The human leucocyte differentiation antigens (HLDA) workshops: the evolv-ing role of antibodies in research, diagnosis and therapy

The 8^th International Workshop on Human Leucocyte Differentiation Antigens （chaired by HZ and managed by BS） was run over a 4-year period and culminated in a conference in December 2004. Here we review the achievements of the HLDA Workshops and provide links to information on CD molecules and antibodies against them, including the 93 new CDs assigned in the 8^th Workshop. We consider what remains to be achieved （including an estimate of the number of leucocyte surface molecules still to be discovered）, and how the field can best move forward.

Serum IgG response to differentiated antigens of Helicobacter pylori

AIM To detect antibodies against Helicobacter pylori spiral and coccoid antigens in human sera. METHODS Blood samples were collected from 278 patients with gastric diseases. A 3 day old culture of H. pylori on chocolate blood agar was used to provide spiral form. ‘Synchronous’ coccoids were cultured in (BHY) (brain heart infusion supplemented with 10% horse serum and 0 4% yeast extract) medium in a chemostat. Antigens from spiral and coccoid form were prepared using acid glycine extraction. Enzyme linked immunosorbent assay (ELISA) was performed to detect serum IgG antibodies against spiral and coccoid forms of H. pylori . RESULTS Seroprevalence of H. pylori infection was higher in patients with gastric ulcer (79%) and gastric cancer (83%) than those with non ulcer dyspepsia (NUD) (44%) and other diseases (45%) ( P <0 05). IgG antibodies against spiral and coccoid antigens were detected in 50 7% (141/278) and 49 6% (138/278) , respectively. CONCLUSION The spiral and coccoid forms of H. pylori coexist in patients infected with the bacterium.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to chronic hepatitis B

AIM： To assess the three polymorphJsm regions within cytotoxic T-lymphocyte antigen 4 （CTLA-4） gene, a C/T base exchange in the promoter region-318 （CTLA-4 -318C/T）, an A/G substitution in the exon 1 position 49 （CTLA-4 49A/G）, a T/C substitution in 1172 （CTLA-4 -1172T/C） in patients with chronic hepatitis B. METHODS： Fifty-one patients with chronic hepatitis B virus infection and 150 healthy subjects were recruited sequentially as they presented to the hepatic clinic. Classification of chronic hepatitis B virus （HBV）-infected patients was as asymptomatic carrier state （26 patients） and chronic hepatitis B （25 patients）. Genomic DNA was isolated from anti-coagulated peripheral blood Bully coat using Miller＇s salting-out method. The presence of the CTLA-4 gene polymorphisms was determined using polymerase chain reaction amplification refractory mutation system （ARMS）. RESULTS： We observed a significant association between -318 genotypes frequency （T＋C-, T＋C＋, T-C＋） and susceptibility to chronic hepatitis B （P=0.012, OR=0.49, 95%CI： 0.206-1.162）. However, we did not observe a significant association for ＋49 genotype frequency （T＋C＋, T＋C- T-C＋） and -1172 genotype frequency （C＋T＋, T＋C- C＋T-） and state of disease. CONCLUSION： Our results suggest that CTLA-4 gene polymorphisms may partially be involved in the susceptibility to chronic hepatitis B.

Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells

Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma （NB）, the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes （CTLs） and natural killer （NK） cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

Relationship between phenotypes of cell-function differentiation and pathobiological behavior of gastric carcinomas

AIM: To reveal the correlation between the functional differentiation phenotypes of gastric carcinoma cells and the invasion and metastasis by a new way of cell-function classification.METHODS:Surgically resected specimens of 361 gastric carcinomas(GC) were investigated with enzyme-, mucin-, and tumor-related marker immunohistochemistry. According to the direction of cell-function differentiation, stomach carcinomas were divided into five functionally differentiated types. RESULTS: (1) Absorptive function differentiation type (AFDT): there were 82 (22.7%) patients including 76 (92.7%) aged 45 years. Sixty-nine (84.1%) cases belonged to the intestinal type. Thirty-eight (46.3%) expressed CD44v6 and 9 (13.6%) of 66 male patients developed liver metastasis.The 5-year survival rate of patients in this group (58.5%) was higher than those with the other types (P【0.01). (2) Mucin secreting function differentiation type (MSFDT): 54 (15%) cases. Fifty-three (98.1%) tumors had penetrated the serosa, 12 (22.2%) expressed ER and 22 (40.7%) expressed CD44v6. The postoperative 5-year survival rate was 28.6%. (3) Absorptive and mucin-producing function differentiation type (AMPFDT): there were 180 (49.9%) cases, including 31 (17.2%) aged younger than 45 years. The tumor was more common in women (62, 34.4%,) and expressed more frequently estrogen receptors (ER) (129, 81.7%) than other types (P【0.01). Ovary metastasis was found in 12 (19.4%) out of 62 female subjects. The patients with this type GC had the lowest 5-year survival rate (24.7%) among all types. (4) Specific function differentiation type (SFDT): 13 (3.6%) cases. Nine (69.2%) tumors of this type derived from APUD system, the other 4 (30.7%) were of different histological differentiation. Sixty per cent of the patients survived at least five years. (5) Non-function differentiation type (NFDT): 32 (8.9%) cases. Nineteen (59.4%) cases had lymph node metastases but no one with liver or ovary metastasis. The 5-year survival rate was 28.1%. CONCLUSION: This new cell-function classification of GC is helpful in indicating the characteristics of invasion and metastasis of GC with different cell-function differentiation phenotypes. Further study is needed to disclose the correlation between the cell-functional differentiation phenotypes and the relevant genotypes and the biological behavior of gastric carcinoma.

No association of the cytotoxic T-lymphocyte associated gene CTLA4 +49A/G polymorphisms with Crohn's disease and ulcerative colitis in Hungarian population samples

AIM： The goal of the current work was to analyse the prevalence of the ＋49A/G variant of the cytotoxic T-lymphocyte antigen 4 gene （CTLA4） in Hungarian patients with Crohn＇s disease （CD） and ulcerative colitis （UC）. METHODS： A total of 130 unrelated subjects with CD and 150 with UC, and 170 matched controls were genotyped for the single nucleotide polymorphism （SNP）. The genotypes were determined by using PCR/RFLP test. RESULTS： The G allele frequency and the prevalence of the GG genotype were 38.1% and 12.3% in the CD group, 40.6% and 18.6% in the UC patients, and 37.4% and 15.9% in the control group, respectively. CONCLUSION： The results of the current study show that carriage of the ＋49G SNP in heterozygous or in homozygous form does not confer risk either for CD or for UC in the Hungarian population.

AstragalosideⅣimproves melanocyte differentiation from mouse bone marrow mesenchymal stem cells

Vitiligo results in an autoimmune disorder destructing skin pigment cells,melanocytes(Mcs).This study aimed to investigate whether Astragaloside IV(AIV)could efficiently induce differentiation of bone marrow mesenchymal stem cells(BMMSCs)into Mcs.BMMSCs were induced and differentiated into Mcs with 0.1,0.2,and 0.4 mg/L AIV during 150-day.Morphologic changes of differentiated cells were observed.Levels of some melanocytic specific genes(TRP-1,TRP-2,MART-1,Mitf)were measured with quantitative polymerase chain reaction(qPCR)at 90,120,and 150 days of induction.After 90-day induction,the differentiated cells with 0.4 mg/L AIV demonstrated the typical morphology of Mcs,positive 3,4 dihydroxyphenylalanine staining,and positive staining of TRP-1,TRP-2,MART-1,and Mitf.After 90-and 120-days’induction with 0.4 mg/L AIV,TRP-1 expression was significantly elevated(p<0.01),and TRP-2 expression was significantly increased in 0.4 mg/L AIV-treated group compared to negative control(p<0.01),0.1 mg/L(p<0.01),and 0.2 mg/L(p<0.01)AIV-treated groups.Moreover,MART-1 expression was significantly up-regulated in 0.4 mg/L AIV-treated group compared to negative control,but without difference compared to 0.1 mg/L(p>0.05)and 0.2 mg/L(p>0.05)AIV-treated groups.During 90 to 150-day induction,there were no significant differences for Mitf levels between AIV-treated groups and negative control(p>0.05).In conclusion,90-day induction with 0.4 mg/L AIV up-regulated TRP-1,TRP-2,and MART-1 expression,indicating that AIV can efficiently induce Mcs differentiation from BMMSCs.These results provide experimental and theoretic evidence for AIV application in clinical vitiligo repigmentation treatment.

LRRC25 plays a key role in all-trans retinoic acid-induced granulocytic differentiation as a novel potential leukocyte differentiation antigen

Leukocyte differentiation antigens （LDAs） play important roles in the immune system, by serving as surface markers and participating in multiple biological activities, such as recognizing pathogens, mediating membrane signals, interacting with other cells or systems, and regulating cell differentiation and activation. Data mining is a powerful tool used to identify novel LDAs from whole genome. LRRC25 （leucine rich repeat-containing 25） was predicted to have a role in the function of myeloid cells by a large-scale ＂omics＂ data analysis. Further experimental validation showed that LRRC25 is highly expressed in primary myeloid cells, such as granulocytes and monocytes, and lowly/intermediately expressed in B cells, but not in T cells and almost all NK cells. It was down-regulated in multiple acute myeloid leukemia （AML） cell lines and bone marrow cells of AML patients and up-regulated after all-trans retinoic acid （ATRA）-mediated granulocytic differentiation in AML cell lines and acute promyelocytic leukemia （APL; AML-M3, FAB classification） cells. Localization analysis showed that LRRC25 is a type I transmembrane molecule. Although ectopic LRRC25 did not promote spontaneous differentiation of NB4 cells, knockdown of LRRC25 by siRNA or shRNA and knockout of LRRC25 by the CRISPR-Cas9 system attenuated ATRA-induced termi- nal granulocytic differentiation, and restoration of LRRC25 in knockout cells could rescue ATRA-induced granulocytic differentiation. Therefore, LRRC25, a potential leukocyte differentiation antigen, is a key regulator of ATRA-induced granulocytic differentiation.

Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

A 1 846 bp cDNA is isolated from a human tonsil cell λgt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.

Five novel monoclonal antibodies to thymic epithelial cell surface antigens in rats

OBJECTIVE: To establish monoclonal antibodies (mAbs) against thymic epithelial cells and study the function of epithelial cells during T-cell differentiation in the thymus. METHODS: Hybridomas secreting mAbs against thymic epithelial cells were derived by immunization of Balb/c mice with two thymic epithelial cell lines, TaD3 and FTE. The distribution of antigens recognized by these mAbs was detected by immunochemical staining and cytofluorographic analysis, and the molecular weight of the antigens by immunoblotting. RESULTS: Five specific monoclonal antibodies (mAb) were obtained. On the basis of their distribution in the thymus determined by immunochemical staining, mAb RE-4D8 was regarded as clusters of thymic epithelium staining (CTES) type IIA: mAb RE-12B2, which showed a unique distribution pattern only in the medulla, was CTES type V: mAb RE-5C6 was CTES type IV: mAb RE-6D6 might be CTES type IIB: and mAb RE-1D4 was classified as type V. The molecular weight (MW) of antigen RE-4D8, RE-6D6 and RE-12B2 were 120 kDa, 220 kDa and 35 kDa, respectively. Antigen RE-1D4 is a novel marker of cortical epithelium, several established thymic epithelial cell lines were classified and their original intrathymic locations were determined by these mAbs. Thymic cell lines, TuD3 and FTE were cortical phenotypes whereas TaD3 had a medullar phenotype. CONCLUSIONS: These mAbs clearly demonstrate the heterogeneity of the thymic epithelium; they could detect antigens not only in the cytoplasm but also on the surface of thymic epithelial cells. Our data suggest that these newly established mAbs may help elucidate the interaction between thymocytes and epithelial cells during T cell maturation.

Study on Cellular Differentiation and Genes Rearrangement of Malignant Lymphoproliferative Disorders

Malignant lymphoproliferative disorders are common diseases characterized by the clonal proliferation of cells derived from the lymphocytes of different developmental stages. The analyses of routine morphology and cytochemistry fail to determine the origin and differentiation stages. The cell differentiation origin of most of malignant lymphoproliferative disorders can be defined by using monoclonal antibodies(McAbs) against the differentiation antigens of lymphocytes, but it is unable to determine the cell origin of a minority of undifferentiated malignancies and also fails to distinguish the malig-

Down-regulation of human leukocyte antigens class I on peripheral T lymphocytes and NK cells from subjects in region of high-incidence gastrointestinal tumor

Background Many types of human tumors can suppress the immune system to enhance their survival. Loss or down-regulation of human leukocyte antigens （HLA） class I on tumors is considered to be a major mechanism of tumor immune escape. Our previous studies found that HLA class I on peripheral-blood mononuclear cells was significantly lower in gastric cancer patients. The present study made an analysis of HLA class I expression on peripheral-blood T lymphocytes and NK cells from subjects of Lijiadian village, a village with high-incidence gastrointestinal tumor. Methods A total of 181 villagers from Lijiadian village and 153 normal controls from the Department of Health Examination Center were enrolled in this study. Using a multi-tumor markers detection system, these villagers were divided into two groups： high-risk group （tumor markers positive group） and low-risk group （tumor markers negative group）. The percentage of T lymphocytes and NK cells and levels of HLA class I on their surface were determined in these subjects by flow cytometry. Results Percentages of T lymphocytes and NK cells in peripheral-blood mononuclear cells did not vary with age. The expression level of HLA class I on peripheral T lymphocytes and NK cells was not affected by age or gender, but was significantly down-regulated in Lijiadian villagers （P 〈0.05）, with the low-risk group, there was a significant reduction of cells （P 〈0.05） in the high-risk group. especially on the surface of NK cells （P 〈0.01）. Compared HLA class I on peripheral T lymphocytes （P 〈0.05） and NK Conclusions HLA class I on peripheral T lymphocytes and NK cells may be involved in tumorigenesis and development of gastrointestinal tumor, and understanding their changes in expression may provide new insights into the mechanism of tumor immunity.

High expression of squamous cell carcinoma antigen in poorly differentiated adenocarcinoma of the stomach: A case report

BACKGROUND Squamous cell carcinoma antigen(SCCA)is regarded as a specific indicator of epithelial malignancies and is widely used in the diagnosis of squamous cell carcinoma(SCC).However,the expression of SCCA in gastric adenocarcinoma has not been studied in detail.CASE SUMMARY A 52-year-old man was admitted to our hospital for a 2.5 cm x 2.5 cm ulcer at the antrum-body junction with dull pain and fullness in the upper abdomen for 2 mo.His pre-surgery serological testing results showed 0.51 ng/mL SCCA(reference interval,<1.5 ng/mL)and 9.9 ng/mL carcinoembryonic antigen(reference range,<4.7 ng/mL).He underwent radical distal gastrectomy and Roux-en Y anastomosis and was diagnosed with poorly differentiated mucinous adenocarcinoma(Lauren classification:Diffuse)by pathological examination of the resected lesion.Immunohistochemistry showed that SCCA was highly expressed in the cytoplasm of cancer cells.After surgery,the patient received an S-1 adjuvant chemotherapy regimen for six cycles containing tegafur,gimeracil,and oteracil potassium.He showed no sign of recurrence or metastasis within 24-mo follow-up.CONCLUSION This is a frontal report of SCCA overexpression in poorly differentiated adenocarcinoma of the stomach.

Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes

AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I tetramers.METHODS： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 heavy chain （A2） and β2-microglobulin （132m） from total RNA extracted from leukocytes of HLA-A2＋ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and 132m proteins were expressed in ~/a oo/i^uain BL21（DE3） and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain 132m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme （BirA） and purified by low pressure anion exchange chromatography on a Q-Sepharose （fast flow） column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4：1. Flow cytometry was used to confirm the expected tetramer staining of CD8^＋ T cells.RESULTS： Recombinant genes for HLA-A＊0201 heavy chain （A2） fused to a BirA substrate peptide （A2-BSP） and mature β2m from HLA-A2＋ donor leukocytes were successfully doned and highly expressed in E. coli, Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of 132m and peptides loaded with either human cytomegalovirus pp65495-503 peptide （NLVPMVATV,NLV; designated as A2-NLV） or influenza virus matrix protein Mp58-66 peptide （GILGFVFTL, GIL; designated as A2-GIL）. Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplicationas revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8^＋ T cells from a HLA-A2^＋ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION： The procedure is simple and efficient for generating peptide-MHC tetramers.

An Immunohistochemical Study of Langerhans Cells,T-Cells and the HLA Antigen in Human Cornea

The distribution of Langerhans cells (LC),T-cell subsets andHLA antigen in 12 normal and 7 morbid corneas,including 4 of suppurativecorneal ulcer and 3 of uveogenic endophthalmitis,was investigated withmonoclonal antibodies.The results revealed that a small amount of LC andT-cell subsets were present in the limbal region of normal corneas,whilelarge numbers of LC and OKT_4^+ were observed in the corneas of suppurativeulcer.HLA-A,B,C antigens were expressed on the epithelial cells keratocytes of the normal corneas, while the HLA-DRantigens wereexpressed on the surface of LC at the limbus.

Changes in tumor-antigen expression profile as human small-cell lung cancers progress

Objective： Our group has previously observed that in patients with small-cell lung cancers （SCLCs）, the expression of a tumor antigen, glioma big potassium （gBK） ion channel, is higher at the time of death than when the cancer is first treated by surgical resection. This study aimed to determine whether this dichotomy was common in other potential lung tumor antigens by examining the same patient samples using our more extensive profile analysis of tumor-antigen precursor protein （TAPP）. We then tested the hypothesis that therapeutic intervention may inadvertently cause this increased gBK production. Methods： SCLC samples （eight surgical resections and three autopsy samples） and three control lungs were examined by quantitative real-time polymerase chain reaction for 42 potential TAPPs that represent potential T-cell-mediated immunological targets. Results： Twenty-two TAPP mRNAs displayed the same profile as gBK, i.e., more mRNAs were expressed at autopsy than in their surgical counterparts. B-cyclin and mouse double minute 2, human homolog of PS3-binding protein were elevated in both autopsy and surgical specimens above the normal-lung controls. When HTB119 cells were incubated with doxorubicin, gBK was strongly induced, as confirmed by intracellular flow cytometry with a gBK-specific antibody. Conclusion： Our findings suggested that more immunological targets became available as the tumor responded to chemotherapy and proceeded toward its terminal stages.

Combined Determination of CEA, Tch and ADA for Differential Diagnosis of Ascites

Objective: To study the value of combined determination of carcinoembryonic antigen (CEA), total cholesterol (Tch) and adenosine deaminase (ADA) in the differential diagnosis of ascites due to different causes. Methods: Sixty-eight cases with ascites were divided into 3 groups based on their etiology, namely malignant ascites, tubercular ascites and non-tubercular benign ascites. CEA, Tch, and ADA were measured and analyzed in different ascites. Results: CEA was significantly higher in malignant ascites than in benign ascites, the sensitivity and specificity for malignant ascites being 50% and 100% respectively. Tch is higher or equal to 1.54 mmol/L in tubercular ascites and lower or equal to 1.18 mmol/L in non-tubercular benign ascites, and Tch level in malignant ascites was frequently between that in tubercular acites and non-tubercular benign ascites. Ascitic fluid ADA activity was higher than 30 U/L in 80% of tubercular ascites, while none of non-tubercular benign ascites reached to such level. Conclusion: CEA, Tch and ADA are valuable for the diagnosis and differential diagnosis of ascitic etiology and combine measurements of these indices can increase the diagnostic efficiency.

Cyst fluid glucose: An alternative to carcinoembryonic antigen for pancreatic mucinous cysts

Pancreatic cystic lesions(PCLs) have been increasingly recognized in clinical practice. Although inflammatory cysts(pseudocysts) are the most common PCLs detected by cross-sectional imaging modalities in symptomatic patients in a setting of acute or chronic pancreatitis, incidental pancreatic cysts with no symptoms or history of pancreatitis are usually neoplastic cysts. For these lesions,it is imperative to identify mucinous cysts(intraductal papillary mucinous neoplasms and mucinous cystic neoplasms) due to the risk of their progression to malignancy. However, no single imaging modality alone is sufficient for a definitive diagnosis of all PCLs. The cyst fluid obtained by endoscopic ultrasound-guided fine needle aspiration provides additional information for the differential diagnosis of PCLs. Current recommendations suggest sending cyst fluid for cytology evaluation and measurement of carcinoembryonic antigen(CEA) levels. Unfortunately, the sensitivity of cytology is greatly limited, and cyst fluid CEA has demonstrated insufficient accuracy as a predictor of mucinous cysts. More recently, cyst fluid glucose has emerged as an alternative to CEA for distinguishing between mucinous and nonmucinous lesions. Herein, the clinical utility of cyst fluid glucose and CEA for the differential diagnosis of PCLs was evaluated.

Differential expression and significance of 5-hydroxymethylcytosine modification in hepatitis B virus carriers and patients with liver cirrhosis and liver cancer

BACKGROUND The relationship between hepatitis B surface antigen(HBsAg)-positive carrier status and liver cancer has been extensively studied.However,the epigenetic changes that occur during progression from HBsAg-positive carrier status or cirrhosis to liver cancer are unknown.The epigenetic modification of DNA hydroxymethylation is critical in tumor development.Further,5-hydroxymethylcytosine(5hmC)is an important base for DNA demethylation and epigenetic regulation.It is also involved in the assembly of chromosomes and the regulation of gene expression.However,the mechanism of action of 5hmC in HBsAgpositive carriers or patients with cirrhosis who develop liver cancer has not been fully elucidated.AIM To investigate the possible epigenetic mechanism of HBsAg-positive carriers and hepatocellular carcinoma(HCC)progression from cirrhosis.METHODS Forty HBsAg-positive carriers,forty patients with liver cirrhosis,and forty patients with liver cancer admitted to the First People's Hospital of Yongkang between March 2020 and November 2021 were selected as participants.Free DNA was extracted using a cf-DNA kit.cfDNA was extracted by 5hmC DNA sequencing for principal component analysis,the expression profiles of the three groups of samples were detected,and the differentially expressed genes(DEGs)modified by hydroxymethylation were screened.Bioinformatic analysis was used to enrich DEGs,such as in biological pathways.RESULTS A total of 16455 hydroxymethylated genes were identified.Sequencing results showed that 32 genes had significant 5hmC modification differences between HBsAg carriers and liver cancer patients,of which 30 were upregulated and 2 downregulated in patients with HCC compared with HBsAg-positive carriers.Significant 5hmC modification differences between liver cirrhosis and liver cancer patients were identified in 20 genes,of which 17 were upregulated and 3 were downregulated in patients with HCC compared with those with cirrhosis.These genes may have potential loci that are undiscovered or unelucidated,which contribute to the development and progression of liver cancer.Analysis of gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes showed that the major signaling pathways involved in the differential genes were biliary secretion and insulin secretion.The analysis of protein interactions showed that the important genes in the protein-protein interaction network were phosphoenolpyruvate carboxykinase and solute carrier family 2.CONCLUSION The occurrence and development of liver cancer involves multiple genes and pathways,which may be potential targets for preventing hepatitis B carriers from developing liver cancer.

钼靶和超声多普勒结合血清肿瘤标志物诊断早期乳腺癌研究

目的:探讨钼靶、超声多普勒和血清肿瘤标志物中血清前列腺特异抗原(PSA)、血清糖类抗原15-3(CA153)、黏蛋白1(MUC1)、人类生长分化因子3(GDF3)单独及联合检测在早期乳腺癌诊断中的价值。方法:选取2018年1月至2021年12月在唐山市人民医院经病理检查确诊的96例乳腺癌患者(乳腺癌组)和同期在本院接受诊治的70例乳腺良性疾病患者(良性病灶组)以及同时选取在本院体检健康的50名体检者(健康对照组),以术后病理检查为“金标准”,比较钼靶、超声多普勒检查以及血清PSA、CA153、MUC1、GDF3单独及6者联合应用对乳腺癌的诊断价值。结果:乳腺癌组96例乳腺癌患者中有78例乳腺超声诊断为恶性,阳性检出率为81.3%;80例钼靶X射线检查诊断为恶性,阳性检出率为83.1%;乳腺癌组的血清PSA、CA153、MUC1及GDF3的水平明显高于良性病灶组和健康对照组,差异均有统计学意义(t_(良性病灶组)=8.783、10.361、11.258、18.965;t_(健康对照组)=9.564、12.658、12.688、20.163,P<0.05);以乳腺癌作为因变量,血清PSA、CA153、MUC1及GDF3为自变量,进行Logistic回归分析,血清PSA、CA153、MUC1及GDF3是乳腺癌的重要危险因素(OR=1.165、1.168、1.472、1.248,P<0.05);受试者工作特征(ROC)曲线分析各指标单独应用时:乳腺超声、钼靶,血清PSA、CA153、MUC1及GDF3的ROC曲线下面积(AUC)(95%CI)、灵敏度和特异度分别为0.723(0.595~0.851)、82.56%和67.32%,0.761(0.636~0.886)、85.79%和65.36%,0.833(0.726~0.941)、81.48%和85.73%,0.837(0.738~0.926)、61.25%和70.17%,0.768(0.648~0.889)、71.49%和80.87%,0.613(0.469~0.758)、52.94%和50.57%;而6项联合应用时AUC(95%CI)、灵敏度和特异度分别为0.958(0.905~0.999)、96.37%和84.83%,其诊断效能更高。结论:钼靶、超声多普勒和血清PSA、CA153、MUC1、GDF3联合检测效能高于单独检测,有助于早期鉴别和诊断乳腺癌。