstay of treatment for PAH is still the use of prostacyclin mimetic compounds and more recently endothelin-receptor antagonists and PDE-5 inhibitors. There are potential advantages in using combinations of these drugs to enhance the clinical benefits that can be obtained when the drugs are used in isolation. The use of strategies that combine treatment with PDE-5 inhibitors with PGI2 analogues and/or ET receptor antagonists have recently been comprehensively reviewed99,100. Of particular interest are fibres that are capable of releasing NO and that are suitable for delivery via inhalation to deliver NO directly to the pulmonary vasculature101,102. These nanofibres have recently been shown to be able to continuously release NO over an 8-hour period and relax pulmonary vessel of rats with pulmonary hypertension in vitro103. These initial observations suggest that nanofibres capable of releasing NO may be of use in the future treatment of patients with PAH.The current pharmacological strategies to augment the supply or duration of the action of NO have a number of limitations mainly relayed to the routes of administration, duration of action and systemic effects. Novel nanoparticles are being developed for the treatment of a number of different conditions 99,100. ONO plays a significant role in the pulmonary circulation. Changes to its synthesis, release or signalling contribute to the pathogenesis of PAH. While targeting NO in has resulted in advances the treatment of the disease, the quality of life and the prognosis for patients with PAH remains poor. There remains a substantial amount of work to be done in order to optimise strategies that target to NO system or utilise the delivery of NO as a pharmacological agent.


Under the high idle condition, gasoline engine emission decreased by 8.13%, 11.35% and 44.91% for CO, HC and NOx, respectively. When tested in the low idle condition, engine emission reduced by 0.43%, 1.14% and 85.67% for CO, HC and NOx, respectively. The diesel engine emissions of CO, NOx and total amount of HC and NOx decreased by 32.26%, 3.28% and 4.66%, respectively. The results illustrate the composite installation exhibits satisfactory emission reduction effect.In this work, a new organic-inorganic composite filter was prepared. The thickness, pore size, air permeability, bursting strength and microstructure were characterized systematically, proving that coatings had regulatory effect on filters physical properties. Benefitting from the distinct coatings containing 5% sepiolite nanofibers after five times dilution, the physical properties of corresponding air filter exhibits the most favorable performance and meet the standard of air filter. When used as fuel filter, it satisfies the fuel filter standard and achieves the best performance after six times dilution. The contrast test on engine emission was taken based on auto filters coated with/without as prepared nanofibers. An obvious decrease in the emission of carbon monoxide (CO), hydrocarbons (HC) and nitrogen oxide (NO Resources and environment have been global ticklers that beset the sustainable development of human society. As the demands progress, the mobile equipments are impelled to the light and high efficiency. In recent years, with atmosphere pollution being more serious, e.g. smog, in large and medium sized cities of China, there are higher requirements on reduction of automobile emission. Especially in aspect of air pollution, according to the standard of EURO V, the particulate emissions of diesel vehicles are expected to decline 80% in comparison with the present. On the other side, the nitrogen oxides emissions of diesel engine shall decrease 68%, according to the standard of EURO VI. The development of high efficiency emission reduction technology has been explored worldwide in response to the challenge of environment pollution1It is known that the harmful substances in the automobile exhaust mainly consist of hydrocarbons, carbon monoxide, nitrogen oxides, sulfur compounds, smoke, etc. Among them, carbon monoxide, hydrocarbons and nitrogen oxides are the main air pollutants3456791011121314151612O30Mg8(OH)4(H2O)4·8H2O and fine microporous channels of dimensions of 0.37 nm × 1.06 nm running parallel to the direction of fibers18192022232425262729Sepiolite is a type of fibrous silicate clay mineral rich of magnesium with a unit cell formula of SiIn our previous research, we have prepared sepiolite nanofibers using high speed airflow technique31The wood pulp was purchased from Henan Huining Trade Co., Ltd. The synthetic fiber was supplied by Zibo Ruibao Materials Co., Ltd. The sepiolite as one of natural mineral materials was purchased from Henan province of China, and the high performance sepiolite mineral nanofibers (SMN) preparation has been studied previously31The preparation process of composite coating material was shown in An Olympus STM6 metallographic microscope and a Hitachi S-4800 scanning electron microscope operated at 25.0 kV and 30 μA were used to study sample morphological features. After cutting the composite filter material into the size of 100*100 mm, quantitative tests of filter paper were carried out employing a precision electronic balance  to weigh the samples. In our study, paper thickness tester was performed via the measurements of the samples according to the China Standard Test Methods . At least 5 samples with the dimensions of 100*100 mm were cut from each test piece, placed between the measuring head and the anvil and given with certain hammer pressure. The samples thickness was delivered to the gear mechanism of watch via the displacement of the measuring rod, where it let the watch hands rotate an angle to obtain the thickness. The thickness was measured at least 3 times at each position and the average value was recorded as the result. Air permeability, a common detection index of air filtration materials, referred to the gas volume through unit area of materials under unit time and pressure, and reflected the gas permeation ability of filters. Air permeability test was carried out according to the China Standard Test Methods . There was no direct conversion relationship between the pore size and filtration precision of filters. However, the filtration precision was reflected indirectly via the aperture test which referred to the principle of capillary flow according to the China Standard Test Methods (QC/T794-2007). Bursting strength was the maximum pressure which was endured evenly increasing in the unit area and denoted by liquid pressure or gas pressure. The tests were performed using electronic paper bursting tester (ZDNP-1) according to the China Standard Test Methods (QB/T Bursting Strength of Paper and Paperboard). According to measurement method of fuel saving technology for automobiles (GB/T 14951-2007), the pollutant emissions of engines were detected. On the other side, the effects on the emission reduction and environmental protection of air filter and diesel filter were also tested, according to the emissions of pollutants from light-duty vehicles limit values and measurement methods in China III, IV stage (GB/T 18352.3-2005).The contrast observation was carried out with the resultant samples, the air composite filter material with 5 times dilution of the coating material and the fuel oil filter material with 6 times dilution of the coating material. In this study, the air filter paper and the fuel oil filter paper were chosen as the substrate, and the microscopic observation was conducted on the outflow surface of raw material samples. The distribution changes of fibers and pore sizes in two types of composite filters were presented in As observed in The as-prepared coating material, possessing the properties of high viscosity and concentration, was not suitable for the direct preparation of the coating. Therefore, the coating material needed to be diluted with a certain amount of water in order to ensure the performance of composite filters. In our study, the as-prepared coating material was denoted as the standard solution with 100% concentration and diluted according to solution/water ratio of 1:1,1:2,1:3,1:4,1:5 and 1:6, corresponding to dilution times of 2, 3, 4, 5, 6, and 7, respectively. The diluted coating materials were coated and dried on the substrate, and tested on the physical performance.The effect of coating materials dilution times on the physical performance of air composite filters is shown in In this method, the material construction mainly depends on the bonding strength between layers, ensuring the tight binding between layers without adopting any inter layer pressure, which yields identical material density. Therefore, the amount of the composite coating after drying can be directly denoted as the change of composite materials thickness. As presented in As is clearly observed in The effect of dilution times of coating material on bursting strength is shown in 2·s) and 313KPa, respectively.Together the effect of different dilution times and coating amount on physical properties of composite materials above, we can see that the physical properties of the coating material could be adjusted through adding a certain amount of composite materials in a certain range. According to the air filter production requirements, each air filter index must be in line with the relevant standards, as shown in 2·s). However, when the dilution times continue increasing, the curve of air permeability variation levels out. As shown in The effect of the coatings dilution times on the physical performance of fuel composite filters is shown in The change trend of different dilution times and coating material amount effect on the physical properties of oil composite filter states that the physical properties of composite filter can be adjusted within a certain range via adding a certain amount of coating material. According to fuel oil filter production requirements, each physical properties index of fuel oil filter must meet the related standard of fuel oil filter as shown in The effect of the addition amount of SMN on the physical performance of air composite filters is shown in 2·s). The figures above present when the addition amount is more than 10%, the air permeability of the filter material decreases below to 223.3 L/(m2·s), which does not meet the standard. As discussed above, the SMN addition amount can adjust the composite filter pore size and increase bursting strength within a certain range, but the air permeability decreases with increasing the SMN addition amount. According to the air filter production requirements, the air filter indexes must comply with the relevant standards, as shown in The effect of the addition amount of SMN on the physical performance of fuel composite filters is shown in As shown in 2·s) and 27 L/(m2·s) respectively. This is ascribed to that the dense fibers of fuel oil substrate show smaller intake capacity to sepiolite. When the addition amount reaches to a certain amount, the SMN forms the blockage to the filter material pores. Fuel filter standard states that the air permeability is not less than 80 L/(m2·s), as shown in From As the analysis above, it is shown that the trends on performance indexes of the composite fuel oil filter material with the addition amount variation of SMN and composite air filter material are alike, which validates the analysis of the SMN dosage effect on the composite filter material and states that the effect of SMN dosage on composite filter material is consistent with the fact. According to the production requirements, performance indexes of fuel oil filter material have to meet the criteria as shown in For the sake of the detection on the effect of organic-inorganic composite filter material on the emission of gasoline vehicles, organic-inorganic composite filters are prepared to a certain type of air filter and fuel filter, which are record as experiment group of air filter and experimental group of gasoline filter. The untreated substrate filter paper is selected to fabricate the control group of air filter and gasoline filter with the corresponding type. According to GB/T 14951-2007, the pollutant emissions of engines are detected, and the contrast test results on the pollutant emission of gasoline vehicles are shown in x are 8.13%, 11.35% and 44.91%, respectively. Under the low idle condition, the reduction rate of CO, HC and NOx is 0.43%, 1.14% and 85.67%, respectively.The design on the physical and chemical properties improvement of vehicle fuel and activating fuel is integrated into the composite filter material. According to the combustion principle of internal combustion engine, it has been stated that the surface tension of the fuel oil decreases and the evaporation property improves, which is benefits to the complete atomization of the fuel oil and promote the oxidation reaction of the fuel oil molecule and oxygen, so as to achieve the goal of emission reduction of HC, CO and NOx in tail gas. Far infrared functional material has the function of releasing far infrared ray. After the far infrared ray is absorbed by fuel oil, the fuel surface tension reduces and the atomization improves, so that it can promote the fuel complete combustion.2 and NOx in combustion products from the combustion chamber are measured, and the combustion efficiency is determined. The expression of combustion efficiency is:A method for judging the combustion efficiency is by means of the combustion product definition. This method depending on the instrument measurement, which is not related to the fuel application, has been worldwide adopted. CO and HC, the form of unburned carbon element, which content can directly reflect the complete extent of the fuel combustion, is an important symbol of the combustion efficiency. By means of gas analysis method, the contents of HC, CO, COIn formula: EI is the mass of the pollutants produced by each kilogram of fuel, g/kg. LHV represents low calorific value of combustion, MJ/Kg.The unburned hydrocarbons (HC) in tail gas emissions are the important symbol to reflect the complete extent of the fuel combustion. As it can be seen from the test data, the content of HC in the engine combustion products of experimental group is less than that in the control group. It shows that the fuel combustion complete extent in the experimental group is higher than that in the contrast group, which explains the combustion efficiency is higher. The CO in the tail gas is a form of unburned carbon element in fuel. The content of CO reflects the complete degree of the fuel combustion and affects the combustion efficiency. The reduction amounts of CO and HC are put into the formula(1), so the combustion efficiency of the experimental group is expressed as:It can be seen from that formula, when the HC and CO harmful gases reduce, the combustion efficiency of the experimental group is higher than that of the control group. The gasoline combustion efficiency increases, the harmful gas emission reduces, and the effect of emission reduction is achieved. It shows that the structure and properties of composite materials match the models.In order to detect the effect of organic-inorganic composite filter material on the pollutant emission of diesel vehicles, organic-inorganic composite filters are prepared to a certain type of air filter and fuel filter, which are record as experiment group of air filter and experimental group of gasoline filter. The untreated substrate filter paper is selected to fabricate the control group of air filter and gasoline filter with the corresponding type. According to GB/T 18352.3-2005, the effects on the emission reduction and environmental protection of air filter and diesel filter are detected, and the contrast test results on the pollutant emission of gasoline vehicles are shown in x in experimental group decreases in comparison with that of the control group. The reduction rate of CO, HC, the sum of HC and NOx is 32.26%, 3.28% and 4.66% respectively, which achieves the effect of emission reduction. CO and HC are two kinds of unburned carbon in the fuel, which content directly reflects the degree of fuel combustion. The values of CO and HC are put into the formula (1), so the combustion efficiency is expressed as:As shown in It can be seen from the formula above that the combustion efficiency is improved obviously, when the emission amounts of CO and HC are decreased. The main reason could be that when the diesel fuel flows through with far infrared radiation ceramics filter, physicochemical properties of diesel are improved and the extent of diesel atomization is enhanced. Accordingly, the diesel is burned completely in the engine, and the emission could be reduced effectively. The experiment results show that the structure design of composite materials is reasonable and exhibits good performance.2·s), and the bursting strength is 305kPa. When the coating material was diluted by 6 times and the SMN addition amount maintained 5%, the physical performance of fuel filter material achieves the best. Meanwhile, the filter material thickness is 0.52 μm, the maximum pore size is 45.38 μm, the average pore size is 416 μm, the air permeability is 128.37 L/(m2·s) and the bursting strength is 416kPa, satisfying all the standard of fuel filter material. In the tests of emission reduction performance on gasoline and diesel vehicles, obvious decrease in the emission amount of CO, HC and NOx has been detected after installation of the composite filter. In particular, the gasoline engine emission of CO, HC and NOx decrease by 8.13%, 11.35% and 44.91% respectively under the high idle condition. Under the low idle condition, the emission of CO, HC and NOx reduce by 0.43%, 1.14% and 85.67%, respectively. On the other hand, the diesel engine emissions of CO, HC and the total amount of HC and NOx decrease by 32.26%, 3.28% and 4.66% respectively. The results demonstrate that the good effect of emission reduction can be achieved by utilizing auto filters fabricated from our nanofibers with composite coating.In our study, a novel type of multi-functional organic-inorganic composite filter material for emission reduction was prepared, and the structure and performance were also studied systematically. The results show that coating amount exhibit positive influence on physical performance of composite filter material. When the coating material was diluted to 5 times and the SMN addition amount was 5%, the composite air filter achieves the best physical performance. Meanwhile, all the indexes of as prepared composite meet the standard of air filter material with the material thickness of 0.554 μm, the maximum pore size of 52.14 μm, the average pore size of 47.63 μm, the air permeability of 270.8 L/.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Additionally, the properties of the atoms inside the dipole potential trap, such as the distribution profile and lifetime, are deduced from the fluorescence images.The primary purpose of this study is to investigate the influence of the vertical guiding laser beam waist on cold atom guiding efficiency. In this study, a double magneto-optical trap (MOT) apparatus is used. With an unbalanced force in the horizontal direction, a cold atomic beam is generated by the first MOT. The cold atoms enter the second chamber and are then re-trapped and cooled by the second MOT. By releasing a second atom cloud, the process of transferring the cold atoms from MOT to the dipole trap, which is formed by a red-detuned converged 1064-nm laser, is experimentally demonstrated. And after releasing for 20 ms, the atom cloud is guided to a distance of approximately 3 mm. As indicated by the results, the guiding efficiency depends strongly on the laser beam waist; the efficiency reaches a maximum when the waist radius ( Over the past several years, there has been a growing interest in the transferring and guiding of atoms by the dipole potential trap. The development of efficient atom guiding and manipulating systems is of great importance for applications related to the transportation of cold atoms from the atom source to experimental regions, such as atomic interferometers ,2, atom The concept of guiding cooled atoms by a red-detuned laser beam was theoretically proposed in 1978 . The fir87Rb was captured in a red-detuned Gaussian beam in this study. By varying the magnification of the beam expander, the effects of the laser beam waist on the atomic guiding efficiency were investigated. Additionally, the analysis of some other characteristics, such as atom cloud temperature, guiding trap lifetime, and effect of laser power, are presented in this report.The two studies mentioned above mainly concentrated on the theoretical analysis and calculation of the influence of the guiding laser beam waist. However, an experimental demonstration, to the best of our knowledge, has not been performed since then. To experimentally verify the influence of the laser beam waist on the guiding efficiency, a cold atom cloud of 87Rb using a red-detuned Gaussian beam, as illustrated in 2 transitions (52S1/2→52P3/2) of 87Rb, where Γ = 2π × 6.07 MHz is the natural linewidth of the D2 line. The 87Rb atoms are trapped and cooled in the left chamber (source chamber) by utilizing the standard three-dimensional magneto-optical trap (3D-MOT) technique  [s is the saturation intensity [2 transitions of 87Rb, is coupled to one of the trapping lasers; thus, the fluorescence image of trapped atoms can be captured by a charge-coupled device (CCD) camera.A second 3D-MOT technique is performed in the right chamber with a similar mechanism to the first one but with the three pairs of retro-reflected trapping lasers inclined by 45° with respect to the vertical direction. After the second MOT is loaded from the atom beam, a vertical 1064-nm Gaussian guiding laser, which is red-detuned with respect to the D-line dublett transitions of rubidium , is usedntensity , and I of about 133 μm and contains about 3.7 × 10w0) of the guiding light was fixed at a constant value (40 μm), and the laser power used in this experiment was varied from 2 to 15 W, corresponding to the potential energy U0 at the center of the beam from 1.7 to 12.9 mK based on the theory in the previous section, to investigate the lifetime of the guided atoms within the potential trap. As illustrated in First, the beam waist diameter (2w0) on the guiding efficiency of the atoms. According to the experimental results mentioned above, the number of atoms in the dipole trap decreases with time. However, most atoms still remain in the trap 20 ms after MOT2 is switched off, so we compared the guiding efficiencies of the atoms in this case for different laser beam waists. The guiding efficiency here is defined as the relative value of the number (Ncap) of guided atoms compared to the number (N) of all atoms trapped in MOT2 (Ncap/N), and the guiding distance at 20 ms is around 3 mm. The fluorescence image of experimental guided atoms and the background-released atoms is shown in Secondly, we set the laser power to a constant value (8.9 W and 14.5 W) and investigated the influence of the laser beam waist (w0) is around 15 μm and 20 μm, for the case of two guiding laser powers. However, as the beam waist increases further, the guiding efficiency slowly decreases. This experimental result can be approximately explained by the two counteracting trends: as the beam waist rises, the atomic ensemble has a larger interacting region with the guiding laser, so the number of guided atoms initially increases; however, the density of light intensity at the focusing point decreases at the condition of constant laser power, which causes the potential trap to decrease in depth. When the latter dominates, the guiding efficiency decreases.As illustrated in w0) is maintained at around 15 μm and 20 μm, respectively, to which the corresponding U0/kB is about 2.2 mK and 2 mK, for the two different laser powers employed in our experiment. This result is also valid according to the finding of References [0/kBT. Here, T = 16.65 μK is the temperature of the atom cloud mentioned before. The variation trend in the guiding efficiency with the beam waist is approximately in good agreement with the theoretical calculations in References ,13, and ferences ,13, thatw0/2σ ≈ 0.07, U0BT, the factor 2 results from the different definition of the waist of the guiding laser and the size of the atom cloud, see Equations (2) and (3) from Reference [w0/2σ ≈ 1 to obtain a guiding efficiency of about 75%, where U0/kBT = 4).However, there still exists a notable disagreement: the ratio of the optimal beam waist to the atom cloud radius in the experiment is much smaller (eference ) than thw0 varies from 10 to 50 μm, as shown by the x-axis of There are a few possible explanations for this. It should be noted that the calculation models from both references are based on the hypothesis that the initial position and velocity distribution are described by independent Gaussian functions. However, in our experiment, this hypothetical condition may be invalid because the guiding laser was constantly switched on from the beginning of the experiment. Compared to the initial atom cloud with a radius (σ) of 133 μm, the beam waist of the guiding laser in the experiment was so small (In this study, using a red-detuned Gaussian laser, we experimentally demonstrated the existence of an optimal laser beam waist for the guiding efficiency of cold atoms. Our experimental results demonstrate that, in the case that cold atoms are released from the MOT and then expanding ballistically in the red-detuned guiding potential trap, the guiding efficiency strongly depends on the beam waist of the laser. We successfully guided atoms for a distance of about 3 mm. The analysis of the experimental data shows that the guiding efficiency first increases with the beam waist radius and then peaks at around 20 μm, which is about 13 and 22% when the input laser powers are 8 and 14.85 W, respectively. However, as a consequence of the increasing guiding laser size, the laser intensity decreases and results in the weakening of the guiding effect. The comparison of our experimental results to the theoretical research by previous researchers is also qualitatively discussed, from which it can be confirmed that an optimal laser beam waist was realized in our research.In contrast, we should also be aware of the disagreement between the calculation and our experimental results regarding the issue of the specific value of the optimal laser beam waist or the relationship between it and the atom cloud size. Therefore, further discussions are crucial. In follow-up investigations of the experimental imperfections, issues such as the reflection caused by the angle of incidence of the cooling laser should be addressed. Moreover, an accurate and comprehensive simulation model, in which the actual distribution of cold atoms when the guiding laser exists is taken into account, must be established. In addition, the influence of the laser beam waist on the guiding efficiency must be well interpreted. Finally, we would like to refer to the significance of our study, since in some applications the light field size is in the micron-scale, which is small compared to the dimensions of a cold atom cloud, such as that guiding cold atoms through hollow core fiber. Therefore, the study of the optimal mode field diameter is of great importance for optimizing the transferring efficiency.


This study aimed to assess the effect of 25-hydroxyvitamin D3 (25OHD) which is a hydroxide of vitamin D3 ingestion on upper respiratory tract infection (URTI).A prospective, randomized, double-blind, placebo-controlled study was performed from December 2015 to September 2016 in the Nihonbashi Egawa Clinic, Kei Medical Office TOC Building Medical Clinic, and Medical Corporation Kaiseikai Kita-Shinyokohama Medical Clinic, in Japan.Four hundred twenty eight participants aged 45-74 years were screened by their serum 25-hydoroxyvitamin D concentration.The participants were randomized to either 25OHD (10 μg/day) or placebo capsule, daily, for 16 consecutive weeks.The primary outcome measure was the incidence proportion of URTI, and the secondary outcome measures were the physical severity score, the quality-of-life (QOL) score, the duration of URTI, and the incidence proportion of new URTI events every four weeks. Data were collected using cold diary Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) during the intervention.Of 428 participants screened, 252 with serum 25-hydroxyvitamn D levels were deficient or insufficient (75 nmol/L or less) were enrolled in this study. Of these, 105 placebo and 110 25OHD group subjects completed the study. For the incidence proportion of URTI, no effect of 25OHD intake was observed. On the other hand, the duration of URTI was shorter in the 25OHD (P = 0.061) compared to placebo. For the incidence proportion of URTI every four weeks, the incidence of new URTI was decreased in both groups over the time of intake. However, when the 25OHD and the placebo were compared, a decrease in the incidence proportion of URTI was seen earlier in the 25OHD. When the total physical severity score and the total QOL score during the study were assessed, they both were significantly improved in the 25OHD compared to placebo.The intake of 25OHD may reduce the duration of URTI, the physical severity, and the QOL when suffering from URTI. In general, the common cold is a group of diseases that present with various upper respiratory tract symptoms, including runny nose, nasal congestion, sneezing, sore throat, and coughing, and is a group of diseases caused by many different types of viruses and bacteria. Normally, the common cold remits spontaneously without treatment . The aveDue to the fact that causes of the common cold are associated with many different types of viruses and bacteria, vaccination is not practical as a preventive measure. For this reason, symptomatic treatment is the main measure for colds.Recent studies have reported that vitamin D affects various components of the immune system. For example, antimicrobial peptide LL-37, which is considered to be important for innate immunity, is a peptide produced by cleavage of human cathelicidin by proteinase 3, and has high antibacterial activity and antiviral activity . It has There have been several studies on vitamin D in the blood and respiratory tract infection, and the relationship between the serum vitamin D level and the incidence of respiratory tract infection has been reported , 11. FroVitamin D in humans is sourced from the synthesis in the skin through exposure to UV light and from intake of food containing vitamin D2 or vitamin D3 such as fish, dairy products, and mushrooms. Synthesized or ingested vitamin D is mainly hydroxylated in the liver at position 25 by CYP2R1 or CYP27A1 that belongs to the cytochrome P450 superfamily, and converted to 25-hydroxyvitamin D .25-hydroxyvitamin D binds to vitamin D-binding protein, and circulates in the bloodstream. Subsequently, 25-hydroxyvitamin D transported to the kidneys is hydroxylated in a cell at the 1α position by CYP27B1 to form 1α, 25 (OH)2 vitamin D , 18. 1α,Although studies and applications of vitamin D and 1α, 25 (OH)2 vitamin D3 derivatives have been accomplished, the application of 25-hydroxyvitamin D3 (25OHD) has not been advanced. Regarding the relationship between serum 25-hydroxyvitamin D level and lower limb muscle strength, Bischoff-Ferrari et al. have reported that 25OHD taken by postmenopausal Caucasian women has a reinforcement effect on lower limb muscle strength , 24. In Hence, in this study, the effect of 25OHD intake on upper respiratory tract infection (URTI) was assessed using placebo as a control.Study implementation: This study was conducted after it was reviewed and approved  by the HUMA R&D Ethical Review Committee consisting of third parties who have no involvement in the study. In accordance with the spirit of the Declaration of Helsinki , the study was implemented within the Ethical Guidelines for Medical and Health Research involving Human Subjects . (UMIN ID: UMIN000020169)The purpose and content of the study were fully explained by the principal investigator to the eligible subjects, and the study was performed only on those who gave consent in writing on their own free will.For this study, the assessment of physical findings, physical measurements, physical examinations, and clinical examinations were conducted at Nihonbashi Egawa Clinic, Kei Medical Office TOC Building Medical Clinic, and Medical Corporation Kaiseikai Kita-Shinyokohama Medical Clinic, where the study subjects were managed and the study implementation system was maintained. The principal investigator controlled the operations related to the study; instructions and explanations to the study subjects, acquisition of written informed consent from the subjects, medical interviews, confirmation and assessment of adverse events, and maintenance of the study implementation system were all performed by the principal investigator. We also planned to give treatment to adverse events as needed. This study was implemented between December 2015 and September 2016.Background surveys including medical history, drinking habit, and eating habit, physical condition checkup, measurements, and clinical examinations were performed as a screening test on the 428 subject candidates who gave written consent, and 252 cases, who met the selection requirements and did not meet the exclusion criteria, were selected as the study subjects.2, and who did not meet the following exclusion criteria, were selected. I. Current smoker, II. Subject with severe disease , III. Subject with disease that would affect the study results , IV. Subject who has a plan to be exposed excessively to the sun  during the study, V. Subject whose fasting glucose level is 110 mg/dL or more, VI. Subject who received hormone replacement therapy for the past six months, VII. Subject who would take other supplements than multivitamins during the study; this excludes person who will stop taking them before the study and will not take any until the study ends, VIII. Subject who took vitamin D supplements for the past three months, IX. Subject who took 600 mg/day or more of calcium supplement for the past three months, X. Subject with hypertension: the systolic blood pressure at rest is 145 mmHg or higher, or the diastolic blood pressure is 95 mmHg or higher, XI. Subject being treated for hypertension, XII. Subject with history of mental illness, person in a state where it is difficult to understand the study content, XIII. Subject who does a highintensity exercise continuously (who does a high-intensity exercise more than three times a week), XIV. Subject in a state that would affect the absorption of the trial supplement: impairment of intestinal absorption, sprue , colitis, gastroenterological surgery, M. Whipple disease , XV. Subject with disease that carries a risk of hypercalcemia: multiple organ granulomatous disease (sarcoidosis), tuberculosis, lymphoma, primary hyperparathyroidism, XVI. Subject with kidney stones, XVII. Subject whose creatinine clearance is 30 mL/min or less , XVIII. Subject who is sensitive or allergic to dairy products, XIX. Subject using drugs: anticoagulants, steroids, parathyroid hormone, thiazide diuretics, anticonvulsants, antipsychotics, drugs for schizophrenia, drugs that affect fat absorption , XX. Subject who used drugs that could affect bone metabolism  for the past one year, XXI. Subject who consumes a non-fat diet or has other extreme dietary habits, XXII. Subject who is on a weightloss program or whose diet is under the supervision of a doctor, XXIII. Subject with symptoms of acute or severe diseases: unintended weight loss, night sweats, and cancer treatment, XXIV. Subject whose alcohol consumption is 140 g/week or more, and XXV. Subject who was judged to be unsuitable for the study by the principal investigator or other investigators.For the selection of study subjects, those who had the capacity to consent to the study, who met the following: I. Healthy Japanese between 45 and 74 years of age at the time of consent, II. Subject whose serum 25-hydroxyvitamin D level is 30 ng/mL (= 75nmol/L) or less, and III. Subject whose BMI is between 18.5 kg/m2 and 24.9 kg/mFor the trial supplement, a hard capsule formulation containing 10 μg of 25-hydroxivitamin D3 (25OHD) was used. Placebo was prepared by replacing 25OHD with crystalline cellulose so that it would not be distinguished from the trial supplement by the color. 25OHD was provided for free by DSM Nutritional Products, Ltd. .For the assessments of the incidence of URTI, the physical severity, and the quality-of-life (QOL), the Japanese version of Wisconsin Upper Respiratory Symptom Survey-21 (WURSS- 21) was used . WURSS-2The next 10 questions (the second to the 11th) are questions about 10 physical symptoms of URTI , and they were answered on a 0-7 Likert scale  to obtain the total score as the physical severity score. The next nine questions (the 12th to the 20th) are questions about nine events associated with QOL , and they were answered on a 0-7 Likert scale  to obtain the total score as the QOL score which is reflected mental severity.The date of occurrence of URTI was the day on which the subject selected 1-7 after selecting 0 two days in a row for the first question, and the date of disappearance was the day, which was two days prior to the time when the subject selected 0 two days in a row after the onset (the day 1-7 was selected last). The events of URTI in this study were the ones whose duration (the period from the date of occurrence to the date of disappearance) was two days or more, and whose highest physical severity score when suffering from URTI was over 10. For the duration of URTI, the physical severity score, and the QOL score, the highest values during the study were evaluated.The study was a randomized, double-blind, placebocontrolled, parallel group comparison study. Hence, a staff who has no involvement in the study prepared an assignment list using random numbers, and after the study subjects were randomly divided into the 25OHD group and the placebo group, 25OHD and placebo were given to the two groups respectively. The subjects for blinding were all those who were involved in the study, and the assignment was unblinded after securing analysis subjectsDuring the study, the subjects were required to fill in the WURSS-21 and to record subjective symptoms and whether they made a deviation from instruction such as intake of medical products and health food in their journals. The duration of the supplement intake of this study was from winter (January 2016) to spring (May 2016).A body measurement, a blood pressure measurement, a pulse measurement, clinical examinations, and the food frequency questionnaire ) were performed at baseline and 16-weeks tests . For theClinical laboratory tests were conducted using a γ-counter, ARC 950 , Modular Analytics , and clinical chemistry analyzer, BioMajesty ™ series JCA-BM 8060 . For 25-hydroxyvitamin D, the 25-hydroxy-vitamin D 125I RIA Kit  based on the RIA 2 antibody method was used. The CV% of the 25-hydroxyvitamin D measurement during the test period was in the range of 8.8% to 12.1%.Primary outcome measure: The primary outcome measure was the incidence proportion of URTI for the period of 16 weeks of supplement intake. The incidence proportion of URTI was calculated by dividing the number of study subjects who developed URTI in each group by the number of study subjects in each group.Secondary outcome measures: The secondary outcome measures were the physical severity score, the QOL score, the duration of URTI, and the incidence of new URTI every four weeks. For the physical severity score, the QOL, and the duration of URTI, the study subjects who developed URTI were assessed.The guideline by the task team of Michael F. Holick et al. says that the risk of osteomalacia and rickets is increased in a deficiency state where the serum 25-hydroxyvitamin D level is 50 nmol/L (= 20 ng/mL) or lower, and that the state where the serum 25-hydroxyvitamin D level is between 50 nmol/mL and 75 nmol/L (= 30 ng/mL) is an insufficiency state where the risk of falling is increased , 29. BasAs an exploratory efficacy analysis not listed in the protocol, the total physical severity score and the total QOL score were assessed. Each total score was calculated by adding all the scores when suffering from URTI during the supplement intake.The safety assessment was conducted based on changes in laboratory values, complaints of symptoms from the study subjects, and conventional assessments for adverse events by a doctor.The efficacy analysis was conducted on the per-protocol set (PPS) who fulfilled the protocol. The PPS was a group that excluded out of the full analysis set (FAS): I. Person whose intake rate of the trial supplement was below 80%, II. Person who had developed URTI on the start date of supplement intake, and III. Person who was judged by the principal investigator to be appropriate for being excluded from the study.The safety assessment was conducted on the FAS.The primary outcome measure, the incidence proportion of URTI during the period of 16 weeks of supplement intake, and a secondary outcome measure, the incidence proportion of new URTI every four weeks were assessed by Fisher’s exact test. For assessments of the other secondary outcome measures, the generalized Wilcoxon test was used for the duration of URTI, Student’s t test was used for the physical severity score and the QOL score.The significance level of the study was set to 5% on both sides, and 10% on both sides were marginally significant. JMP® 12  was used for all the analyses.The flowchart of this study is shown in A screening test was performed on the 428 subject candidates who gave consent, and 252 subjects were selected. These 252 cases were randomly assigned to the 25OHD (126 cases) and the placebo (126 cases). Among them, two cases (one case in the 25OHD and one case in the placebo) withdrew their consent before taking the trial supplement and dropped out. As a result, 250 cases started taking the trial supplement. During the intake of the trial supplement, one case in the 25OHD and two cases in the placebo withdrew their consent for the reason that they could not come to the final test due to personal reasons, and dropped out. Therefore, 124 cases in the 25OHD and 123 cases in the placebo completed the predetermined study schedule. Out of the subjects included in the study, one case in the placebo group, whose credibility on the efficacy evaluation and the intake of the trial supplement was questioned based on the responses to the dietary survey, and one case in the placebo, who turned out to meet the exclusion criteria because he had started treatment of hypertension before the supplement intake, were excluded from the FAS, and therefore, the FAS had 125 cases in the 25OHD and 123 cases in the placebo. Among the FAS, out of the subjects who completed the study schedule, one case in the 25OHD and one case in the placebo, who did not comply with the restrictions, and 13 cases in the 25OHD and 15 cases in the placebo, who had developed URTI on the start date of supplement intake, were excluded from the PPS. Therefore, 110 cases in the 25OHD and 105 cases in the placebo were in the PPS. The intake rate of the trial supplement was 99.8% in the 25OHD and 99.4% in the placebo. The number of subjects for safety assessment was 248 cases in the FAS .The background information about the study subjects in the PPS in this study is shown in For the serum 25-hydroxyvitamin D levels, the subjects who were determined to be in the deficiency state (50 nmol/L or less) were 61 subjects in the 25OHD and 60 subjects in the placebo, and the subjects who were in the insufficiency state (between 50 nmol/L and 75 nmol/L) were 49 subjects in the 25OHD and 45 subjects in the placebo; the difference was not significant between the two groups (P = 0.891).The results of the dietary survey are shown in There was no item that significantly changed during the period of 16 weeks of the trial supplement intake in the 25OHD or in the placebo, and there was also no item that showed a significant difference between the two groups. Hence, it was speculated that there was no great change in dietary habits during this study.In this study, the number of subjects who developed URTI during the supplement intake was 41 subjects in the 25OHD and 43 subjects in the placebo respectively. The total number of URTI events during the intake was 52 cases in the 25OHD and 66 cases in the placebo respectively.The incidence of URTI during the period of 16 weeks of supplement intake, which was the primary outcome measure, was 37.3% in the 25OHD and 41.0% in the placebo, and no statistically significant difference was observed between the two groups (P = 0.675). The physical severity score was 26.7 ± 11.6 in the 25OHD and 31.2 ± 16.3 in the placebo, and although it was lower in the 25OHD, no statistically significant difference was observed (P = 0.154). The QOL score was 27.6 ± 17.2 in the 25OHD and 31.3 ± 19.3 in the placebo, and although it was lower in the 25OHD, no statistically significant difference was observed (P = 0.346).For the duration of URTI, the 25OHD had lower numbers (median: 10 days) compared to the placebo (median: 13 days) (P = 0.061) .Because 25-hydoxyvitamin D activation (hydroxylation) occurs mainly in the kidneys, an analysis of the duration of URTI was performed with creatinine clearance and gender, which are renal function markers, as adjustment factors. As a result, the supplement effect was significant (P = 0.046), and the shortening effect of the intake of 25OHD on the duration of URTI was suggested.Although the incidence proportion of new URTI every four weeks decreased both in the 25OHD and in the placebo over the season, no statistically significant difference in the incidence was observed between the two groups. However, the decrease in the incidence occurred earlier in the 25OHD than in the placebo, which was likely the result of 25OHD intake .The incidence proportion of URTI, the physical severity score, the QOL score, the duration of URTI, and the incidence proportion of new URTI every four weeks in the 25-hydroxyvitamin D deficiency group, whose serum 25-hydroxyvitamin D levels were 50nmol/L or less at the time of screening and in the 25-hydoxyvitamin D insufficiency group, whose serum 25-hydoxyvitamin D levels were between 50 nmol/L and 75 nmol/L, are shown in Tables 3 and 4.For the incidence of URTI, no significant lowering effect of 25OHD intake on the incidence was observed in the 25-hydroxyvitamin D deficiency group or insufficiency group. However, in the 25OHD, the incidence proportion of URTI was lower in the 25-hydroxyvitamin D deficiency group compared to the insufficiency group, and it was considered that a high serum 25-hydroxyvitamin D level is effective in preventing the onset of URTI.For the incidence proportion of new URTI every four weeks, a decrease in the incidence was observed both in the D deficiency group and the insufficiency group over the time of the study. However, no significant difference was observed between the 25OHD and the placebo in each group of subjects. Looking at the difference in the incidence proportion of URTI between the 25OHD and the placebo, the difference at 9-12 wk was the greatest  in the deficiency group. On the other hand, the difference between the 25OHD and the placebo was the greatest  at 5-8 wk in the insufficiency group, and a decrease in the incidence of URTI in the 25OHD occurred earlier in the insufficiency group than in the deficiency group.For the physical severity score and the QOL score, no statistically significant difference due to the 25OHD intake was observed in the deficiency group and in the insufficiency group. However, in the deficiency group, the QOL score was lower (P = 0.167) compared to the placebo. On the other hand, in the insufficiency group, the physical severity score was lower (P = 0.192) compared to the placebo.In order to comprehensively evaluate the physical severity of URTI and the decrease in QOL due to URTI, the total physical severity score and the total QOL score during the intake of the trial supplement were calculated respectively. The results are shown in The total physical severity score was significantly lower (P = 0.0496) in the 25OHD compared to the placebo. The total QOL score was significantly lower (P = 0.019) in the 25OHD compared to the placebo. These results suggested that the intake of 25OHD improves the physical severity and the QOL when suffering from URTI.Although significant changes were seen in some items, the principal investigator judged that they all were changes without any clinical significance. For reference, the changes in serum 25-hydroxyvitamin D, 1α, 25 (OH)2 vitamin D, intact PTH, serum calcium, and urinary calcium by the supplement intake are shown in 256 adverse events occurred in 73 subjects in the 25OHD, and 339 occurred in 74 subjects in the placebo. The adverse events that occurred include symptoms such as the common cold and a stomachache. Those symptoms were all mild, and they disappeared during the study. For all the adverse events, the principal investigator denied the connection with the trial supplement.As seen in the above results, there was no problem in the safety of the trial supplement under the conditions of this study.URTI is considered to cause serious economic losses, and it is believed that the prevention of this infection and the improvement in symptoms are economically important even from the perspective of public health. Vitamin D3 has been examined for the impact on the protective effect against a cold and influenza, while 25OHD has not been examined. Therefore, in this study, the effect of intake of 25OHD on URTI was examined.As a result, there was no effect of 25OHD intake observed in the incidence proportion of URTI during the period of 16 weeks of supplement intake, which was the primary outcome measure. However, at the time of 9-12 weeks after the start of intake when it was considered that the serum 25-hydroxyvitamin D level was increasing due to the intake of 25OHD, a considerable difference from the placebo group was seen in the incidence proportion of URTI. This suggests the possibility of preventing URTI by raising the serum 25-hydroxyvitamin D level. This can be explained also by the fact that a decrease in the incidence of URTI was seen earlier in the group of subjects whose serum 25-hydroxyvitamin levels were insufficient compared to the group of subjects whose serum 25-hydroxyvitamin D levels were deficient.For the duration of URTI, it was suggested that the duration is shortened by 25OHD intake, and it was also suggested that the physical severity and the QOL when suffering from URTI are improved by 25OHD intake.The shortening effect on the duration of URTI and the improvement of the physical severity and the QOL, which are thought to be the results of 25OHD intake, can be associated with protection from viruses and bacteria and proliferation inhibition after infection due to 25OHD intake. It is speculated that protection from viruses expected by 25OHD intake is associated with antimicrobial peptide LL37 and secretory immunoglobulin A (sIgA).It has been reported that LL37, an antimicrobial peptide whose production is enhanced by 1α, 25(OH)2 vitamin D3, a hydroxylated metabolite of 25OHD, activates TLR3 and TLR9, which are involved mainly in virus recognition among the Tolllike receptors (TLRs), pattern recognition receptors , 31. TLRIn other words, it is assumed that the intake of 25OHD increased the production of LL37 and showed antiviral activity.The fact that the reduction of symptoms and the shortening of the duration of disease can be expected by suppressing viral growth shows that anti-influenza virus drugs, Oseltamivir and Zanamivir also have effects on the shortening of the duration of influenza and the reduction of severity by suppressing the growth of influenza viruses , 33.In addition, since there is a report that taking Oseltamivir and Zanamivir prophylactically protects against the onset of influenza, suppressing viral growth is considered to not only delay the incidence of URTI but also shorten the duration of URTI and reduce the severity -36. In tIt has been reported that there is a correlation between serum 25OHD and salivary sIgA, and salivary sIgA is expected to increase by way of raising the serum 25-hydroxyvitamin D level . In factIt is believed that these presumed functions of 25OHD are exerted when 25OHD is hydroxylated by CYP27B1. However, since the blood level of 25OHD is about 1000 times higher than the blood level of its hydroxide, 1α,25 (OH)2 vitamin D, 25OHD itself may also be exerting the functionality.The influence of vitamin D intake on respiratory infection (RTI) has evaluated by meta-analysis. Several meta-analyses have demonstrated the reduction of the incidence and shortening the duration of RTI by intake of vitamin D -15.TablMeanwhile, in meta-analysis by Vuichard Gysin D et al., there is no preventive effect of RTI or shortening of the duration of disease by ingestion of vitamin D in healthy subjects . Such diThe result of this study suggests that the intake of 25OHD will be a new option to reduce the burden of URTI with respect to physical severity and QOL and to accelerate the healing of URTI.There are some research limitations in this study. First, it seems that the number of cases in this study was too small to statistically significantly prove the efficacy of 25OHD intake. Secondly, immune markers such as antimicrobial peptides and salivary sIgA are not measured. Thirdly, because the epidemic of URTI is dependent upon the season, it is difficult to distinguish it from the effects of 25OHD intake. Fourthly, since the clinical examinations were conducted only twice: before the intake and after the intake, the behavior during the intake could not be assessed. Fifthly, the evaluation of URTI may be biased, because subjects recorded their subjective symptoms of URTI using WURSS-21.Based on the above facts, it is considered that there is a need to conduct a study with larger number of samples, measure immune markers, laboratory examination, and conduct examinations at multiple points of time in the future.Conflict of interest: This study was conducted with funds from FANCL Corporation (FANCL). 25OHD used in this study was provided for free by DSM Nutritional Products, Ltd. YS, YI, and KY are employees of FANCL. Based on the results of this study, FANCL has applied for a patent. YS and YI are inventors in patent law. FANCL has paid a supervision fee to HO. There are no other conflicts of interest to be noted.Acknowledgment: We would like to express sincere gratitude to the 25OHD3 research group established in Medical Corporation Foundation Kenkoin Clinic  who provided valuable advice for preparing this paper. YS, YI, KY, and HO designed this study; KE conducted research; YS analyzed data; and YS, YI, KY and HO wrote the paper. YS had primary responsibility for final content. All authors read and approved the final manuscript.Ethical standard: This study was conducted according to the spirit of the Declaration of Helsinki  and the Ethical Guidelines for Medical and Health Research involving Human Subjects . All procedures were approved by the HUMA R&D Ethical Review Committee. Written informed consent was obtained from all participants. No animals were used in this study.