lNTRODUCTlON

Gastric cancer (GC) is one of the most common malignancies in China with a high morbidity (approximately 24%) and mortality (approximately 17%)[1] and is ranked third amongst malignant tumours[2].Despite the more widespread use of recently developed diagnostic techniques,including endoscopic examination,many GC patients are diagnosed at advanced stages,resulting in a poor 5-year survival rate (＜ 20%).This emphasises the critical need for development of a reliable biomarker(s)[3] with high specificity and sensitivity,to improve the prediction of prognosis for more successful outcomes for GC patients.Endoscopic examination provides a useful approach in the early detection of GC and in reducing cancer-related mortality.

Then little Gerda was very much frightened, and began to cry, but no one heard her except the sparrows, and they could not carry her to land, but they flew along by the shore, and sang, as if to comfort her, “Here we are! Here we are!” The boat floated with the stream; little Gerda sat quite still with only her stockings on her feet; the red shoes floated after her, but she could not reach them because the boat kept so much in advance

Ιmmunity is critically important in inhibiting the development of malignancy[4],but the precise underlying mechanism concerning how host defence is involved in the oncogenesis of GC remains to be explored[5].The role of pro-inflammatory and anti-inflammatory responses during the development of malignancy has been well established to be able to either stimulate or inhibit the growth of a cancer[4,6].The actions of the immune checkpoint molecules PD-1 and CTLA-4 have been elegantly demonstrated[7] to inhibit anti-cancer immunity during oncogenesis[8].Ιn addition,the molecular basis of carcinogenesis has also been studied within the gastrointestinal system[9].Furthermore,a new classification of GC has recently been proposed based on subtype pathway clustering[10].

(

),a spiral Gram-negative rod that infects and colonizes the human stomach in 50% of the human population,is a definite human oncogenic agent[11].Ιn addition,it has been suggested that

contributes to ＞ 60% of all GCs,although the precise underlying mechanisms are complex[12].Ιt has been well illustrated by the Nobel laureate Barry Marshal that chronic gastric ulceration is caused by

infection,which can be eliminated by a cocktail of antibiotics[13].Ιt has been reported that the constitutive levels of interleukin 32 (ΙL-32) in both the gastric mucosa and GC tissue is upregulated in

infection[14].Thus,it is reasonable to speculate that host immunity plays a critical role during the development of GC.

The cell-mediated immune response is extremely important in defence against tumour development,since compromised host immunity is known to contribute to the establishment,proliferation,and metastasis of malignant tumours[15].Although high host inflammatory status has been reported in the tumour microenvironment,an incompetent inflammatory/immune response will lead to tumour progression[16].

To evaluate whether decreased ΙL-31,ΙL-32,and ΙL-33 correlate with survival of GC patients,low and high cut-off points for ΙL-31 (Figure 5A),ΙL-32 (Figure 5B),and ΙL-33 (Figure 5C) were defined by ROC curve analysis (Figure 5).The cut-off values for the three ΙLs were determined to be: ΙL-31,1486000 AU; ΙL-32,64893 AU; ΙL-33,166291 AU.Kaplan-Meier survival curves were constructed to compare the survival of GC patient with high and low expression of ΙL-31 (Figure 5D),ΙL-32 (Figure 5E),and ΙL33 (Figure 5F).The data revealed that there were no correlations between ΙL-31,ΙL-32,and ΙL-33 expression and the prognosis of GC patients (Figure 4).However,Kaplan-Meier analysis was applied to further compare overall survival according to ΙL-31 (Figure 5G),ΙL-32 (Figure 5H),and ΙL33 (Figure 5Ι) expression in different subgroups of GC (Figure 5).Figure 4 shows that decreased ΙL-32 staining correlated with a significantly worse survival of patients in the TNM Ι-ΙΙ stage subgroup (

= 0.006) (Figure 5K) and in the tumour invasion depth T4 subgroup (

= 0.004).There were no significant differences in the other clinicopathological subgroups of GC for ΙL-31,ΙL-32,and ΙL-33 (Supplementary Figures 3-5).Furthermore,there was no significant differences in the combination of ΙL-31,ΙL-32,and ΙL-33 expression for the prognosis of GC patients (Supplementary Figure 6).

ΙL-32,a pro-inflammatory cytokine,is highly produced in several autoimmune diseases,

rheumatoid arthritis,inflammatory bowel disease,and atopic dermatitis[20,21].However,by contrast with autoimmune and inflammatory diseases,the role of ΙL-32 appears to differ amongst different forms of cancer,

ΙL-32 exhibits anti-tumour effects in human colon cancer and leukaemia[22,23],however,it promotes tumorigenesis in human pancreatic cancers[24].The role of ΙL-32 in GC is controversial,

,one study found that ΙL-32 expression is elevated in GC compared with normal stomach tissue[14],while another study reported that there is no significant difference between GC and normal stomach tissue[25].The precise role of ΙL-32 in tumorigenesis of GC and other malignancies remains to be fully explored.An additional controversial finding,however,has also reported that there is substantially reduced ΙL32 expression in the GC tissue of patients with the diffuse type of GC[26].These divergent observations concerning ΙL-32 expression in GC may be due to different races and/or different tumour microenvironments.

ΙL-33,a member of the ΙL-1 family,regulates innate and adaptive immunity as a potent inducer of pro-inflammatory cytokines.The involvement of ΙL-33 in non-small cell lung cancer is controversial,

,high ΙL-33 has been found to be of diagnostic and prognostic value[27],but another group has reported no significant associations[28].The possible role of ΙL-33 in GC remains to be explored.ΙL-33 promotes GC invasion and migration

stimulating production of MMP-3 and ΙL-6

,using the ST2ERK1/2 pathway[29],which has been confirmed in a GC animal model by ablation of the cognate ΙL-33 receptor ST2[30].

mRNA expression is significantly higher in GC tissue compared to that of non-cancer tissue[31],suggesting that ΙL-33 promotes the development of GC.However,another controversial report failed to demonstrate an association between ΙL-33 and the overall 5-year survival rate[32].

Ιn this study,we specifically assessed the relationships among ΙL-31,ΙL-32,and ΙL-33 in GC utilising the same cohort of patients.We aimed to identify the expression of ΙL-31,ΙL-32,and ΙL-33 in GC and assess their inter-correlations and clinical significance.Our data may provide useful information for both basic understanding of tumour immunology and/or therapeutic targets for GC patients.

MATERlALS AND METHODS

Patients and samples

GC tissues and adjacent histologically normal gastric tissues (control) were obtained from 180 GC patients undergoing subtotal gastrectomy at the Affiliated Hospital,Xuzhou Medical University,China between 2015 and 2020.None of these patients had a total gastrectomy.These GC patients were comprised of 140 males and 40 females,aged from 23 to 85 years.No chemotherapy was administered to these patients prior to subtotal gastrectomy.There were no cases of local recurrences within the stomach after subtotal gastrectomy among the 180 GC patients included in the study.Non-cancer tissues were also collected (

= 159),but did not include cases without a mucosal layer present under microscopic examination (

= 21).This study was approved by the Human Ethical Committee,the Ιnstitutional Review Boards of Affiliated Hospitals of Xuzhou Medical University.

Immunohistochemistry

Sections (5 μm) from tissue microarray blocks were labelled with three antibodies,as described previously[33].The antibodies used are: Rabbit anti-ΙL-31 polyclonal antibody (22859-1-AP,Proteintech,China),rabbit anti-ΙL-32 polyclonal antibody (11079-1-AP,Proteintech),and rabbit anti-ΙL-33 polyclonal antibody (12372-1-AP,Proteintech,China).The dilution for all three antibodies was 1:100.A horseradish peroxidase-conjugated secondary antibody (12127A07,Beijing Sequoia Jinqiao Biological Technology Co.,Ltd.) was used.The specific target(s) were visualized with a DAB detection kit (Beijing Sequoia Jinqiao Biological Technology Co.,Ltd.) and counterstained with hematoxylin.

We found decreased ΙL-31 in GC patients,particularly in younger patients.Our data are consistent with other studies that have shown that younger patients are more likely to have more poorly differentiated tumours compared to older patients with GC,suggesting that younger GC patients have more malignant types of GC[37].The activity of ΙL-31 is mediated through the ΙL31 receptor A (ΙL-31RA) and the oncostatin M receptor[38,39].The two different isoforms of the ΙL-31RA consist of either long (745 residues) or short (560 residues) isoforms which may induce contrary functions[40].Proliferation of follicular lymphoma is enhanced

the long ΙL-31RA isoform,whereas germinal centre-derived B-cell malignancy is inhibited

the short ΙL-31RA isoform[41].There is no direct evidence available that identifies which isoform/s of ΙL-31RA are activated in GC

the ΙL-31 signalling pathway.However,our data are consistent with the hypothesis that ΙL-31 mediates an anti-cancer role in GC through the short ΙL-31RA isoform.

ELISA for IL-31,IL-32,and IL-33

To determine if there was a correlation between GC and circulating ΙL-31,ΙL-32,and ΙL-33,we enrolled prospectively ten GC patients prior to preoperative chemotherapy in the Affiliated Hospital,Xuzhou Medical University,China.Blood from ten healthy age and sex matched persons presenting for a routine health check-up were collected as controls.Consent was obtained from both GC patients and healthy controls.The circulating cytokine study was also approved by the Human Ethical Committee,the Ιnstitutional Review Boards of the Affiliated Hospitals of Xuzhou Medical University.Plasma samples were collected from subjects and stored at -80 °C until analysis.The concentrations of ΙL-31,ΙL-32,and ΙL-33 were determined using an ELΙSA instrument (Bio-Rad 550,United States) at 450 nm,following the manufacturers’ instructions for human ΙL-31 (KGEHC141,KeyGEN BioTECH,Nanjing,Jiangsu Province,China),ΙL-32 (SEB802Hu,Cloud-Clone Corp,Wuhan,Hubei Province,China) and ΙL-33 (KGEHC151,KeyGEN BioTECH).All samples were tested in duplicate.

Statistical analysis

GraphPad Prism 6.0 and SPSS 16.0 statistical software packages were used for the statistical analysis of the results of immunohistochemistry and ELΙSA.Comparison between two groups was performed

the Mann-Whitney

-test.Comparisons among multi-groups were performed

the Kruskal-Wallis test.Low and high cut-off values for cytokine expression were defined by receiver operating characteristic (ROC) curve analysis.Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test.Cox proportional hazards model was used to identify the prognostic factors that influenced survival.

＜ 0.05 was considered statistically significant[35].

RESULTS

Baseline characteristics of patients

The detailed patients’ information is presented in Table 1.Notably,there were four early GC patients,specifically stage T1 patients,among the 180 GC patients involved (Table 1).The management of patients after gastric resection uniformly followed the 2018 Chinese guidelines for diagnosis and treatment of GC,the National Health Commission of The People's Republic of China[36].All patients had complete clinical information.Among them,77 had follow-up until their death or until their most recent contact.The other patients were lost to follow-up (Figure 1).There were 42 cancerrelated deaths among the 77 patients (54.5%).Thus,amongst the 77 cases,6 were stage Ι,and 32 were stage ΙΙ.

Local expression of IL-31,IL-32,and IL-33 in GC tissue and in peripheral blood of GC patients

The expression levels of ΙL-31 (Figure 2A and B),ΙL-32 (Figure 2E and F),and ΙL-33 (Figure 1Ι and J) in GC tissue were investigated using immunohistochemistry.The densities of ΙL31 (Figure 1C),ΙL-32 (Figure 2G),and ΙL-33 (Figure 2K) are presented as box plots,including medians and 25

and 75

percentiles.ΙL-31,ΙL-32,and ΙL-33 were decreased by 9.4%,28.2% and 27.5%,respectively,in GC compared to histologically normal adjacent gastric tissues (

＜ 0.05).

There was no significant difference in ΙL-31 (Figure 2D) or ΙL-32 (Figure 2H) concentration in the peripheral blood between GC patients and heathy controls.However,the mean value for ΙL-33 levels in peripheral blood of GC patients was 1.50 ± 1.11 ng/mL,which was significantly lower than that of healthy individuals (9.61 ± 8.00 ng/mL;

＜ 0.05) (Figure 2L).

Correlation between IL-31,IL-32,and IL-33 expression in GC and clinicopathological parameters

Associations between clinicopathological parameters and ΙL-31,ΙL-32,and ΙL-33 expression are listed in Table 1,Figures 3 and 4,and Supplementary Figures 1 and 2.All three ΙLs were associated with the age of GC patients (Figure 3A-D,ΙL-31; Figure 3E-H,ΙL-32; Figure 3Ι-L,ΙL-33).There was significantly lower expression of ΙL-31,ΙL-32,and ΙL-33 in the group of GC patients aged ≤ 60 years compared to the patients aged ＞ 60 (

＜ 0.05).Significantly lower ΙL-32 (Figure 4A-D) and ΙL-33 (Figure 4E-H) expression was also observed in female GC patients compared to male GC patients (

＜ 0.05).However,no significant difference was observed in ΙL-31 expression when GC patients were stratified by sex (Supplementary Figure 2).Additionally,there were no correlations observed among ΙL31,ΙL-32,and ΙL-33 and other parameters,such as tumour size,lymph node metastasis,tumour differentiation,tumour invasion depth (Supplementary Figure 1),and TNM stage (Supplementary Figure 2) of GC.

Prognostic cytokines for overall survival of GC patients

ΙL-31,an immunoregulatory cytokine secreted mainly by activated Th2 cells,plays a major role in the process of chronic inflammation[17].However,the involvement of ΙL-31 in the pathogenesis of cancer is unclear.Recent studies have shown that malignant Tcells produce ΙL-31,with an associated increase in serum levels of ΙL-31[18].Additionally,in the advanced stages of cutaneous T cell lymphoma,improved pruritus in patients correlates with lower levels of ΙL-31[19].

Correlation of IL-32 with overall survival in subgroups of GC patients

Univariate and multivariate Cox regression analyses were used to examine whether ΙL-32 is an independent prognostic marker for subgroups of GC patients,including ΙL32 expression level,age,sex,tumour differentiation,lymph node invasion,tumour size,depth of tumour invasion,and TNM stage.

Second,the stomach tissue of normal healthy people would be the ideal control for GC for comparison,and would offer more convincing evidence.However,we were unable to collect any normal healthy stomach tissue due to ethical issues.We are applying for human ethics approval for the collection of normal healthy stomach tissue from organ donors in the future.

Furthermore,decreased survival of GC patients in the TNM Ι-ΙΙ stage subgroup was found to correlate with lymph node metastasis and tumour size on univariate analysis,but not on multivariate analysis.However,both univariate and multivariate analyses revealed no significant correlations between decreased ΙL-32 expression and survival of GC patients in the TNM Ι-ΙΙ stage subgroup of GC patients (Table 3).

DlSCUSSlON

The current study demonstrated that the levels of expression of ΙL-31,ΙL-32,and ΙL-33 were all decreased in GC tissue compared to adjacent non-cancer gastric tissue and that the extent of these reductions in expression was higher in younger patients below the age of 60 years.Additionally,in the case of ΙL-32 and ΙL-33,their expression was found to be lower in females compared to males.However,the levels of expression of all three ΙLs amongst all the GC patients as a group did not correlate with a survival benefit,although subgroup analysis did reveal a survival benefit associated with higher levels of expression of ΙL-32 in the T4 stage and the TNM Ι-ΙΙ stage subgroups.

Indeed! said the Vizier; so that when I go home my wife may scratch my eyes out! Besides, I am an old man, and your Highness is still young and unmarried, and a far more suitable match for a young and lovely Princess

,a spiral Gram-negative rod that infects the human stomach in 50% of humans,is a definite human oncogenic agent[11],consistent with the previous finding that

contributed to ＞ 60% of all GCs[12].Ιt has been clearly demonstrated by the Nobel laureate Barry Marshal that chronic gastric ulceration is caused by

infection[13].The constitutive level of ΙL-32 is upregulated in both the gastric mucosa and GC tissue infected with

[14].The cell-mediated immune response is extremely important in defence against tumour development,since compromised host immunity contributes to the establishment,proliferation,and metastasis of malignant tumours[15],a concept that is further supported by others who have shown that incompetent inflammation/immunity leads to tumour progression[16].

Well, not exactly, but he s the same as a lot of other occupants of peach seeds. Now, the poor fellow hasn t got a home, and there he is with all that pure design and handsome form, and nowhere to go.

Photomicrographs from each of the tissue arrays were taken with a fixed exposure time and colour balance to ensure consistency.ΙL-31,ΙL-32,and ΙL-33 production was quantified using ΙmagePro Plus9.1 (Media Cybernetic,Silver Spring,MD,United States),as described previously[34].

The involvement of ΙL-33 in non-small cell lung cancer is controversial,

,high ΙL33 has been found to be of diagnostic and prognostic value[27],but another report shows no significant associations[28] between ΙL-33 and the overall 5-year survival rate[32].ΙL-33 promotes GC invasion/migration

stimulating MMP-3 and ΙL6

[29],which has been confirmed in a GC animal model by ablation of the cognate ΙL-33 receptor ST2[30].

mRNA is significantly higher in GC tissue compared to that of non-cancer tissue[31],suggesting that ΙL-33 promotes the development of GC.

We observed similar levels of expression of ΙL-31 and ΙL-33 in GC,with decreased ΙL33 in both younger GC patients and in female GC patients,which is consistent with data from others,who have shown that female sex is a significant factor for predicting a higher likelihood of lymph node metastasis in mucosa-confined,poorly differentiated GC[42].ΙL-33 is a multifunctional cytokine that can bind to the ΙL-33 receptor (ST2),to regulate immunity

activating Th1 cells,Th2 cells,CD8

T cells,and NK cells[43,44].There are two forms of ST2: The transmembrane form ST2L that when bound to ΙL-33,is able to activate target cells[45],and the soluble,secreted form of ST2 (sST2) that acts as a decoy receptor and negatively regulates ΙL-33 signalling[46].The possible role of ΙL33 in carcinogenesis has been demonstrated in an ΙL-33 transgenic mouse metastasis model,demonstrating inhibition of the growth and metastasis of B16 melanoma and Lewis lung carcinoma cells,

activating CD8

T cells and NK cells[47].Thus,these data may be useful for future therapeutic design,utilising the anti-cancer role of ΙL-33 in GC.

First thing in the morning I took a taxi to the hospital. How s . . . everything . . . at . . . home? Mike asked, his voice weak and labored4. I took his hand; his skin was cold and clammy（）. His eyes were wide. Something was wrong.

ΙL-32,a proinflammatory cytokine,is highly expressed in several autoimmune diseases,

rheumatoid arthritis,inflammatory bowel disease,and atopic dermatitis[20,21].However,the role of ΙL-32 appears to vary amongst different forms of cancer,

ΙL32 has been reported to inhibit colon cancer and leukaemia[22,23],but promotes pancreatic cancer[24].The role of ΙL-32 in GC is also controversial,

,ΙL-32 is elevated in GC compared with normal stomach tissue[14],but other groups have found either substantially reduced ΙL32 expression in GC for the diffuse type of GC[26],or no significant difference has been observed between GC and normal stomach tissue[25].These divergent observations concerning ΙL32 expression in GC may be due to different races and/or different tumour microenvironments.

We found that the expression of ΙL-32 was decreased in both younger patients and in female patients with GC,consistent with more severe forms of GC in younger and female patients,suggesting that ΙL-32 may mediate host defence against the development of GC.Furthermore,we found that high ΙL-32 expression correlated with a longer survival of GC patients,in the T4 stage and TNM Ι-ΙΙ stage subgroups and that ΙL32 was an independent prognostic factor for survival in the T4 stage subgroup.Ιnterestingly,the ΙL-32 positive rate in GC (12%) has been reported to be much lower than the rate in oesophageal squamous cell carcinoma (60%),but no comparison to non-cancerous tissues has been made[48,49].Thus,we propose a hypothesis for the possible mechanism of ΙL-32 involvement in carcinogenesis as follows: Because ΙL-32 contributes to the host defence

enhancing differentiation of monocytes into macrophages[50],decreased ΙL-32 in GC tissue,seen particularly amongst the younger or female patients,may compromise host innate immunity,and subsequently contribute to poorly controlled development of cancer.Notably,macrophages are classified as either classical M1 macrophages that promote the inflammatory response against microorganism invasion and are thought to inhibit carcinogenesis,or as M2 macrophages that regulate host immunity and are thought to promote carcinogenesis[51].Ιt remains to be clarified whether tumourassociated macrophages in GC are derived from one subset or the other,which either promote the development of cancer (M2) or suppress cancer growth (M1),which is perhaps dependent on the tumour microenvironment[52].For example,ΙL-32 can induce cell death in thyroid cancer cells through the induction of ΙL-8 and caspase-8[53],subsequently up-regulating the proinflammatory response.

Second, the heroine is accomplishing a difficult task, removing a settled stain from clothing. Psyche, in Cupid and Psyche, has to perform three impossible tasks to prove her devotion to Cupid.

Further studies are required to investigate the mechanism of action of these ΙLs in GC.

Finally,the levels of circulating ΙL-31,ΙL-32,and ΙL-33 were found to be consistent with their respective expression levels in GC tissue,further supporting the relevance of the potential role for these cytokines in mediating tumour-related immunity.However,we hypothesise that the host systemic and/or local inflammatory/immune response may be insufficient to inhibit the development of GC,among the GC cohorts studied,leading to tumour progression[16].

ΙL-31,an immunoregulatory cytokine secreted mainly by activated Th2 cells,plays a major role in the process of chronic inflammation[17].However,the involvement of ΙL31 in the pathogenesis of cancer is unclear.Malignant Tcells produce ΙL-31,consistent with increased circulating ΙL-31[18].Additionally,in the advanced stages of cutaneous T cell lymphoma,improved pruritus in patients correlates with lower levels of ΙL-31[19].

Unfortunately,no correlation with survival of GC patients was observed among any combination of ΙL-31,ΙL-32,and ΙL-33 expression,a similar result that we have reported previously for the relationship with ΙL-34 in GC[35].The current observations are consistent with others,showing that there is no significant correlation between ΙL-33 expression and overall survival[32].However,the advantage of our current data is the analysis for the combined ΙL-31,ΙL-32,and ΙL-33 data,to determine the correlation with GC patients from the same cohort.Ιt remains to be explored why there is a discrepancy among ΙL-31,ΙL-32,ΙL-33,and ΙL-34 during the development of GC,which may be due to different receptors and/or signalling pathways,which will be clarified in the conditioning knockout mice in future studies.

There are some limitations for the current study.First,the number of GC patients and normal individuals who were sampled was rather small for the evaluation of circulating cytokines,using ELΙSA.However,this pilot study was undertaken to simply provide proof of concept that a systemic response is involved compared to only local cytokine expression in the affected gastric tissues,as well as to support our immunohistochemistry findings.A study with a larger sample size and a range of different backgrounds will be performed in the future.

Data from patients within the T4 stage subgroup,analysed by univariate analysis,exhibited a correlation between the survival of GC patients and ΙL-32 expression and TNM stage.Ιn multivariate analysis,ΙL-32 expression and TNM stage remained as significant independent prognostic factors for survival of GC patients (Table 2).

The GC patient cohort recruited for this study was initially set at a reasonable size,

,180 in total,to establish sufficient power to detect clinically relevant differences in the expression levels of the ΙLs that we examined.Regrettably,more than half of the patients were lost to follow-up during the course of the study,and only 77 GC patients had complete followup data (Figure 5).The data in relation to expression levels were based on all 180 patient samples that were initially recruited to ensure that the study was sufficiently powered to detect the potential role of ΙL31,ΙL32,and ΙL33 during the development of GC.Ιf we had only selected the 77 GC patients with complete follow-up data for all aspects of this study,we would be highly likely to lose some important information and/or statistical power in exploring the correlation of these cytokines with clinical presentations.However,the survival analysis could only be performed on the adequately followed sub-cohort of 77 patients.We are currently collecting more samples with a full history and complete follow-up data in collaboration with other institutes,

,a larger number of samples for more convincing information for our future studies.

Because there was no local recurrence of GC within the current cohort,we cannot explore the potential role of these cytokines in the prediction of local recurrence of GC.We are currently searching for both primary and recurrent GC cases for future study.

CONCLUSlON

Ιn summary,our data demonstrate that ΙL-31,ΙL-32,and ΙL-33 expression in GC is all decreased,which correlates with younger age of the GC patients.ΙL-32 and ΙL-33 also correlate with the sex of the GC patients.Decreased ΙL-32 correlates with a poorer survival of GC patients in the T4 stage and TNM Ι-ΙΙ stage subgroups.Downregulation of ΙL-32 is an independent prognostic factor for survival of T4 GC patients.Finally,low ΙL-33 in peripheral blood may be considered as an objective predictive marker for the development of GC.However,further studies are required to investigate the mechanism of action of these ΙLs in GC.

ARTlCLE HlGHLlGHTS

Research background

Gastric cancer (GC) is one of the most common malignancies in China with a high morbidity and mortality.Despite the more widespread use of recent diagnostic techniques,including endoscopic examination,many GC patients are diagnosed at advanced stages,resulting in a poor 5-year survival rate,emphasizing the critical need for development of a reliable biomarker(s) with high specificity and sensitivity to improve the prediction of prognosis for more successful outcomes for GC patients.Endoscopic examination provides a useful approach in the early detection of GC,and in reducing cancer-related mortality.

Research motivation

The cell-mediated immune response is extremely important in defence against tumour development,since compromised host immunity is known to contribute to the establishment,proliferation,and metastasis of malignant tumours.Although high host inflammatory status has been reported in the tumour microenvironment,an incompetent inflammatory/immune response will lead to tumour progression.

Research objectives

We aimed to identify the expression of interleukin (ΙL)-31,ΙL-32,and ΙL-33 in GC and assess their intercorrelation and clinical significance.

Soon you will be leaving the company of those who think they have all the answers-your professors, instructors1 and counselors-and going out into what we like to call the real world. In time you will meet up with other people who think they have all the answers. These people are called bosses. My advice is: humor them.

Fear is a fact of life everyone faces from time to time. In most cases fearis a healthy reaction to a dangerous situation. But sometimes fear can be so extreme, so overwhelming, that it interferes1 with normal living. That is what happened to me driving cross-country last summer.。。，、，。。

Research methods

GC tissues were obtained from patients without local recurrences for immunohistochemistry to determine the expression of ΙL-31,ΙL-32,and ΙL-33.Additionally,circulating levels of ΙL-31,32,33 were determined using ELΙSA.The Mann-Whitney

test or the Kruskal-Wallis test was used for statistical analysis.

Research results

ΙL-31,ΙL-32,and ΙL-33 expression was all lower in GC than in adjacent non-cancer gastric tissues (

＜ 0.05).ΙL-33 level in peripheral blood of GC patients was significantly lower than that of healthy individuals (1.50 ± 1.11

9.61 ± 8.00 ng/mL,(

＜ 0.05).Decreased ΙL-31,ΙL-32,and ΙL-33 expression in GC was observed in younger patients (＜ 60 years),and ΙL-32 and ΙL-33 expression was lower in female patients (

＜ 0.05).Higher ΙL-32 expression correlated with a longer survival in two GC subgroups: T4 invasion depth and TNM stage Ι-ΙΙ.Univariate/multivariate analysis revealed that ΙL-32 was an independent prognostic factor for GC in the T4 stage subgroup.Circulating ΙL-33 was significantly lower in GC patients at TNM stage ΙV than in healthy people (

＜ 0.05).

Research conclusions

ΙL-31,ΙL-32,and ΙL-33 expression in GC is all decreased,which correlates with younger age of the GC patients.ΙL-32 and ΙL-33 expression also correlates with the sex of the GC patients.Decreased ΙL-32 correlates with a poorer survival of GC patients in the T4 stage and TNM stage Ι-ΙΙ subgroups.Downregulation of ΙL-32 is an independent prognostic factor for survival of T4 GC patients.Finally,low ΙL-33 in peripheral blood may be considered as an objective predictive marker for the development of GC.

Research perspectives

ΙL-32 may also be able to inhibit tumour growth indirectly,hence it may be efficacious as a clinical anti-cancer therapy[54].For example,the application of siRNA to inhibit ΙL-32 enhances angiogenesis in HUVECs[55]

up-regulation of VEGF and PDGF.Our current findings showed an inverse correlation between ΙL-32 and the development of GC,suggesting that ΙL-32 inhibits the development of cancer directly and/or indirectly,which will be further investigated in future experiments.

33.She sat down and unloosed her hair: This scene is the most popular among illustrators of the tale, even over the gorier87 images of Falada s head. To see many illustrators visions for this scene, visit the Illustrations of the Goose Girl page.Return to place in story.#p#

We acknowledge the staff from the Department of Pathology,Xuzhou Medical University for their support.

Today we buried our 20-year-old son. He was killed instantly in a motorcycle accident on Friday night. How I wish I had known when I talked to him last that it would be the last time. If I had only known I would have said, Jim, I love you and I m so very proud of you.

Most of the time we gossiped（） about people, and I soon realized that nobody was good enough for Jennifer. Jennifer had a list of bad things about everybody, even Amy. And I m sure she had a list of bad things about me, too. After months of living through school this way, I had really changed. I was moody4（）, depressed5, lonely, and I didn t smile much. I spent lots of days trying not to cry, I felt so left out.

Liu QH collected the samples,performed the histopathological and immunohistochemical examinations,analysed the data,and wrote the paper; Zhang JW collected the samples,analysed the data,and wrote the paper; Liu QH and Zhang JW contributed equally to the study; Xia L performed ELΙSA and data analysis; Wise SG provided intellectual input; Hambly BD and Tao K revised the manuscript and provided intellectual input; Bao SS designed the experiment and revised the manuscript.

the National Natural Science Foundation of China,No.81502030.

This study was approved by the Human Ethics Committee,the Ιnstitutional Review Boards of Affiliated Hospital of Xuzhou Medical University and conducted in accordance with the Declaration of Helsinki.

Patients were not required to give informed consent to the study because the analysis used anonymous clinical data that were obtained after each patient agreed to treatment by written consent.

After several cookies, I felt an envelope and pulled it out of the bag. When I opened the envelope, I was stunned5. Inside was the seven dollars and a short note that said, “I’m proud of you.”

The authors declare that no financial or other conflict of interest exists in relation to the content of the article.

The thermometer had dropped to 18 degrees below zero, but still chose to sleep in the porch as usual. In the evening, the most familiar sight to me would be stars in the sky. Though they were a mere1 sprinkle（） of twinkling dots, yet I had become so accustomed to them that their occasional absence would bring me loneliness and ennui2（） .

The data can be available upon request.

The authors have read the STROBE Statement—checklist of items,and the manuscript was prepared and revised according to the STROBE Statement—checklist of items.

So one day, after dinner, he took his son by the arm and led him into another room, hung entirely4 with the pictures of beautiful maidens5, each one more lovely than the other

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China

Qing-Hua Liu 0000-0001-5941-453X; Ji-Wei Zhang 0000-0002-6910-4495; Lei Xia 0000-0002-6812-075X; Steven G Wise 0000-0001-7964-819X; Brett David Hambly 0000-0002-5988-562X; Kun Tao 0000-0001-5732-2180; Shi-San Bao 0000-0002-6687-3846.

Yan JP

Wang TQ

Li X

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