Radix dichroae (UERD) and to provide experimental support for a safe clinical dose for anticoccidial treatment of broiler chickens. Radix dichroae root extracts were prepared using the ultrasonic extraction method. The anticoccidial activity of the oral solution prepared from the ultrasonic extract of Radix dichroae roots was tested in broiler chickens following oral infection with a field isolate of E. tenella. Ninety Lingnan yellow broiler chickens (14 days old) were randomly divided into nine groups (n = 10), including six UERD oral solution treatments , a toltrazuril group (0.10%), an E. tenella-infected control group, and a healthy control group. All groups were inoculated orally with 7 × 104 sporulated E. tenella oocysts (Guangdong strain) except for the healthy control group. The chickens in the seven drug-treated groups were administered a UERD oral solution or toltrazuril in drinking water for 7 days. The anticoccidial efficacy of the UERD oral solution was evaluated by the bloody diarrhoea severity level, relative body weight gain (rBWG), lesion score, oocyst per gram (OPG), and anticoccidial index (ACI). Compared with the infected control group, there were no significant differences in the groups treated with UERD oral solution or toltrazuril with regard to the lesion changes in the caecal regions (P > 0.05); however, the blood contents, OPG, and oocyst score in three UERD oral solution treatment groups  were significantly reduced, and the bloody diarrhoea was also alleviated. The ACI in three UERD oral solution treatment groups  was higher than that in the toltrazuril group (ACI = 127.0), and the rBWG in the 1.50% UERD oral solution treatment group (95.0%) was similar to that in the healthy control group (100%), which was also 12.5% higher than that in the toltrazuril group (82.5%). The findings of this study demonstrated that the UERD oral solution (0.50% ～ 2.50% dose range) showed better prevention, anticoccidial efficacy, and growth promotion effects than toltrazuril (0.10%), and the 1.50% dose level of UERD oral solution in water is the clinically recommended dose according to the present study conditions.This study was conducted to determine the optimal dose of the oral solution of the ultrasonic extract of  Eimeria, and the most severe harm to chickens is always caused by Eimeria tenella (E. tenella) [Radix dichroae (RD) is the dried root of Dichroa febrifuga Lour., which belongs to the family of Saxifragaceae and is widely distributed in humid mountainous and other subtropical areas, such as the Sichuan, Guizhou, and Yunnan provinces in China. Radix dichroae is a famous herb and has been traditionally utilized as an antimalarial, expectorant, and antifebrile compound in China for centuries according to previous reports by Zhang and Huang [β-dichroine and α-dichroine were the main anticoccidial effective components of Radix dichroae, with β-dichroine having more effective pharmacological activity than α-dichroine [β-dichroine and has been used as an antiparasitic feed additive for the prevention of coccidiosis in poultry production, which has been approved by the European Union and the US Food and Drug Administration [Radix dichroae has anticoccidial properties in chickens when used as a single herb or as the main herb in an herbal formulation complex during treatment; however, the content of the active ingredients  in the crude extract of Radix dichroae in the above coccidiostats (single Radix dichroae or Radix dichroae complex) has always been lower. Therefore, in this study, the total alkaloids as active ingredients were extracted from the dried roots of Radix dichroae using a modified phytomedicine extraction separation technique to increase the content of β-dichroine therein. Moreover, considering that the constituents in the oral solution could more easily access and interact with the parasites than the constituents remaining in the powder solid, the production technique was optimized to prepare an oral solution with the ultrasonic extract of Radix dichroae (UERD) to improve the treatment effects and ease of use in clinical settings. Therefore, the objective of this study was to determine the potential anticoccidial activity of UERD oral solution; a pathological model of chicken coccidiosis was reproduced after E. tenella (Guangdong strain) was inoculated artificially into chickens to further explore the efficacy of the oral solution prepared with UERD, as well as to determine the optimal dose in clinical practice. Employing a standard parasitological procedure as an alternate phytomedicine and prescription would contribute to the control of chicken coccidiosis.Chicken coccidiosis is an intestinal disease caused by intracellular protozoan parasites belonging to the genus tenella) –3, resulnd Huang  and Kirand Huang . The anaichroine , 7. Furtstration . Howeverstration , 11 and stration . Moreovestration , 14, andRadix dichroae roots were purchased from Lanzhou Huanghe Chinese medicine market and identified as the dried root of Radix dichroae in the genus Bergenia of the family Saxifragaceae by Professor Yun Li from Gansu University of Chinese Medicine. Voucher specimens were deposited in the Herbarium of the College of Pharmacy  for Radix dichroae (voucher number: GUCM 621222130517114 LY). Two hundred grams of dried Radix dichroae roots was ground into powder using an electric grinder, and the extraction of the Radix dichroae roots was carried out using an ultrasonic extraction method. Two percent hydrochloric acid was used as the extraction solvent, and the solid-liquid ratio was 1 : 6 . Ultrasonic wave power was used at 80 W, for 1 h extraction at 50°C, and 3 extraction stages were used. The supernatants collected from each stage were pooled together, the impurities were removed with a small amount of chloroform, and then the pH value was adjusted to 10 with strong ammonia. The solution was extracted 3 times with chloroform, and the recovered chloroform extracts containing the extract of Radix dichroae were concentrated, dried, and ground into a powder. The end product was approximately 25.80 g of dried extract powder, for an overall yield of 12.90% (w/w). The average contents of total alkaloid and β-dichroine therein were 4.79% and 0.18%, respectively. The optimal production conditions for the oral solution preparation of UERD were 120 mg UERD powder dissolved in 100 mL of a solution, by a prepared solution of 1.5% Tween-80 and 0.5% sodium benzoate in distilled water  to obtain the test solution with a pH value of 5–6 that was stored at 4°C until use. The positive control drug was toltrazuril  Animal Health Co. Ltd, China), administered in drinking water at the recommended dose of 0.10% (v/v).Ninety one-day-old Lingnan yellow broiler chickens obtained from the Hualong Commercial Hatchery of Lanzhou were reared in a coccidia-free isolation facility and fed commercial food that contained no anticoccidial drugs or antibiotics for 14 days ad libitum during the study under the following conditions: temperature (23 ± 2°C), relative humidity 55 ± 15%), and ventilation (air exchange rate of 18 cycles/h). To confirm that the chickens were free of infection prior to experimental inoculation, faecal samples were analyzed by salt flotation and light microscopy to ensure the absence of oocysts. All animal care procedures were performed in accordance with the Veterinary Laboratory Biosafety Guidelines  [%, and veE. tenella (Guangdong strain) used in this study were isolated from chickens that died from E. tenella infection in 1996 in Huadu, Guangdong province, China, as confirmed by microscopic examination and sequence analysis of the rRNA gene internal transcribed spacer regions, and maintained in the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, CAAS. The oocysts were propagated in the broiler chickens without E. tenella infection by oral infection, and the faeces were collected on postinfection (PI) days 6, 7, 8, and 9. The unsporulated oocysts were sporulated by placement in a 2.5% potassium dichromate solution at a suitable humidity and temperature (28°C). Sporulated oocysts were cleaned with water and counted by the McMaster technique mentioned by Foreyt [The oocysts of y Foreyt .n = 10). At 14 days of age, the chickens in groups IC , DC (positive drug control group), A, B, C, D, E, and F were orally infected with 7.0 × 104 sporulated E. tenella oocysts. The clinical signs were observed and recorded every day during the experiment. Signs of infection were defined as the presence of oocysts in the faeces with bloody diarrhoea according to the method of Holdsworth et al. [A total of ninety Lingnan yellow broiler chickens (14 days old) free from coccidian infection were weighed individually and randomly divided into nine groups ; additionally, all surviving chickens were weighed individually before culling. The individual and mean body weight gains were calculated for the period of days 14–22. The relative body weight gain (rBWG) was calculated using the following formulae: BWG = final body weight － initial body weight; BWG rate (%) =  ÷ initial body weight × 100%; rBWG (%) =  × 100%. Clinical observations of bloody diarrhoea and mortality for all the chickens were recorded daily throughout the experimental period. The survival rate (%) =  × 100%. Faecal droppings were examined visually for bloody diarrhoea during the days 4–7 after infection according to the method of Morehouse and Baron [Each chicken was weighed on day 14 before nd Baron  and assind Baron . The ranOn days 5, 6, and 7 after infection, the total daily faeces of each group were collected, and the daily oocyst production was determined using a McMaster chamber . The surThen, the caecal contents were ground, and the oocyst counts in the pooled caecum contents of each chicken were determined using the McMaster's method. The result was expressed as oocysts per gram (OPG) based on the method of JIAO . The oocThe anticoccidial index (ACI), established by Merck Sharp & Dohme , was calP < 0.05. The results are expressed as the means ± SD.The data were analyzed using SPSS 17.0 software . The bloody diarrhoea was statistically compared by the Kruskal-Wallis H test, and the results were expressed as median frequencies . The parameters of mean body weight for each time point, body weight gain, and mean lesion score were conducted using one-way analysis of variance, followed by a LSD (least significant difference) test, Dunnett's post hoc test, or Duncan's multiple range test, which were used to test the significance between different groups at the level of E. tenella oocysts, chickens took 3 days to demonstrate clinical symptoms of infection to different degrees, including depression, loss of appetite, drowsiness, feather disorders, fear of cold, and huddling. Bloody diarrhoea was observed in all infected groups from postinfection day 4 to 7. The most severe bloody diarrhoea was found in group IC (infected/nontreated group), which had the highest bloody score than that of any of the other groups (bloody score (median) = 4, P < 0.05), followed by group A (bloody score (median) = 4, P < 0.05), group F (bloody score (median) = 2), and the toltrazuril group (bloody score (median) = 2). The degree of bloody diarrhoea and faecal score in four of the groups treated with the UERD oral solution  were lighter and milder among the 7 drug-treated groups, and the bloody score in the 5.00% UERD oral solution group was similar to that in the toltrazuril group. The broiler chickens administered UERD oral solution and toltrazuril showed lower bloody scores than those in the infected/nontreated group , and the physical condition and feed intake improved obviously. Three chickens in the positive infected control group (group IC) died from coccidial infection; and in the 0.10% toltrazuril group and four UERD oral solution treatment groups , one chicken died from E. tenella infection in each group  were the highest among groups infected with sporulated E. tenella oocysts. The group medicated with 0.10% toltrazuril (group DC) had the lowest output of oocysts with an OPG of 3.11 × 106. The OPG and oocyst values in groups B, C, and D  and group DC (0.10% toltrazuril) were significantly lower than those in group IC (infected/nontreated group). However, chickens administered UERD oral solution at doses of 3.50% and 5.00% showed similar OPG and oocyst values to those of the infected control group (group IC).As shown in P < 0.05). Moreover, chickens in group F treated with 5.0% UERD oral solution had a similar and lower rBWG compared with group IC.The rBWG of group C administered 1.50% UERD oral solution (95.0%) was similar to that of group HC (100%), which was 12.5% higher than that of group DC . The chickens in the groups treated with 0.50, 1.50, and 2.50% UERD oral solution and 0.10% toltrazuril had no significant difference in the mean final weights compared with group HC, while there was a significant difference compared with group IC . The highest ACI value was observed in group C , followed by group D  and group B . Moreover, the ACI values in these three UERD-treated groups were superior to toltrazuril treatment (ACI = 127.0). However, with the increasing dose of UERD oral solution, the ACI values in group E  and group F  decreased significantly, and the lowest ACI value from the six UERD-treated groups was observed in group A (ACI = 99.1) .Radix dichroae is a famous herb used as an antimalarial, expectorant, and antifebrile medicine in Chinese medicine for centuries, and its antimalarial pharmacological activities were researched by Jang et al. in 1948 [Radix dichroae and the β-dichroine found therein could promote the proliferation of spleen T lymphocytes and B lymphocytes and had better immune-enhancing activity for macrophages in mice [Radix dichroae roots possessed lower toxicity and showed dose-dependent toxicity in the liver, kidneys, spleen, and lungs at high doses after a long course of administration [Radix dichroae (UERD) in chickens to determine whether UERD oral solution can be used as an effective and safe Chinese medicine under conventional dose conditions and also to determine if it is suitable for long-term clinical use against coccidiosis.Coccidiosis is one of the biggest challenges faced by the global poultry industry and results in annual losses, including decreased productivity and the usage of anticoccidial drugs and vaccines, estimated at approximately 3 billion US dollars worldwide , 23, 24. in 1948 . Moreovestration . Hence, Radix dichroae. Additionally, it easily causes gastrointestinal dysfunction, which in turn greatly reduces its anticoccidial effect, causing substantial waste of the original medicinal materials. Therefore, the development of an oral suspension for the ultrasonic extract of Radix dichroae can ensure the utilization of the highest drug content and meet the fluidity requirements for oral administration. Second, oral solution preparation has the advantages of stable quality, fast absorption, a good curative effect, and a safe and hygienic clinical application, which is convenient for absorption and utilization in chickens and thus can better exert anticoccidial activity. E. tenella is the most pathogenic chicken coccidian and mainly parasitic in the caecal region, where active infection is characterized by diarrhoea and massive caecal haemorrhage. Infection with E. tenella typically leads to swelling and damage to the caecal wall, which alters the normal function of the chicken intestine [E. tenella and when three chickens died of coccidiosis infection. Signs of infection were alleviated by drinking water supplementation with UERD oral solution and toltrazuril. Compared with the infected control group, the degree of severity of the caecal lesion was significantly improved in groups medicated with UERD oral solution and toltrazuril, and the oocyst value and OPG were both reduced in the drug-treated groups . The findings in this study indicated that UERD oral solution at doses of 0.50, 1.50, and 2.50% in broiler chickens could significantly increase the rBWG compared with that in the infected control group after challenge with sporulated E. tenella. In addition, the amount of bloody faeces, the OPG, and the oocyst score also improved in chickens after administering UERD oral solution at the recommended dose mentioned above. It can be confirmed that the ultrasonic extract from a single herbal Radix dichroae root extract had a curative effect against E. tenella infection in broiler chickens. However, chickens medicated with the 0.25% and 5.00% UERD oral solutions tended to have a lower rBWG compared with the other drug-treated groups, while having a similar rBWG to the infected control group; this reduced rBWG might be inferred as the effect caused by the lower doses of UERD oral solution, which contain inadequate amounts of the active ingredients (such as β-dichroine). However, for the higher dose of UERD oral solution, the toxic reaction from excessive active ingredients might hamper the growth of the chickens and cause high OPG values (mentioned above).Considering that poultry has a different gastrointestinal structure compared with ruminants, it is impossible for chickens to completely digest and absorb the crude extract of ntestine . In thisE. tenella-infected control group, the UERD oral solution administered by drinking water at high doses might produce drug toxicity, which would be harmful to chickens in the clinic. In this study, the ACI values from all the drug-treated groups were all less than 160, which may be related to the virulence of the E. tenella strain selected in the test (Guangdong strain). The field isolate of E. tenella (Guangdong strain) obtained from the State Key Laboratory of Veterinary Etiological Biology has been used in research on chicken coccidiosis for a long time by Li et al. [4 oocysts of sporulated E. tenella within one month. In general, mortality ranging from 5% to 10% in the infection control group is considered optimal in the experimental design for anticoccidial drugs. However, in this study, after challenge with 7 × 104 oocysts of sporulated E. tenella (Guangdong strain), the mortality rate in the infection control group reached 30%, and we can infer from this result that the chickens in eight drug-treated groups tended to be inoculated with a relatively high dose of sporulated E. tenella oocysts. Although lower ACI values were expressed in the toltrazuril and six UERD oral solution treatments in the present study, the relationship between the drug dose and the anticoccidial effect could still be obtained from the results; that is, the UERD oral solution administered through drinking water according to the recommended doses of 0.50% (ACI = 143.7), 1.50% (ACI = 151.0), and 2.50% (ACI = 144.3) had better anticoccidial effects than 0.10% toltrazuril (ACI = 127.0). Considering the drug cost in the clinic and the growth performance of broiler chickens, 1.50% UERD oral solution in drinking water should be the clinically recommended dose for broiler chickens.The lesion score, OPG, and ACI values are commonly applied parameters used to evaluate the anticoccidial efficacy of animal drugs. In this study, the highest lesion score and lowest ACI value were observed in the infected control groups, followed by the 0.25%, 3.50%, and 5.00% UERD oral solution treatments, which showed lower lesion scores and higher ACI values. Moreover, similar lesion scores and oocyst values but higher ACI values were observed in 3 UERD oral solution treatment groups  and in the 0.10% toltrazuril treatment group, in which 0.10% toltrazuril had lower OPG and ACI values. These results indicated that the UERD oral solution and toltrazuril produced better protective effects on the intestinal mucosa, and the concentration of UERD oral solution within the range of 0.50% ～ 2.50% demonstrated better anticoccidial efficacy than 0.10% toltrazuril in the present study. In addition, although the anticoccidial effects of the two highest UERD oral solution treatments (3.50% and 5.00%) were better than those of the i et al. . The virE. tenella (Guangdong strain) while reducing bloody diarrhoea, oocyst output, and lesion scores in the caecal region. It was demonstrated that UERD oral solution within a 0.50% ～ 2.50% dose range had better anticoccidial and growth promoting effects than toltrazuril in chickens infected with sporulated E. tenella oocysts. The oral solution prepared with the ultrasonic extract of Radix dichroae roots was suitable for administration in drinking water, and its good efficacy after 7 days of treatment revealed that the UERD oral solution was appropriate for the prevention, therapy, and intermittent treatment of E. tenella-infected chickens.In this study, a higher protection rate was found in three UERD oral solution treatments  compared with the 0.10% toltrazuril treatment. The addition of the UERD oral solution could increase the body weight and survival rate of broiler chickens infected with


Normal blood cells are formed in the bone marrow by a process called hematopoiesis. This process is supported by a network of non-hematopoietic cells including connective tissue cells, blood vessel cells and bone-forming cells. However, these cells can also support the growth of cancer cells, i.e., hematological malignancies  and cancers that arise in another organ and spread to the bone marrow. Two of these cancer-supporting normal cells are bone-forming osteoblasts and a subset of connective tissue cells called mesenchymal stem cells. One mechanism for their cancer support is the release of proteins that support cancer cell proliferation and progression of the cancer disease. Our present study shows that both these normal cells release a wide range of proteins that support cancer cells, and inhibition of this protein-mediated cancer support may become a new strategy for cancer treatment.Mesenchymal stem cells (MSCs) and osteoblasts are bone marrow stromal cells that contribute to the formation of stem cell niches and support normal hematopoiesis, leukemogenesis and development of metastases from distant cancers. This support is mediated through cell–cell contact, release of soluble mediators and formation of extracellular matrix. By using a proteomic approach, we characterized the protein release by in vitro cultured human MSCs (10 donors) and osteoblasts (nine donors). We identified 1379 molecules released by these cells, including 340 proteins belonging to the GO-term Extracellular matrix. Both cell types released a wide range of functionally heterogeneous proteins including extracellular matrix molecules , several enzymes and especially proteases, cytokines and soluble adhesion molecules, but also several intracellular molecules including chaperones, cytoplasmic mediators, histones and non-histone nuclear molecules. The levels of most proteins did not differ between MSCs and osteoblasts, but 82 proteins were more abundant for MSC  and 36 proteins more abundant for osteoblasts. Finally, a large number of exosomal proteins were identified. To conclude, MSCs and osteoblasts show extracellular release of a wide range of functionally diverse proteins, including several extracellular matrix molecules known to support cancer progression , and the large number of identified exosomal proteins suggests that exocytosis is an important mechanism of protein release. The cellular elements of the bone marrow microenvironment consist of hematopoietic cells together with various stromal cells that support normal as well as leukemic hematopoiesis and metastatic disease ,2. The hThe extracellular matrix is also important for regulation of hematopoiesis. The matrix proteins are important both for communication between bone marrow cells  and for regulation of hematopoietic cell migration and localization ,3. The sSeveral studies suggest that MSCs and osteoblasts are important in carcinogenesis and therefore also for chemosensitivity and/or cancer progression. First, as discussed in a previous review  MSCs seeOsteoblasts were derived from nine and MSC from 10 healthy individuals; the characteristics of these donors are presented in We were able to quantify 1255 proteins for the various MSCs. The number varied between 470 and 1004 proteins for individual cell donors; 795 proteins (63%) were quantified for at least five MSC donors and more than 400 proteins were quantified for at least nine of the 10 donors ; 571 (46.8%) proteins could be quantified for at least five donors  after 48 h of culture in serum-free medium , and the viable cells were relatively small compared with cells of the other MSC donors. Based on light microscopy the large majority of cells were viable and adherent both during at the end of culture for all the other MSC donors. Cells from MSC4 and MSC9 had to be cultured for an extended time period of 13 days before reaching 70% confluence, but in contrast to MSC6, cells from these two MSC donors had similar morphology  as the other MSCs both at the time of medium change and at the time of supernatant harvesting . Thus, MThe OB4 donor differed from the other osteoblast donors with regard to the protein release, but these cells did not differ with regard to morphology or cell proliferation during culture .We identified a total of 1379 proteins for the MSCs and the osteoblasts. The released proteins were very heterogeneous both for the MSCs and the osteoblasts, and more than 90% of them did not differ significantly between the two cell types when comparing the overall results see . A detaiMany previous studies of the human MSC protein profiles have investigated relatively few cell donors and used alternative proteomic methods and thereby detecting fewer proteins . Three pThe results described above clearly show that both MSC and osteoblasts derived from different donors vary with regard to the number of detectable proteins, and both cell types are able to release a high number of structurally as well as functionally diverse proteins. However, the data presented in We investigated the release of selected protease and protease inhibitors  for three MSC donors; these cells were purchased from another distributor and were cultured in an optimal serum-containing medium . When usp < 0.05 and at the same time a significant fold change (FC) (p < 0.05) determined by Z-score statistics; or (ii) being detected either in one or none of the osteoblast but at least seven of the MSC populations. These proteins are listed in We identified 82 proteins that were released at significantly increased levels by MSCs, i.e., either (i) being detected for at least three MSCs and three osteoblast donors and the corresponding statistical comparison showing p < 0.05 plus FC significance <0.05, or (ii) expressed by ≤1 MSC but at least eight osteoblast populations; see Osteoblasts showed increased release of only 36 proteins when using similar criteria as described for MSCs  p = 0.0007) and the unequal distribution of abundantly expressed proteins between the two cell types (p = 0.0011) illustrate that these proteins were not identified by coincidence alone.A total of 118  proteins showed abundant expression when comparing MSCs and osteoblasts; both the total number of abundantly expressed  plus those proteins that were detected only or mainly for MSC . The most significant GO terms identified for proteins increased in MSC reflected mainly differences with regard to extracellular matrix, cell adhesion, cell migration , and the GO term analysis was based on those among these 746 proteins that differed significantly between MSCs and osteoblasts  and no protein network could be identified (data not shown). For osteoblasts 48 proteins differed significantly, and we could also identify several protein interaction networks . However, it is a relevant and possibly important question because differences between male and female MSCs have been described previously and may be important especially with regard to MSC aging . For MSCnetworks .http:/www.exocarta.org/exosome_marker-new), and 21 of them are among the top 25 proteins. None of these proteins showed any difference between MSCs and osteoblasts. Although we did not isolate and characterize exosomes in our present study, this large protein number strongly suggests that extracellular protein release through exosomes is important for the communication between MSCs/osteoblasts and their neighboring cells.Exosome release is one of the mechanisms for communication between cells, and 683 of the identified proteins belonged to the GO term exosome . SeventyA total of 206 proteins included in the term Protease reached detectable levels in our study , and mosNonleukemic stromal cells support both normal and leukemic hematopoiesis, and probably also cancer metastases, through their release of soluble mediators and extracellular matrix molecules as well as through cell–cell contact . Both noWe investigated enriched MSCs and osteoblasts derived from bone marrow, and we observed differences between individuals with regard to proliferation (MSCs), survival (MSCs) and protein release profile  and the larger number of increased expression in MSC compared with osteoblasts (82 vs. 36) is significantly different from what would be expected by coincidence. Furthermore, both GO term and network analyses as well as the detailed descriptions of individual proteins show that the abundant proteins are highly selected and do not include or include very few proteins from large protein classes/subclasses, e.g., exosomal proteins, histones and non-histone nuclear proteins.Both MSCs ,19,43 anSeveral extracellular matrix molecules and other molecules expressed on or released from the cell surface of bone marrow stromal cells are important for the regulation of normal hematopoiesis; this includes a wide range of various extracellular matrix molecules, adhesion molecules and other cell surface molecules released in biologically active soluble forms as well as various soluble mediators ,2,3,5. SStudies of several malignancies suggest that the extracellular matrix is important for the development and chemosensitivity of human malignant cells. Firstly, an extracellular matrix profile identified a subset of breast cancer patients with increased risk of progression ,69. ThisA wide range of proteins involved in important intracellular processes were also released both from MSCs and osteoblasts. Histones provide structural stability to the chromatin and are transcriptional regulators, but histones may also be released into the extracellular space either freely, as DNA-bound nucleosomes or as a part of extracellular traps . These eAs discussed above, cells can release proteins to the extracellular space through various mechanisms, including proteolysis of cell surface proteins and release through exosomes . We cannEven though our MSC and osteoblast donors showed a limited heterogeneity with regard to the overall protein release profile, some differences were observed, and these differences also included the release of proteins important for the function of the stem cell niches/normal hematopoietic stem cells . PreviouIn the present study we have investigated bone marrow MSCs and osteoblasts derived from healthy individuals. An important question is therefore how the secretoma of MSCs and osteoblasts is modified by the crosstalk between these stromal cells and the malignant in their common bone marrow microenvironment. The communication between these cells is probably mediated both by direct cell–cell contact, modulation of the extracellular matrix and the release of soluble mediators ,89. As iConditioned medium from MSCs are now considered as a possible therapeutic tool ,36,92, aThe effects of soluble mediators derived from MSC and osteoblasts have been studied more in detail in AML. By using a coculture model where stromal and AML cells were separated by a semipermeable membrane it was possible to study the effect of the crosstalk between the two cell types mediated by soluble mediators. These studies showed that the stromal cell secretoma could increase proliferation and/or promote survival of the malignant cells; this was true both for normal MSCs, osteoblasts and fibroblasts but the mechanisms behind this effect differed between patients ,13,95. HTo conclude, bone marrow MSCs and osteoblasts release a wide range of functionally divergent proteins that are involved in the communication between cells, the regulation of normal as well as leukemic hematopoiesis and possibly also development of metastases from distant tumors. However, many proteins involved in the intracellular regulation of fundamental cellular functions are also released, and the possible extracellular function of these proteins should be further investigated.Cryopreserved human bone marrow MSCs  derived from 10 healthy donors and human osteoblasts  derived from nine healthy donors were purchased from PromoCell GMBH  . The cel5 cells were further expanded in T25 flasks  with Mesenchymal Stem Cell Growth Medium  and Osteoblast growth medium  with supplement-39615 , respectively. The culture medium was exchanged after 24 h. When the cells had reached 70% confluence, the culture medium was exchanged with serum-free IMDM medium . The cultures were thereafter incubated for 48 h before supernatants were collected and centrifuged at 3000× g for 10 min before being aliquoted and stored at −80 °C. These conditioned media samples were later concentrated by ultra-filtration using Amicon Ultra-15 3kDa cutoff centrifugal filters  prior to proteomic sample preparation. The filters were conditioned with 4 mL H2O (HPLC grade) before adding the conditioned medium, which was centrifuged for 30–45 min at 3220× g at 4 °C. dH2O (4 mL) was then added for washing, using the same settings and repeated twice. Protein concentration was measured using the Qubit Protein Assay . The study was approved by the Regional Ethics Committee (REK Vest 350/2017). MSCs and osteoblasts (1 mL) were thawed at 37 °C according to a highly standardized procedure recommended by the manufacturer, and approximately 1.5 × 10The conditioned media samples containing 10 μg of protein were buffered with 50 mM Tris/1 mM CaCl, reduced with 20 mM DTT (7 min at 95 °C), alkylated with 50 mM iodoacetamide , and digested overnight at 37 °C with 1:50 enzyme: substrate ratio of sequencing grade trypsin . Following digestion, samples were acidified with methanoic acid and desalted using HLB Oasis SPE cartridges . Samples were eluted with 80% acetonitrile in 0.1% methanoic acid and concentrated (speedvac). The peptides were stored at −80 °C until use. A quality control sample was also made by pooling conditioned media from the MSC and osteoblast samples, in addition to conditioned media from in vitro cultured primary acute myeloid leukemia (AML) cells. This quality control sample was analyzed after every tenth sample in the following LC-MS analysis to document reproducible instrument performance.The final cultures were prepared in IMDM medium alone with supplementation of serum, phenol red, lipids, growth factors or proteins. We could not detect albumin or immunoglobulin  in our proteomic analyses of conditioned media; these proteins are detected at high levels in normal serum but could not be detected in our culture supernatants.All cultures were regularly evaluated by light microscopy during and at the end of the culture period; the large majority of cells were viable  for all donors during culture but not for MSC6 at the end of culture.About 0.5 μg protein as tryptic peptides dissolved in 2% acetonitrile (ACN), 0.5% formic acid (FA), were injected into an Ultimate 3000 RSLC system  connected online to a linear quadrupole ion trap-orbitrap (LTQ-Orbitrap Elite) mass spectrometer  equipped with a nanospray Flex ion source . The sample was loaded and desalted on a pre-column  at a flow rate of 6 µL/min for 5 min with 0.1% TFA .2O and 100% ACN respectively. The gradient composition was 5% B during trapping (five minutes) followed by 5–7% B over one minute, 7–32% B for the next 129 min, 32–40% B over 10 min, and 40–80% B over 5 min. Elution of very hydrophobic peptides and conditioning of the column were performed during 20 min isocratic elution with 80% B and 20 min isocratic elution with 5% B respectively. The eluting peptides from the LC-column were ionized in the electrospray and analyzed by the LTQ-Orbitrap Elite. The mass spectrometer was operated in the DDA-mode (data-dependent-acquisition) to automatically switch between full scan MS and MS/MS acquisition. Instrument control was through Tune 2.7.0 and Xcalibur 2.2.Peptides were separated during a biphasic ACN gradient from two nanoflow UPLC pumps (flow rate of 200 nL/min) on a 50 cm analytical column . Solvent A and B were 0.1% FA (vol/vol) in dH6 in the linear ion trap with maximum allowed ion accumulation time of 300 ms. The 12 most intense eluting peptides above an ion threshold value of 3000 counts and charge states 2 or higher were sequentially isolated to a target value of 104 and fragmented in the high-pressure linear ion trap by low-energy CID (collision-induced dissociation) with normalized collision energy of 35% and wideband-activation enabled. The maximum allowed accumulation time for CID was 150 ms, the isolation was maintained at 2 Da, activation q = 0.25, and activation time of 10 ms. The resulting fragment ions were scanned out in the low-pressure ion trap at normal scan rate, and recorded with the secondary electron multipliers. One MS/MS spectrum of a precursor mass was allowed before dynamic exclusion for 40 s. Lock-mass internal calibration was not enabled.Survey full scan MS spectra (from m/z 300 to 2000) were acquired in the Orbitrap with resolution R = 240,000 at m/z 400 after accumulation to a target value of 10The spray and ion-source parameters were as follows. Ion spray voltage = 1800 V, no sheath and auxiliary gas flow, and capillary temperature of 260 °C.The LC-MS raw files were searched in MaxQuant , against the concatenated forward and reversed-decoy Swiss-Prot Homo sapiens database version downloaded 5 September 2019 and an implemented contaminant database by the Andromeda search engine ,100. Oxit-test and Z-statistics [Perseus  was used to process and filter the MaxQuant results . Hierarcatistics  were useatistics . Cytoscaatistics ,106.Normal human MSCs and osteoblasts show constitutive extracellular release of a wide range of proteins, and there is a considerable overlap between the two cell types with regard to their protein release profiles. However, MSCs and osteoblasts differ especially with respect to their release of collagens as well as certain other extracellular matrix proteins. Both cell types release several proteins that support the growth of normal hematopoietic stem cells as well as hematopoietic and non-hematopoietic malignant cells.


ADH1B (rs1229984) and ALDH2 (rs671), alcohol consumption, the effect of a combination of the two polymorphisms, and breast cancer risk were studied in a population of East-Asian women. In this study, 623 breast cancer cases and 1845 controls, aged 40 or above, were included. The association between ALDH2 polymorphism and breast cancer risk was validated in 2143 breast cancer cases and 3977 controls. Alcohol consumption increased the risk of breast cancer regardless of ADH1B and ALDH2 genotypes. The rs671 polymorphism of ALDH2 was independently associated with increased breast cancer risk . The ADH1B rs1229984 polymorphism, and combined effects of the rs671 and rs1229984 polymorphisms, did not reveal any significant association with breast cancer. Stratification by menopausal status revealed that rs671 gene polymorphisms were significantly associated with breast cancer only in postmenopausal women . This is the first study to demonstrate an independent association between ALDH2 gene variants and breast cancer in Asian women. Further studies are warranted to further elucidate the etiology of breast cancer as it relates to alcohol consumption in Asian women.The associations between genetic polymorphisms in  In 2007, the International Agency for Research on Cancer (IARC) classified alcoholic beverages as group 1 carcinogens for the pharynx, larynx, esophagus, liver, colorectum, and female breast based on evidence on the toxicity of ethanol from epidemiological and animal studies2. Following alcohol intake, the enzyme alcohol dehydrogenase (ADH) oxidizes alcohol to acetaldehyde, which is highly toxic and carcinogenic to cells and tissues, and is detoxified to acetate by aldehyde dehydrogenases (ALDH)3. Thus, gene variants involved in alcohol metabolism and detoxification are also thought to affect the genetic susceptibility to alcohol-associated cancers. Among these, polymorphisms in the ADH1B and ALDH2 genes are known to play a pivotal role in cancer development in combination with alcohol consumption4. The active ADH1B allele results in approximately 40 fold higher metabolism of alcohol to acetaldehyde compared to the inactive allele5. In cases with mutant ALDH2 genes, ALDH2 enzyme activity is suppressed and acetaldehyde metabolism in the liver is impaired; subsequent toxic reactions cause symptoms including facial flushing, nausea, headache, or palpitations after alcohol intake; those with deficient variants of ALDH2 genes are likely to either consume less alcohol or be highly addicted7.Alcohol consumption is a major risk factor that adversely affects the health of people worldwide2; however, the gene variants involved in alcohol metabolism or detoxification have only been inconclusively associated with breast cancer risk9. Considering increases in both alcohol consumption in women10 and incidence of breast cancer and related mortality in Asian countries11, in combination with the higher distribution of variant genes involved in alcohol metabolism or detoxification in the Asian population12, new studies exploring the associations and interactions among alcohol consumption, related ethanol-metabolizing genes, and breast cancer are warranted.In general, epidemiological and animal studies have consistently correlated high alcohol intake with increased breast cancer risk in womenADH1B (rs1229984) and ALDH2 (rs671), alcohol consumption, and their combined effects and breast cancer risk in an East-Asian population.Thus, in the present study, we investigated the association among genetic, functionally established single nucleotide polymorphisms (SNPs) in The general characteristics of the patients with breast cancer from the National Cancer Center (NCC) and age-matched controls from the Korean Genome Epidemiology Study (KoGES) are described in Table ALDH2 rs671 polymorphism, ADH1B rs1229984 polymorphism, and breast cancer risk. Compared to the non-drinkers, the ever drinkers and those who consumed 12 g alcohol or more per day exhibited increased association with breast cancer . The rs671 genotype, although unassociated with breast cancer in an adjusted model, was significantly associated with breast cancer risk after the model was additionally adjusted for daily alcohol intake in patients with a GA or AA genotype compared with those presenting a GG homozygote  and per increment of A . The ADH1B rs1229984 polymorphism did not exhibit any significant association. The combined effects of the ALDH2 rs671 and ADH1B rs1229984 polymorphisms are presented in Appendix Table 2. No significant association was observed between the combination of the two polymorphisms and breast cancer risk.Table ALDH2/ADH1B polymorphism. In those homozygous for the GG allele of rs671, the effects of ever consuming alcohol or daily alcohol intake were associated with increased risk of breast cancer . In terms of the rs1229984 variant of the ADH1B gene, those homozygous for TT exhibited a significantly increased risk of breast cancer in both ever drinkers and those who daily consumed >12 g day alcohol ; however, in women with a TC or CC genotype, no association was observed between alcohol consumption and breast cancer. When we combined the ALDH2/ADH1B polymorphisms, those homozygous for the major allele for both rs671 and rs1229984 (GG and TT) presented increased risk of breast cancer in ever drinkers  and those who consumed 12 g alcohol or more per day .Table P-value = 0.001 for ever alcohol drinkers; OR = 1.53, 95% CI = 1.08–2.18, P-value = 0.018 for those consuming ≤12 g ethanol/day; OR = 2.87, 95% CI = 1.52–5.40, P-value = 0.001 for those consuming>12 g ethanol/day, compared with non-drinkers). Nevertheless, polymorphisms in the rs671 gene were significantly associated with breast cancer only in postmenopausal women with dominant alleles  and in the additive model , but not for premenopausal women.Stratification by menopausal status revealed that alcohol consumption was significantly associated with breast cancer in premenopausal women  and an increment in the A allele increased breast cancer risk by 1.14-fold .After investigating 2143 breast cancer cases and 3977 controls, the polymorphism in the ALDH2 gene (rs671) increase the risk of breast cancer independently, after adjusting for other covariates such as alcohol consumption, in Asian women, and in particular in postmenopausal women. In this study, both ever consuming alcohol and high dose alcohol consumption (>12 g/day) increased the risk of developing breast cancer. However, the association between alcohol consumption and breast cancer was more prominent in those with ADH1B His/His or those that were homozygous for the major allele in both ADH1B and ALDH2.This is the first study suggesting that variants of the 13. Previous studies have reported inconsistent results, wherein they evaluated whether menopausal status modifies the effect of the association between alcohol consumption and breast cancer risk14. In the present study, this association was observed only in premenopausal women. A possible explanation for the nonsignificant result in postmenopausal women could be that fewer women ever consumed alcohol or high alcohol content drinks; thus, the number was insufficient to statistically power our results. Considering that several studies suggest a modified effect of known genetic and environmental factors such as MPO gene polymorphism, family history, obesity, or alcohol use by menopausal status15, a detailed approach stratified by menopausal status with larger sample size is warranted.The association among ever consuming alcohol, high alcohol consumption (>12 g/day), and breast cancer in all women observed in the present study was in accordance with that in previous studies and reviewsADH1B and ALDH2 are the major enzymes involved in alcohol metabolism in humans. The role of the ALDH2 gene has been widely studied in East-Asian countries because of the low prevalence of its variants in European populations. It has been reported that variants of the ALDH2 gene are correlated with an increased susceptibility to numerous cancers, particularly of the upper digestive organs, such as esophageal or gastric cancer17; however, for other cancer types, the results were generally nonsignificant or inconsistent18. For breast cancer, only three studies have investigated the role of the ALDH2 gene (rs671); however, none of them reported a significant association. In all three studies, the controls were recruited from women who visited the same hospital as the patients21. Although controls did not have cancer or other systemic diseases, and the proportion of alcohol consumption was in accordance with that in our study population, their inclusion may not substitute for the general population due to Berkson’s bias, a type of selection bias22. In our study population, the controls belonged to a population-based cohort, presumably free of Berkson’s bias, thus indicating a better reflection of females in general, and even after adjusting for alcohol consumption, polymorphisms in the ALDH2 gene independently increased breast cancer risk. The association between ALDH2 gene variants and breast cancer was validated in independent cases and all sampled control populations. In addition, compared with the previous studies in which the number of breast cancer patients and controls was less than 50021, the present study has a higher number of patients and 1:3 matching was used to increase the statistical power. In combination with the possible side effects of paclitaxel, a chemotherapeutic agent, due to alcohol’s role as a solvent23 women with the GA/AA genotype of ALDH2 would be highly susceptible to not only breast cancer but also side effects of breast cancer treatment, and are therefore a population with a potentially high risk for delterious health effects.ADH1B action results in higher blood acetaldehyde levels, which can increase DNA damage and lead to cancer24. In people with ADH1B His/His, the activity of ADH1B is enhanced, followed by rapid accumulation of acetaldehyde25. In epidemiological studies, ADH1B polymorphism has been associated with tumors of the upper digestive organs4 including esophageal cancer26. In terms of breast cancer, ADH1B itself did not present any association in most previous studies 28. One study reported a marginally protective association for ADH1B IVS1 + 896 A > G with breast cancer risk; however, the same SNP considered in this study did not exhibit any association29. Another study reported an interactive effect of ADH1B and alcohol consumption on breast cancer risk27. In the present study, although we did not identify a significant association between ADH1B polymorphism and breast cancer risk, a decreasing trend was observed in those with the C allele, in particular the homozygotes of the C allele, which was consistent with previous studies29. Another study reported that the effects of the ADH1B and ALDH2 genes were mediated via alcohol consumption and decreased hepatocellular carcinoma risk30, which was not consistent with a previous meta-analysis16. In a study by Lie et al., the population included chronic hepatitis B carriers and the effect of alcohol was higher30 than seen in the present study or other studies conducted in the Korean population31. Considering the subgroup of women with possibly higher alcohol susceptibility, the effects of the ADH1B and ALDH2 genes on alcohol drinking behavior and breast cancer risk might require further investigation.ALDH2 and ADH1B polymorphism status, despite the presence of variants and alcohol intake, specifically a high dose of alcohol intake, increased the risk of breast cancer. Among the variants studied, those with a TT genotype for rs1229984 exhibited higher risk of breast cancer in ever drinkers or in those who consume more than 12 g of ethanol per day. These results are contradictory to those from a study in German women, wherein a reduction in breast cancer risk associated with 12 g or more alcohol consumption per day was observed27. The frequency of the His/Arg allele of ADH1B gene varies among populations, specifically the Asian and European populations32, and is based on diet or other lifestyle factors; moreover, other interacting genes may presumably lead to different results between studies.In the present study, the association between alcohol consumption and breast cancer risk stratified by ADH1B and ALDH2 present strong association with alcohol dependence. People with the A allele of rs671 display less alcohol dependence, possibly owing to the unpleasant effects of a higher flush response33, and those with the rs1229984 His allele also show a decreased risk of alcohol dependence, especially in Asian populations34. A genome-wide association study for alcohol consumption revealed that the rs671 A allele was associated with decreased prevalence of drinkers and number of drinks, and that the rs1229984 T allele was associated with fewer drinks but not with drinking status35. In this study population, women who carried the GG allele of rs671 consumed alcohol with >12 g ethanol/day more often compared to those with the GA/AA genotype , as in previous studies35; however, their distribution was similar regardless of rs1229984 genotype. Previous studies included both men and women 35, and sex-differences in terms of alcohol intake pattern35 and alcohol metabolism may affect inconsistencies in the results.It has been reported that both ADH1B and ALDH2 may reduce the effects of alcohol and its toxic metabolites such as acetaldehyde, the effects of alcohol drinking such as intoxication and the recovery period may result in increased alcohol exposure.Women who carried both the GG and TT genotypes for rs671 and rs1229984, respectively, presented a higher risk of breast cancer when they had ever consumed alcohol or more than 12 g ethanol per day, suggesting that for those with increased activity of the enzymes involved in alcohol metabolism and detoxification, the effects of alcohol consumption on breast cancer risk are increased. In these populations, alcohol would be eliminated faster than in other populations. Although highly active ALDH2 gene and breast cancer in postmenopausal women and no association in premenopausal women20, our results reveal an increased association between the Lys (A) allele of rs671 and breast cancer in postmenopausal women. The previous study included few premenopausal women, only 226 cases and 209 controls and only 60 cases of postmenopausal women carrying the Lys allele20; this may explain the lack of association. Regarding the ADH1B gene, other studies have demonstrated no significant association with breast cancer in postmenopausal women36 and those aged 5027, and these observations were consistent with our results.Although a study conducted in Korea revealed marginally decreased association between the Lys allele of the ALDH2 and ADH1B genes is comparable with that in the general population37. Second, recall-bias could affect the results for measurements of alcohol consumption and other covariates. Some patients were recruited while receiving treatment, which may also affect the results. Third, the effects of other genes related to alcohol metabolism, such as Cytochrome P450 2E1, were not considered. Fourth, although this study included a larger sample size compared with previous studies on the association between ALDH2, ADH1B, alcohol consumption, and breast cancer, the statistical power may still be limited for elucidating gene–gene and gene–alcohol consumption interactions, and stratified analysis. In addition, some of the non-significant results from our subgroup analysis could be attributed to the low power of this study. Fifth, some variables that may affect both alcohol intake and breast cancer such as diet and exercise, were not considered because of limited available information and may have been incompletely adjusted for.This study has certain limitations. First, the existence of selection bias should be considered. To increase comparability, cases and controls were individually matched by age. Although external validity may be limited, the prevalence of variants in ALDH2 gene and breast cancer in Asian women with results validated from a large number of cases and controls. Alcohol consumption increased breast cancer risk, but for cases homozygous for the major alleles of ADH1B or/and ALDH2 polymorphisms, the increment was more prominent, suggesting that this was a high-risk population for breast cancers associated with alcohol consumption. Further studies with a larger sample size could help confirm the association between alcohol intake and gene interaction and breast cancer risk.This study is the first to reveal an independent association between variants of the in situ breast cancer, as confirmed by histology, at the NCC in Korea between February 2013 and October 2016. Healthy controls were selected from the KoGES, a population-based cohort constructed by the National Research Institute of Health (NIH), and the Centers for Disease Control and Prevention of Korea. Full details of the KoGES are described elsewhere38. Female control participants with a history of cancer were excluded, and 3986 women were considered for the study. Among these, 3977 controls provided information on alcohol consumption. All participants were from Korea, and written informed consent was obtained before enrolment. This study was approved by the National Cancer Center Institutional Review Board (IRB No. NCC 2015-0177). All procedures were performed in accordance with the Declaration of Helsinki.In total, we selected 750 women recently diagnosed with either invasive or All cases and controls were asked to complete a set of interviewer-assisted questionnaires on demographics, medical history, reproductive factors, and lifestyle . The subjects were questioned about alcohol consumption, their present drinking status, as well as the total duration of alcohol consumption for both present drinkers and those with a history of alcohol intake. In addition, the type of alcoholic beverage, weekly intake frequency, amount of alcohol consumed on each occasion, and serving sizes were evaluated. The subjects were classified according to drinking status (never and ever). Among ever drinkers, subjects were further classified according to alcohol intake: ≤12 g pure alcohol/day and >12 g pure alcohol/day based on the amount of ethanol in a standard drink.40. Of the 750 breast cancer cases and 3977 controls, 5 cases were excluded owing to a low call rate of <97% (n = 1) and family relationships (n = 4); after quality control, 745 cases and 3977 controls were evaluated. SNPs were excluded for low quality based on SNPolisher analysis , low call rate <95% in either the cases or controls (n = 371), deviation from Hardy–Weinberg equilibrium  in the controls (n = 713), and monomorphic characteristics in our population . In total, 688,196 markers were evaluated for quality control.Genomic DNA was extracted from peripheral blood leukocytes and genotyped using the Korea Biobank Array with 833,000 SNPs. The Korea Biobank Array is a chip optimized for genetic studies in the Korean population. Full details on Korea Biobank Array are described elsewhereALDH2 and ADH1B genes, respectively, were analyzed. The cases and controls were individually matched by age at a 1:3 ratio, and ultimately 623 breast cancer cases and 1845 controls aged 40 or above were included in the final analysis. Discrepancies between the evaluated cases and controls were 0% and 0% for rs671 and 0% and 0.6% for rs1229984, with a P-value of 0.922. These SNPs had minor allelic frequencies of 15.5% and 25.2% and met the Hardy–Weinberg equilibrium in the control samples.The SNPs rs671 and rs1229984, in the ALDH2 polymorphism and breast cancer. Demographic and lifestyle information, including on alcohol intake, was unavailable for these patients. Peripheral blood DNA was genotyped using an Affymetrix Genome-wide Human SNP Array 6.0 . As ALDH2 (rs671) was not included in the array, genotype imputation for rs671 was performed. The SHAPEIT (v2.r837) was used for prephasing, and imputation was performed using IMPUTE2 (2.3.2). Data from the 1000 Genome Project phase 3 East-Asian Ancestry sample (n = 504) were applied as a reference panel. The quality control process and imputation was conducted as follows: among all subjects, those with a sex mismatch (n = 3) were excluded. No subjects showed a low call rate or family relationships. SNPs were excluded in cases of low call rate <95% , deviation from Hardy–Weinberg equilibrium  in the controls (n = 897), or monomorphic traits in the population (n = 1710). In total, 513,738 markers were subjected to quality control. After imputation, the quality score was R2 = 0.57. The age distribution of the 2143 breast cancer patients and 3977 controls is presented in Appendix Table Data from 2143 breast cancer patients who were recruited as part of the KoGES were evaluated according to their sex, age, and genotype to validate the association between The relationship between alcohol consumption and breast cancer risk was assessed with a simple logistic regression and a multiple logistic regression adjusted for age at menarche, age at first full term pregnancy, age at menopause, body mass index, smoking status, and rs671/rs1229984 genotype. The relationship between rs671 genotype, rs1229984 genotype, and breast cancer risk was additionally assessed with a simple and a multiple logistic regression adjusted for age at menarche, age at first full term pregnancy, age at menopause, body mass index, smoking status, and alcohol consumption. To assess the association between each SNP and breast cancer, the codominant, dominant, and additive models were considered. In addition, the combined effects of rs671 and rs1229984 genotype on breast cancer were assessed.ALDH2 and ADH1B genotypes and alcohol intake status were collectively studied to evaluate their combined effect on breast cancer risk. Stratified by ALDH2, ADH1B, and a combination of ALDH2/ADH1B genotypes, the association among alcohol consumption itself, daily alcohol intake, and breast cancer was evaluated with odds ratio (OR) and P-value. In addition, after stratification by menopausal status, the relationship among alcohol consumption, rs671, and/or rs1229984 genotypes and breast cancer risk was assessed.The ALDH2 polymorphism and breast cancer risk, the OR and P-values were presented, and adjusted for age between 2143 breast cancer cases and 3977 controls  from KoGES. These analyses were performed with PLINK v.1.0741 and R statistical software version 3.2.2 .To validate the association between Informed consent was collected for all cases and controls. This study was approved by the National Cancer Center Institutional Review Board (IRB No. NCC 2015-0177).Supplementary Table 1-3.


A 79-year-old female with a past medical history of multiple myeloma, diabetes mellitus and chronic kidney disease presented to the hospital with generalized weakness. The patient was found to have a firm abdominal wall mass with no underlying skin changes or drainage on physical examination. Computerized tomography of the abdomen and pelvis without contrast revealed a large mass-like structure in the anterior abdominal wall in the subcutaneous region extending into the abdomen through the mesentery with juxtaposition and partial involvement of the left hepatic lobe and distal stomach. Solitary extramedullary plasmacytomas are extremely rare tumors that occur outside of the bone marrow in the absence of systemic involvement. This case reports details the finding of an extramedullary plasmacytomas, which accounts for less than 5% of plasma cell neoplasms. Out of all the hematologic malignancies in the United States, multiple myeloma accounts for approximately 17% . MultiplExtramedullary involvement is an uncommon occurrence in multiple myeloma with studies citing findings of extramedullary plasmacytomas (EMP) in 7% at diagnosis, and 6% incidence later ,6. ExtraWe present a case of a 79-year-old female with a chief complaint of abdominal pain ongoing for two months. The pain was described as ongoing, increasing in intensity, non-radiating, generalized, and diffuse with no other associated symptoms. She also complained of generalized weakness during this time. The patient did not report fever, chills, weight loss, night sweats, headache, chest pain, dysuria, melena, diarrhea, or constipation. She reported a medical history of multiple myeloma diagnosed 2 years ago and currently in remission, chronic kidney disease, diabetes, and an episode of complicated sigmoid diverticulitis. She reported a surgical history of subtotal colectomy with ileorectal anastamotic reconscrution, ventral hernia repair and left nephrectomy. The physical exam demonstrated an abdominal wall mass that was firm with no overlying skin changes or drainage.Blood work found a leukocytosis of 15,800 leukocytes per microliter and a hemoglobin of 8.8 g/dL. Liver function was preserved with an alkaline phosphatase of 128 U/L, alanine transferase and aspartate transferase of 15 U/L and 23 U/L, respectively, total protein of 6.7 g/dL, and albumin of 3.4 g/dL was done given the hepatic involvement seen on tomography.Computed Tomography of the abdomen and pelvis without IV contrast reported a large mass-like structure was seen in the anterior abdominal wall in the subcutaneous region extending into the abdomen and through the mesentery with juxtaposition and partial involvement of the left hepatic lobe and distal stomach. The mass measured 17.9 x 15.9 x 8.5 cm and was of soft tissue density with circumscribed margins, 38 hounsfield units, without adjacent inflammatory changes. Additional masses were seen abutting the posterior right hepatic lobe and spleen, and there was a prominent mass in the mid-mesentery figure .A core needle biopsy performed by interventional radiology confirmed the presence of monoclonal CD138+ plasmacytoid cells consistent with a plasma cell neoplasm.Medical oncology was consulted and arranged for outpatient follow up with positron emission tomography to evaluate for active areas of relapse and to begin a triple therapy regimen with daratumumab, pomalidomide and dexamethasone.Multiple myeloma (MM) is a malignant process that involves a neoplastic proliferation of plasma cells characterized by bone marrow involvement, extensive skeletal destruction, renal dysfunction, and elevated total serum protein levels. As a systemic illness, this disease typically presents with systemic symptoms including anemia, bone pain, elevated renal function tests, and weight loss in the majority of patients.Extramedullary involvement is uncommon, with studies citing findings of extramedullary plasmacytomas (EMP) of 7% at diagnosis, and 6% incidence later in the course of multiple myeloma ,6,7,8. EA subtype of extramedullary disease is cutaneous multiple myeloma and has been associated with particularly aggressive biological behavior and short survival . Common Diagnosis of extramedullary plasmaycotoma is confirmed through needle biopsy of the affected tissue with histopathology demonstrating plasmacytoid cells and immunohistochemistry staining for CD138+. As the presence of extramedullary plasmacytomas in multiple myeloma, both at diagnosis and later during the disease course, has been associated with poor outcomes, extramedullary recurrence warrants an escalation of therapy with proteasome inhibitor therapy found superior to that of thalidomide alone . In certIn our report, we provide evidence of the need to consider a broad differential and to evaluate for recurrence when assessing solid masses in the setting of known hematologic malignancies.The presence of an extramedullary mass is particularly concerning for patient prognosis in the setting of known hematologic malignancy. Consideration of extramedullary plasmacytoma in patients who present with subcutaneous masses demonstrated on exam and with a history of multiple myeloma should raise a high index of suspicion for recurrence and treatment failure. The findings warrant prompt subsequent histologic and immunohistory chemistry confirmation with biopsy, genotyping and escalation to 2nd and 3rd line agents in treatment. Further studies are necessary to determine the optimal course of treatment for patients with this particular presentation given the known and previously documented poor prognosis.


Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms. Ontogenetic shifts are common in nature , and canOntogenetic shifts are well-known in the venom composition of animals such as snakes ,17,18,19Crotalus oreganus ssp.) suggested that protease activity in venoms begins to increase between 50 and 60 cm in snout–vent length (SVL) [Crotalus adamanteus there is evidence for a shift in venom composition at a length of 102 cm, the size that indicates sexual maturity in the species [Studies on both the evolution and biomedical application of venom often categorize animals as juvenile or adult based on size cutoffs, such as size at reproductive maturity ,33,34,35th (SVL) , while i species . Prior w species ,36, the  species , and theCrotalus horridus) and eastern diamondback rattlesnake  are heavy-bodied pit vipers native to the southeastern United States that reach adult body sizes over 1 m in length. Both species undergo ontogenetic shifts in venom composition [C. adamanteus) and five timber rattlesnakes  including representatives of Type A (neurotoxic) and Type B (hemorrhagic and hemotoxic) phenotypes [The timber rattlesnake ; the other three cases showed evidence of a gradual shift that would take longer than a single active season, although KW1753 (Type A C. horridus) was more rapid than others , but this individual also showed a rapid change , we likely missed between ⅓ and ½ of the ontogenetic shift experienced by MM0027, suggesting that earlier sampling may have led to detecting a gradual shift in this animal.We detected statistically significant ontogenetic shifts in overall venom composition in all individual snakes in this study using permutational mutivariate analysis of variance (PERMANOVA) , Table 1C. adamanteus and both venom types of C. horridus occupied unique areas of venom phenotype space, and the major directions of within-individual variation in each species (or venom type) tend to have a similar pattern of orientation. For example, within-individual variation in the three C. adamanteus is oriented largely along NMDS axis 2, whereas type B C. horridus venom varies along NMDS axis 1. Meanwhile, the two type A C. horridus vary over an apparently smaller overall space, and that variation is oriented diagonally along We used non-metric multidimensional scaling of binned high performance liquid chromatography (HPLC) retention time blocks to allow between-individual comparison and visualize the position of all samples from all snakes in a single multivariate space . Such coThe clear implication of these analyses is that ontogenetic shifts in the complete venom profile are not wholly discrete processes. Instead, the change in composition from a neonate to adult venom tends to occur across a longer period of time. It is important first to acknowledge our small overall sample size of eight individual snakes (two species) as a limitation in our ability to generalize our findings as representative of each species, and particularly to rattlesnakes as a group. However, we are confident in the power of our repeated samples to provide useful information about the individual with high resolution, and similar gradual shifts across those individuals over which we had the most experimental control encourages the view that gradual ontogenetic shifts are commonplace and worth further exploration. The passing of time, which is likely a proxy for increases in body size, led to increasing Aitchison distance from the neonate baseline phenotype with 90% of observed change being completed in 213–989 days . Post-hoC. adamanteus, as proposed by Margres et al. [The slow tempo of compositional change over time and body size in at least half of our samples challenges the notion that ontogenetic shifts occur discretely in s et al. . There as et al. . First, s et al. ,34. The s et al.  used a fGradual shifts in overall venom composition could result from many gradually shifting peaks, or multiple discretely shifting individual peaks whose shifts occur at different time points. Our repeated sampling within the same individuals permitted peak-by-peak analysis of compositional shifts, allowing an estimate of the overall percentage of venom proteins that shift expression levels and determine if both gradual and discrete modes of expression variation operate among venom genes. These analyses showed that ontogenetic shifts involved large numbers of venom proteins, with between 55% and 89% of the peaks changing significantly in relative abundance over time . We founC. adamanteus and C. horridus described by Wray et al. [The repeated-samples nature of our study allowed for the control necessary to call peaks in a highly standardized fashion for each individual, providing the resolution to visualize slow shifts in peak abundance over time and classify their mode of shift based on our statistical framework. Previously, proteomic analysis of y et al.  identifiMargres et al.  discusseR2 = 43%, p = 0.01; KW1576: R2 = 55%, p = 0.0003; KW1753: R2 = 18%, p = 0.04; KW1726: R2 = 62%, p = 0.001; MM0071: R2 = 23%, p = 0.07; KW1594: R2 = 15%, p = 0.18). These significant correlations underscore the patterns of maturation in both these snake species, where both testosterone concentrations and venom distance from the juvenile venom phenotype increase as snakes mature. We expected both venom to change and testosterone to increase into adulthood, as ontogenetic changes in venom composition are known from both species studied [Our results do not meet the prediction of a strong linear relationship between the change in venom composition and the testosterone concentration across the course of our study , and a h studied ,40,45 as studied . As a re studied ,48,49,50 studied . TherefoThe proximate physiological causes of intra-individual venom variation have received very little attention, despite the potential of such information as a tool in both studies of venom gene regulation and in proper care of animals used in venom production for medical uses. Our investigation of testosterone as a possible physiological trigger of venom variation is among the first to assess the proximate factors inducing the extreme changes seen in animal venoms ,51. GeneC. horridus , where there were significant differences in diet composition between both the 2nd and 3rd body size quartiles (p = 0.006), as well as between the 3rd and 4th , although failure to detect differences among smaller size classes could be due to low power . The 4th size quartile present in the diet study actually lies outside the body size range of the snakes whose venom we sampled in this study, meriting examination of the taxon-specific patterns of abundance variation in the 1st through 3rd size quartiles. There is a propensity toward the consumption of larger prey taxa over time in C. adamanteus, evidenced by the C. adamanteus diet exclusively (100%) consisting of mice and rats at the 512–782 mm range whereas rabbits and squirrels appear in the 2nd size quartile (783–1053 mm), and rabbits double in abundance between the 2nd and 3rd quartiles.We found evidence that ontogenetic changes in diet reported in prior studies occurred across body size in n nature , with siEchis vipers and Sistrurus rattlesnakes [C. adamanteus and C. horridus may be expected to predict the mode and tempo of venom ontogeny, but like the mode and tempo of venom shifts, the nature of dietary variation is complex, with the overall nature of these shifts underscored by apparent gradual and discrete shifts in the abundance of individual prey types. Our ability to link dietary shifts to the mode of venom shifts is likely limited by the differential scope of the data collected, as we compare range wide diet data with venom sampled from individuals from a single region in the southeastern U.S. Given the evolutionary lability of snake venoms such as those of C. adamanteus [As dietary breadth has been previously shown to impact venom composition in taxa such as lesnakes ,12, the amanteus , it may amanteus ,53,54, aamanteus .Ontogenetic shifts in snake venom are more the rule than exception, changing the functional profile of venom in both biologically and biomedically important ways ,27,55,56Bothrops atrox) provides a demonstration of the utility of considering body size information when making inferences about venom ontogeny [Crotalus to Bothrops leads us to hypothesize that these animals may have simply had the most opportunity for greater compositional change in venom, whereas the larger animals were already close to their terminal adult venom phenotype at first collection.We therefore suggest that one of the most important implications of our work is the need to record more detailed life history data, particularly body size, as venom is collected. The only comparably long-term study to ours involving another large pit viper  were obtained from three C. adamanteus and five C. horridus. Individuals were either originally caught in Florida or Georgia . We then loaded 15 μg of venom protein onto a Prominence HPLC apparatus . Samples were run through a Phenomenex Aeris widepore column  maintained at 25 °C. Samples were eluted with solution A as 0.1 mM TFA in water and solution B as 0.06 mM TFA in acetonitrile, and absorbance at 220 nm was measured over 150 min with the following run parameters: 5 min at 10% B, 110 min increasing from 10 to 55% B, 5 min increasing from 55 to 75% B, 5 min at 75% B, 5 min decreasing from 75–10% B, and 5 min at 10% B. Venom peak areas were quantified using Shimadzu Lab Solution v2 software, combining default peak calls by the iPeakFinder algorithm with manual curation of the peak baseline and filtering of peaks making up less than 0.1% total area as noise. Peaks were called uniquely per individual snake, by aligning the serial HPLC profiles from all samples from a single individual and calling peaks that were present. The relative abundance of HPLC peaks in each sample will be henceforth referred to as venom composition.n = 9); for these samples, 6.5 μL of serum was extracted as above and redissolved in 71.5 μL of assay buffer and were run singly. All other samples were run in duplicate, and the values of each duplicate averaged for use in analysis. Samples falling below the limit of detection (n = 5) were set at the lower limit of detection for analysis (0.001 ng/mL). The mean intra-assay coefficient of variation was 1.96 ± 1.74% and the inter-assay coefficient of variation was 4.7%.We used a commercially available EIA kit  to measuC. horridus [C. adamanteus [C. horridus fed on shrews, mice, voles, chipmunks, rats, squirrels, and rabbits, while C. adamanteus fed on only mice, rats, squirrels, and rabbits. We tested for differences in the overall proportion of these prey classes between adjacent size class quartiles using chi-square tests.We accessed previously published diet studies for both horridus  and C. aamanteus  to assesamanteus ,64. In oAll statistical analyses were performed in R v. 3.6.0 . Venom pUpon detecting significant shifts in the overall venom profile, we followed with modeling the clr transformed abundance of each peak versus time. This peak-by-peak analysis provided a post-hoc test of which peaks varied in abundance significantly over the course of the study and their mode of change, where mode  was determined as above using linear and non-linear regression. Non-linear fits were again obtained using the nlsLM function from the minpack.lm package .Last, we determined whether testosterone concentrations were closely associated with the change in venom composition as snakes grew via linear regression. Given the correlative nature of these tests, the best evidence for a potential role of testosterone would be a particularly strong linear relationship between the change in testosterone concentration between the focal and initial reads and the change in venom composition as measured by Aitchison distance. Other forms of a relationship would be inconclusive, as testosterone is expected to be higher in adult than juvenile snakes.


Allergy to olive pollen is one of the primary causes of allergic asthma in Spain. Even though allergen immunotherapy (AIT) has shown clinical benefits in patients sensitized to different allergens, studies in asthmatic patients sensitized to olive pollen are insufficient.To assess the effectiveness and safety of an ultra-short course of AIT with an L-tyrosine-adsorbed and monophosphoryl lipid A-adjuvanted olive pollen and olive/grass pollen extract (Pollinex Quattro®) in patients with allergic asthma in the real-world setting.Retrospective, controlled study including patients with asthma, with and without allergic rhinitis, caused by sensitization to olive pollen from 11 centers in Spain. Patients received out-of-season (October–March) treatment with AIT in addition to their pharmacological treatment (active group) or pharmacological treatment (control group). Effectiveness variables, including unscheduled visits to the healthcare center, emergency room admissions, symptoms of asthma and rhinitis , and use of medication to treat asthma and rhinitis during the subsequent pollen season were compared between treatment groups.P = 0.001 and P = 0.006, respectively). Severity and control of asthma symptoms remained unchanged , rhinitis type improved (P = 0.025), and severity remained unchanged in the active compared to the control group. The use of short-acting beta-agonists and inhaled corticosteroids to treat asthma symptoms decreased in the active vs. the control group . Twelve (13.5%) and two (2.2%) patients in the active group experienced local adverse reactions , and systemic adverse reactions (hypertensive crisis and low-grade fever) to AIT, respectively; none was serious.Of 131 study patients, 42 were treated with their usual asthma medication (control group) and 89 were treated with AIT (active group), either Pollinex Quattro® 100% olive pollen  or 50% olive pollen/50% grass pollen . Patients’ demographic and clinical characteristics were similar between groups. The mean (SD) number of unscheduled visits to a healthcare center and emergency room admissions due to allergy symptoms was 2.19 (1.40) and 0.43 (0.63) in the control group, and 1.09 (1.25) and 0.11 (0.51) in the active group (AIT with Pollinex Quattro® specific for olive pollen and olive/grass pollens resulted in reduced visits to the healthcare center and emergency room and the use of asthma medication during the pollen season, indicating that this treatment was safe and effective in treating asthma in patients sensitized to these pollens. Even though these treatments provide temporary control of symptoms, they are unable to alter disease progression.,Even though Pollinex Quattro® is prescribed to treat asthma due to olive pollen allergy, evidence is limited, raising the need for additional clinically relevant data.This was an observational, retrospective, multicenter study including patients with seasonal allergic asthma, with or without rhinitis, caused by olive or grass/olive pollens. Patients aged 5–65 years treated at any of the 11 participating Spanish centers, were assigned to one of the two cohorts of the study according to treatment. Cohort 1, or active treatment group, included patients receiving, besides their usual control medication to treat their allergic disease, allergen immunotherapy (Pollinex Quattro®) for the first time, according to the routine clinical practice, between October 2016 and March 2017, or between October 2017 and March 2018. Cohort 2, or control treatment group, included patients who visited the allergist for the first time after the usual out-of-season period of AIT treatment (October–March) and, therefore, received conventional pharmacological treatment according to the routine clinical practice and served as controls. Patients with poor treatment adherence were excluded from the study. Data were obtained from medical records between September 2018 and July 2019. All included patients and legal representatives of patients <18 years signed a written informed consent before any information was recorded. The study was conducted in accordance with the Helsinki Declaration and the local Personal Data Protection Law; the study protocol was approved by the local Ethics Committee .Pollinex Quattro® is an injectable suspension for subcutaneous administration composed of purified allergen extracts of 100% olive pollen or 50% olive pollen/50% grass pollen, modified by treatment with glutaraldehyde into allergoids, adsorbed onto microcrystalline tyrosine (MCT) and adjuvanted with monophosphoryl lipid A (MPL) at 50 μg/mL. AIT composition was selected according to the routine practice considering cutaneous sensitization, determined using a skin prick-test, detection of specific IgE in serum, anamnesis, and exposure to different pollens according to patients’ region of residence. This AIT is administered outside of the pollen season in four injections (ultra-short regimen) of 1 mL each, containing increasingly higher doses of the allergen .,,,The objectives of this study were to assess the effectiveness (main objective) and safety (secondary objective) of AIT with Pollinex Quattro® in the real-world setting. Regarding effectiveness, the primary endpoint of this study was the number of unscheduled visits to a physician or healthcare center due to allergic disease during the pollen season. Secondary endpoints included the number of visits to the emergency room due to allergy symptoms, classification of asthma (type and severity), and use of medication to treat asthma, measured during the pollen season (2017 or 2018). In patients with allergic rhinitis, the classification of rhinitis symptoms and the use of medication to treat allergic rhinitis were also considered. Asthma was classified according to the “Guía Española para el Manejo del Asma” (GEMA), the Spanish version of the Global Initiative for Asthma (GINA) classification,The sample size was calculated based on the hypothesis that the proportion of patients in the control treatment group who would likely visit a healthcare center due to allergic asthma symptoms during the pollen season, estimated to be 75%, would be reduced by at least 25%  in the active treatment group. With a 2:1 study design, including one patient receiving conventional pharmacological treatment (control treatment) for every two patients receiving AIT (active treatment) (2n in the active group:1n in the control group), and considering that 10% of patients would be non-assessable, a sample of 165 and 85 patients in the active and control treatment groups, respectively, was deemed necessary to detect differences between the two groups with a power of 80% and a significance level of 5%.All analyses were performed based on the sample of assessable patients who fulfilled the selection criteria and presented with data about immunotherapy. Categorical variables were described as frequencies and percentages, and quantitative variables as the mean and standard deviation (SD) and/or the median and range. Categorical variables were compared using the Chi-square test or the Fisher test for subgroups of patients; quantitative variables were compared using the Student's T-test and their non-parametric counterpart, the Mann–Whitney test. In addition to analyses using all assessable study patients, effectiveness variables were compared in patient groups based on the composition of Pollinex Quattro® (50% olive pollen/50% grass pollen vs. 100% olive pollen) and the period of treatment administration (October–December and January–March). The significance threshold for all bivariate analyses was set at a two-sided α = 0.05. All analyses were performed using the statistical package SPSS version 19.P; not significant), with the exception of rhinitis diagnosis and sensitization to molds, with a significantly different prevalence between the control and active groups . Regarding AIT composition, 43 (48.3%) and 46 (51.7%) patients were administered Pollinex Quattro® composed of 100% olive pollen and Pollinex Quattro® composed of 50% olive pollen/50% grass pollen, respectively. The period of administration of AIT was from January to March in 68.5% of patients, and from October to December in the remaining 31.5%.Of the 134 patients recruited for this study, three were non-assessable due to lack of data, resulting in a study population of 131 patients, of which 93 (71%) were adults (>18 years), 23 (17.5%) were adolescents (12–17 years), and 15 (11.4%), were children (5–11 years). Of the 131 assessable patients, 89 were treated with AIT (active group) (mean [SD] age of 31.6 [16.6]) and 42 were treated with their usual asthma medication (control group) (mean [SD] age of 28.8 [14.3]) years. Patients’ demographic and clinical characteristics are summarized in P = 0.001) unscheduled visits to a healthcare center and a mean (SD) of 0.11 (0.51) and 0.43 (0.63) (P = 0.006) emergency room admissions due to allergy symptoms during the pollen season, respectively (P < 0.001 for both comparisons). Consequently, the probability of visiting the healthcare center or emergency room was lower for patients in the active group compared to patients in the control group; healthcare center visits ; emergency room visits .Patients from the active and control groups experienced a mean (SD) of 1.09 (1.25) and 2.19 (1.40)  (P = 0.025) (P = 0.858)  A–B. In c= 0.025) C. Howeve= 0.858) D.Fig. 2DP = 0.001 and P = 0.031). Likewise, the use of long-acting beta-agonists (LABAs), leukotriene receptor antagonists (LTRAs), and oral corticosteroids tended to be lower in the active group, albeit differences lacked statistical significance  and inhaled corticosteroids was significantly lower in the active group  compared to the control group    , GEMA classification of asthma, and use of medication to treat asthma, were similar regardless of composition of Pollinex Quattro® and treatment period. Effectiveness variables were similar between patients treated with a Pollinex Quattro® composition of 100% olive pollen and 50% olive pollen/50% grass pollen (riables) . The proriables) .Table 2EA total of 12 (13.5%) and two (2.2%) patients in the active group experienced 13 local and two systemic adverse reactions (ARs), respectively, none of which was serious. Systemic ARs included hypertensive crisis, which resolved in 20 min without needing any treatment, and low-grade fever. Local ARs included local edema (n = 9), injection site swelling (n = 2), erythema (n = 2), unspecified delayed local reaction (n = 1), pruritus (n = 1), local hives (n = 1), and heat (n = 1) . Of the In this retrospective controlled study including patients with allergic asthma sensitized to olive pollen, with and without allergic rhinitis, we provided real-world evidence regarding the effectiveness and safety of Pollinex Quattro®. Patients treated with AIT experienced a low incidence of adverse reactions and experienced a lower number of unscheduled visits to the healthcare center and emergency room during the pollen season compared to patients receiving their usual pharmacological treatment. Differences in asthma classification revealed a trend towards improvement, albeit not statistically significant, with a relevant concomitant decrease in the use of SABAs and inhaled corticosteroids. Pollinex Quattro® showed similar effectiveness outcomes irrespective of the treatment period and composition.Results from this study, showing that the out-of-season course of four Pollinex Quattro® injections improved allergic asthma during the pollen season, were obtained using different effectiveness variables collected from patients’ medical records. Asthma symptoms, measured using the GEMA classification, showed a trend towards a decrease in the active group, albeit not significant. AIT is contraindicated in patients with severe and uncontrolled asthma and therefore, patients receiving AIT lacked severe symptoms and had good asthma control, likely attenuating the observed effects of AIT. In contrast, the use of SABAs and inhaled corticosteroids to treat asthma symptoms significantly decreased in the active group, while rhinitis medication remained unchanged. According to the routine clinical practice in Spain, rhinitis medication is prescribed in advance for the entire pollen season and doses are not adjusted, unlike medication to treat asthma, which is gradually adjusted according to symptom control. The absence of dose adjustments according to rhinitis symptom control during the pollen season likely explain the lack of differences in use of rhinitis medication between active and control treatment groups. The remaining variables, including unscheduled visits to the healthcare center and emergency room, were, in contrast to the previous patient-reported variables, registered events and therefore objective and robust measures of treatment effectiveness. Both variables significantly decreased in the active vs. the control group, with a concomitant decrease in their associated odds ratio. To our knowledge, this is the first study in patients sensitized to olive pollen assessing unscheduled visits to the healthcare center and the emergency room, as previous studies assessed other immunological and clinical parameters.35,,,Similarly to previous studies with Pollinex Quattro®, effectiveness outcomes remained unchanged regardless of the treatment period (October–December vs. January–March), allowing a flexible out-of-season administration throughout an extended period of time.H1 pathway of the unspecific immune response, enhancing the immunogenicity of the allergoid.,The administration of Pollinex Quattro in only four out-of-season injections promotes treatment adherence and, despite the ultra-short course, this AIT showed a good effectiveness profile during the following pollen season. The unique formulation of Pollinex Quattro® in an allergoid by chemical modification with glutaraldehyde and combined with two different adjuvants, MCT and MPL, enables its ultra-short injection course. MCT slows the distribution of the allergoid after the subcutaneous injection and acts as a biodegradable adjuvant, while MPL stimulates the T,,,,,,While specific guidelines for the use of AIT to treat allergic rhinitis symptoms have been developed, recommendations regarding prescription of AIT in patients with allergic asthma are still controversial, and the European Academy of Allergy and Clinical Immunology (EAACI) is still developing specific guidelines.Results from this study should be interpreted in the context of the limitations associated with retrospective studies, including missing data across variables and time points. In addition to the effect of missing values, the study was smaller than the estimated sample size, further decreasing the data available for analysis and its statistical power. Nevertheless, most of the comparisons yielded statistically significant results. This retrospective study was conducted in the real-world setting and consequently, the number of available variables was limited to those assessed and recorded in the routine practice. Additionally, due to its retrospective nature, daily symptoms and medication use were not registered, potentially resulting in an overestimation of treatment effects. Pollinex Quattro® is generally administered before three-to-five consecutive pollen seasons and therefore, despite its long-term efficacy in previous studies, longer studies are likely necessary in patients sensitized to olive pollen.Our results obtained from patients with allergic asthma, with and without allergic rhinitis, show that AIT specific for olive or for grass and olive pollen is effective and safe in the real-world setting. Despite the short study duration, one course of treatment with Pollinex Quattro® resulted in less unscheduled visits to the healthcare center and emergency room and use of medication during the pollen season, with a low incidence of associated adverse reactions, suggesting that patients sensitized to olive pollen and polysensitized to olive and grass pollen may benefit from this treatment.This work was supported by Allergy Therapeutics Ibérica .All authors have reviewed and approved the final version of the manuscript.All authors contributed to the conception and design of the study, or acquisition of data, or analysis and interpretation of data; all authors critically revised the article for important intellectual content and gave their final approval of the version to be submitted.All included patients and legal representatives of patients <18 years signed a written informed consent before any data was recorded. The study was conducted in accordance with the Helsinki Declaration and the local Personal Data Protection Law (LOPD 15/1999); the study protocol was approved by the local Ethics Committee CEIM/CEI Provincial de .I hereby state that the manuscript, including related data, figures and tables, has not been previously published and has not been submitted to any other journal (in any other language or any other type of publication), either by me or any of my co-authors.The anonymized datasets collected during the current study are available from the corresponding author on reasonable request.The authors declare no conflict of interest.