CD19+CD24hiCD38hiBregs involved in downregulate helper T cells and upregulate regulatory T cells in gastric cancer.

Regulatory B cells (Bregs) play a critical role in inflammation and autoimmune disease. We characterized the role of Bregs in the progression of gastric cancer. We detected an increase in Bregs producing IL-10 both in peripheral blood mononuclear cells (PBMCs) and in gastric tumors. Multicolor flow cytometry analysis revealed that a subset of CD19+CD24hiCD38hi B cells produces IL-10. Functional studies indicated that increased Bregs do not inhibit the proliferation of CD3+T cells or CD4+ helper T cells (Th cells). However, Bregs do suppress the secretion of IFN-γ and TNF-α by CD4+Th cells. CD19+CD24hiCD38hiBregs were also found to correlate positively with CD4+FoxP3+ regulatory T cells (Tregs). Neutralization experiments showed that Bregs convert CD4+CD25- effector T cells to CD4+FoxP3+Tregs via TGF-ß1. Collectively, these findings demonstrate that increased Bregs play a immunosuppressive role in gastric cancer by inhibiting T cells cytokines as well as conversion to Tregs. These results may provide new clues about the underlying mechanisms of immune escape in gastric cancer.

Epigenetically downregulated Semaphorin 3E contributes to gastric cancer.

Axon guidance protein Semaphorin 3E (Sema3E) promotes tumor metastasis and suppresses tumor cell death. Here, we demonstrated that Sema3E was decreased in gastric cancer. Its levels were inversely associated with tumor progression. Levels of Sema3E were associated with low p300 and high class I histone deacetylase (class I HDAC). Ectopic expression of Sema3E inhibited proliferation and colony formation of gastric cancer cell lines in vitro and xenografts in vivo. Sema3E overexpression inhibited migration and invasion of gastric cancer cells, which was associated with induction of E-cadherin and reduction of Akt and ERK1/2 phosphorylation. We suggest that silencing of Sema3E contributes to the pathogenesis of gastric cancer.

Vitamin D deficiency contributes to the reduction and impaired function of naïve CD45RA⁺ regulatory T cell in chronic heart failure.

The effect of vitamin D pertinent to cardiovascular health on the heart itself is considered to shift toward an anti-inflammatory response in chronic heart failure (CHF); however, its underlying mechanism is not completely understood. In this study, we demonstrated that plasma 25(OH)D level, negatively associated with NT-ProBNP, correlated with the decreased Treg in CHF compared to the patients with other cardiovascular diseases and healthy and older donors. Naïve Treg cell (CD4(+)CD45RA(+)Foxp3(lo)T) subset, rather than whole Treg cells, contributes to the reduction of Treg in CHF. 1,25(OH)2D treatment maintained partial expression of CD45RA on CD4(+)T cell after αCD3/CD28 monoclonal antibodies activation and ameliorated the impaired CD4(+)CD45RA(+)T cell function from CHF patients through upregulating Foxp3 expression and IL-10 secretion in vitro. Low level of vitamin D receptor (VDR) was detected in CD4(+)CD45RA(+)T cell of CHF than control, while 1,25(OH)2D treatment increased the VDR expression to exert its immunosuppression on T cell. The results of this study might provide tangible evidence to our knowledge of the impact of vitamin D supplementation on naïve Tregs, which may offer new means of preventing and treating CHF.

Assessment of a five-color flow cytometric assay for verifying automated white blood cell differentials.

BACKGROUND: White blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review. However, this last step is time consuming and requires experienced personnel. We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials. METHODS: A total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials. WBC differentials of 622 samples were also determined using both a cell counter and FCM. These results were then confirmed using manual microscopic methods. RESULTS: The probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%. The resulting WBC differentials were well correlated between FCM and the cell counter (r > 0.88, P < 0.001), except in the case of basophils. Neutrophils, lymphocytes, and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r > 0.80, P < 0.001). The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%, respectively) were higher than those of the cell counter method (44.92% and 11.11%, respectively). The specificities of FCM were all above 85%, substantially better than those of the cell counter method. CONCLUSION: These five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.

[Characteristics and monitoring of minimal residual disease in patients with T acute lymphoid leukemia by flow cytometry with two 4-color combinations of fluorescent antibodies].

The purpose of this study was to establish a method for the monitoring of minimal residual disease (MRD) in bone marrow samples of the children with T acute lymphoid leukemia (T-ALL), and to evaluate its value in clinical application. The immuno-phenotype of the leukemic cells were detected by flow cytometry with two sets of 4-color combinations of antibodies against TdT/CD5/cCD3/HLA-DR(+)CD19(+)CD33 and CD34/CD5/cCD3/HLA-DR(+)CD19(+)CD33 in 32 cases of de novo T-ALL and were compared with the results in 10 normal controls. The antibody combination in regions of the two-parameter plots where the leukemic cells appeared were different from the normal cells was screened as the effective combination which was used to monitor MRD in the bone marrows of the T-ALL children after the inductive treatment. The results indicated that the respective effective frequencies of antibodies against TdT/CD5/cCD3/HLA-DR(+)CD19(+)CD33 and CD34/CD5/cCD3/HLA-DR(+)CD19(+)CD33 were 90.6% and 62.5%. 32 cases of childhood T-ALL were successively screened for antibodies combinations of interest and were identified in 100% (32/32) of these cases. After inductive treatment, the positive rate in 129 times of MRD monitoring was 19.4% (25/129) by flow cytometry and 5.43% (7/129) by FAB morphology. It is concluded that monitoring MRD in patients with T-ALL by flow cytometry with two 4 color combinations of fluorescent antibodies is an quick and effective method. The sensitivity of this method is high and it may be of important significance for the treatment and prognostic evaluation in childhood T-ALL.

CD74 and macrophage migration inhibitory factor as therapeutic targets in gastric cancer.

AIM: To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor (MIF)/Toll-like receptor 4 (TLR4) in gastric cancer. METHODS: CD74, MIF and TLR4 expression in the paraffin-embedded sections of gastric cancer from 120 patients were detected by immunohistochemical staining. Knock down of CD74 expression in gastric cancer cell line MKN-45 was performed by lentivirus transduction and detected by Western blotting. MKN-45 cell proliferation assay under the stimulants was measured by the cell counting kit 8 (CCK8) assay and MIF concentration in the culture medium was detected by enzyme-linked immunosorbent assay. Surface staining of CD74 in the MKN-45 cell line under the stimulation of lipopolysaccharide (LPS) was measured by flow cytometry. MIF, CD74 and TLR4 co-localization in the MKN-45 cell line was performed by the immunoprecipitation. RESULTS: CD74, MIF and TLR4 were found to be expressed in gastric cancer and increased significantly in the advanced stage, and were also associated with lymph node metastasis. Correlation analysis revealed that CD74 was positively correlated with MIF (r = 0.2367, P < 0.01) and both proteins were also associated with TLR4 (r = 0.4414, r = 0.5001, respectively, P < 0.01). LPS can significantly promote MKN-45 cell proliferation (3.027 ± 0.388 vs 4.201 ± 0.092, P < 0.05), induce MIF production (54.333 ± 2.906 pg/mL vs 29.667 ± 3.180 pg/mL, P < 0.01) and cell surface expression of CD74 (75.6% ± 4.046% vs 9.4% ± 0.964%, P < 0.01) at LPS concentration of 1 µg/mL compared to medium control. Knockdown of CD74 or using anti-CD74 and MIF antagonist ISO-1 significantly reduced LPS-induced MKN-45 cell proliferation (4.201 ± 0.092 vs 3.337 ± 0.087, 4.534 ± 0.222 vs 3.368 ± 0.290, 4.058 ± 0.292 vs 2.934 ± 0.197, respectively, P < 0.01). MIF, CD74 and TLR4 could co-localize in the MKN-45 cell line. CONCLUSION: Upregulation of MIF, CD74 and TLR4 are associated with increasing clinical stage and provide an opportunity as novel gastric cancer chemoprevention and/or treatment strategy.

Immunomodulatory effects of dietary supplementation with a milk-based wolfberry formulation in healthy elderly: a randomized, double-blind, placebo-controlled trial.

Wolfberry (fruit of Lycium barbarum) has been prized for many years in China for its immunomodulatory property and its high specific antioxidant content. However, clear clinical evidence demonstrating the effect of wolfberry dietary supplementation is still lacking. After our earlier report showing that a proprietary milk-based wolfberry formulation (Lacto-Wolfberry) enhances in vivo antigen-specific adaptive immune responses in aged mice, the present study aimed at demonstrating the effect of dietary Lacto-Wolfberry supplementation on immune functions in the elderly, especially vaccine response known to decline with aging. A 3-month randomized, double-blinded, placebo-controlled study was conducted on 150 healthy community-dwelling Chinese elderly (65-70 years old) supplemented with Lacto-Wolfberry or placebo (13.7 grams/day). Immune response to influenza vaccine was assessed in the study, along with inflammatory and physical status. No serious adverse reactions were reported during the trial, neither symptoms of influenza-like infection. No changes in body weight and blood pressure, blood chemistry or cells composition, as well as autoantibodies levels were observed. The subjects receiving Lacto-Wolfberry had significantly higher postvaccination serum influenza-specific immunoglobulin G levels and seroconversion rate, between days 30 and 90, compared with the placebo group. The postvaccination positive rate was greater in the Lacto-Wolfberry group compared to the placebo group, but did not reach statistical significance. Lacto-Wolfberry supplementation had no significant effect on delayed-type hypersensitivity response and inflammatory markers. In conclusion, long-term dietary supplementation with Lacto-Wolfberry in elderly subjects enhances their capacity to respond to antigenic challenge without overaffecting their immune system, supporting a contribution to reinforcing immune defense in this population.

Gastric cancer cells induce human CD4+Foxp3+ regulatory T cells through the production of TGF-ß1.

AIM: To elucidate the molecular and cellular features responsible for the increase of regulatory T cells (Tregs) in gastric cancer. METHODS: The frequencies of CD4(+)Foxp3(+) Tregs and the level of transforming growth factor-ß1 (TGF-ß1) were analyzed from 56 patients with gastric cancer by flow cytometry and enzyme-linked immunosorbent assay respectively. Foxp3 gene expression was analyzed by real-time polymerase chain reaction. The gastric cancer microenvironment was modeled by establishing the co-culture of gastric cancer cell line, MGC-803, with sorting CD4(+) T cells. The normal gastric mucosa cell line, GES-1, was used as the control. The production of TGF-ß1 was detected in supernatant of MGC and GES-1. The carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay was performed to evaluate the proliferation characteristics of induced Tregs. Neutralizing anti-TGF-ß1 antibody was added to the co-culture system for neutralization experiments. RESULTS: The level of serum TGF-ß1 in gastric cancer patients (15.1 ± 5.5 ng/mL) was significantly higher than that of the gender- and age-matched healthy controls (10.3 ± 3.4 ng/mL) (P < 0.05). Furthermore, the higher TGF-ß1 level correlated with the increased population of CD4(+)Foxp3(+) Tregs in advanced gastric cancer (r = 0.576, P < 0.05). A significant higher frequency of CD4(+)Foxp3(+) Tregs was observed in PBMCs cultured with the supernatant of MGC than GES-1 (10.6% ± 0.6% vs 8.7% ± 0.7%, P < 0.05). Moreover, using the purified CD4(+)CD25(-) T cells, we confirmed that the increased Tregs were mainly induced from the conversation of CD4(+)CD25(-) naive T cells, and induced Tregs were functional and able to suppress the proliferation of effector T cells. Finally, we demonstrated that gastric cancer cells induced the increased CD4(+)Foxp3(+) Tregs via producing TGF-ß1. Gastric cancer cells upregulated the production of TGF-ß1 and blockade of TGF-ß1 partly abrogated Tregs phenotype. CONCLUSION: Gastric cancer cell can induce Tregs development via producing TGF-ß1, by which the existence of cross-talk between the tumor and immune cells might regulate anti-tumor immune responses.

Elevated expression of Foxp3 in tumor-infiltrating Treg cells suppresses T-cell proliferation and contributes to gastric cancer progression in a COX-2-dependent manner.

The transcription factor Foxp3 plays a key role in CD4(+)CD25(+) regulatory T (Treg) cell function. A correlation has been shown between survival and the frequency of tumor-infiltrating Foxp3-positive Treg cells in cancer patients. However, few studies have characterized the regulation of Foxp3 expression and function in Treg cells, which are known to comprise distinct subsets, with different roles in the complex tumor microenvironment. Here, we show that significantly more Foxp3-positive Treg cells accumulated in gastric tumors. In addition, we found increased expression of Foxp3 protein per cell in tumor-infiltrating Treg cells. Moreover, elevated Foxp3 expression in tumor-infiltrating Treg cells was associated with the TNM stage in gastric cancer patients. Importantly, further investigation within the tumor microenvironment showed that expression of Foxp3 in Treg cells correlated with expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Furthermore, Treg cells with higher levels of Foxp3 were able to suppress the proliferation of autologous CD4(+)CD25(-) T cells. The suppression of the effector T-cell response was reversed by COX inhibitors and PGE(2) receptor-specific antagonists. Our data demonstrate a mechanism by which tumor-infiltrating Treg cells with increased Foxp3 expression can mediate immune suppression via COX-2/PGE(2) production in the gastric cancer microenvironment. Thus, we provide new insights into overcoming regulatory T-cell activity, which may be beneficial for the treatment of human gastric cancer.

Ser234Leu missense mutation in the A1 domain of factor V causing moderate factor V deficiency in a Chinese family.

AIMS: To investigate the molecular defects in a Chinese pedigree with inherited factor V (FV) deficiency. METHODS: Laboratory studies including activated partial thromboplastin time (APTT), prothrombin (PT), and thrombin time (TT) were tested in a patient and his family members. FV antigen (FV:Ag) and FV activity (FV:C) were measured by both ELISA and one-stage clotting assays. All the exons, exon-intron boundaries and promoter regions of FV gene were analysed by direct sequencing. The detected mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA and the wild-type and mutant FV proteins were expressed in COS-7 and CHO cells. RESULTS: The proposita, a 52-year-old Chinese man, had no spontaneous bleeding syndrome. It was found that he had prolonged APTT and PT, 52 s and 22.8 s, respectively, a FV:C of 5.5% and a FV:Ag of 33.1%. Gene analysis showed the proposita was a compound heterozygote of FV mutations, carrying Ser234Leu and Arg413Cys. The FV antigen and activity levels of the Ser234Leu and Arg413Cys mutants are lower than wild type both in cell lysates and in culture media. Protein degradation inhibitor experiment in transfected COS-7 cells showed that Ser234Leu and Arg413Cys degraded intracellularly through the lysosomal pathway. CHO cells expressing either the wild-type or the mutant FV were subjected to immunofluorescence staining with the indicated antibodies and organelle markers, indicating that Ser234Leu and Arg413Cys can be transported to Golgi partially. CONCLUSIONS: We identified the molecular pathological mechanism of the novel C785T mutation causing type I inherited FV deficiency for the first time.

CD4(+)CD25(+)CD127(low/-) regulatory T cells express Foxp3 and suppress effector T cell proliferation and contribute to gastric cancers progression.

Increased populations of regulatory T cells (Tregs) impair anti-tumor immunity. Recently, the transcription factor Foxp3 has been reported to play a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. However, Foxp3 is a nuclear protein and is of limited value in the isolation of Tregs, which is a major reason that many functionally relevant aspects of Treg cells are still unknown. Here, we have characterized CD4(+)CD25(+)CD127(low/)- as the surface marker of regulatory T cells in gastric cancer. 88.1-96.1%of CD25(+)CD127(low/-) T cells expressed Foxp3, the frequency of CD4(+)CD25(+)CD127(low/-) regulatory T cells in the peripheral blood of gastric cancer patients was significantly higher than that in healthy controls. Increased CD4(+)CD25(+)CD127(low/-) regulatory T cells were also present in the tumor microenvironment, such as those found in the ascites fluid, tumor tissue or adjacent lymph nodes. Particularly those Treg cells associated with the TNM stage. In addition, we found that CD4(+)CD25(+)CD127(low/-) Tregs suppressed effector T cell proliferation and also correlated to advanced stage of gastric cancer. Thus, CD4(+)CD25(+)CD127(low/-) can be used as a selective biomarker to enrich human Treg cells and also to perform functional in vitro assays in gastric cancer.

[Effect of pax5 gene blocked by RNAi on biological characteristics of B cell malignant lymphoma].

This study was aimed to investigate the biological characteristics of B hematological tumor cells such as proliferation, immunological phenotype and apoptosis by silencing pax5. The specific pax5 small hairpin RNA (shRNA) was synthesized by in vitro transcription. For evaluating the inhibition efficiency, the expression change at mRNA and protein levels were assessed by real-time RT-PCR and Western blot respectively. To detect the biological characteristics of pax5-silenced hematological tumor cells, the immunological phenotype, apoptosis and cell proliferation were measured by using real-time RT-PCR, MTT assay and flow cytometry respectively. The results showed that two shRNA were synthesized, both of which were effective to block pax5 expression. After being blocked by RNAi the immunological phenotype of pax5-silenced lymphoma cells was changed, the expressions of CD19 mRNA and protein were reduced, but the expression of IgM was not changed. As compared with control group, the effect on proliferation and apoptosis of lymphoma cells not could be detected after pax5 silencing. It is concluded that the pax5 plays important role in late differentiation of B cells, and may participate in signal transduction of lymphoma cells, but the effect on proliferation and apoptosis of lymphoma cells were not detected after RNAi, which need to be elucidated further.

Effect of flow rate, negative pressure, and duration of modified ultrafiltration on hemodynamics and inflammatory mediators.

The objective of this study was to evaluate the effect of flow rate, negative pressure, and duration on modified ultrafiltration (MUF). Eighty children weighing less than 10 kg with congenital heart disease were randomly divided into four groups: group C (conventional MUF); group H (high flow rate MUF); group P (high negative pressure MUF); and group L (long duration, high flow rate MUF). The changes in body weight, hematocrit, and hemodynamics were recorded. Tumor necrosis factor and interleukin-6 were measured before bypass, bypass stop, and MUF cessation. The durations of MUF in groups H and P were significantly shorter than in the other two groups; the volume filtered in group L was much greater than in the other three groups. The changes of bodyweight, heart rate, blood pressure, and hematocrit were similar in all groups. The increased extent of inflammatory mediators was a little lower in group L. Modified ultrafiltration can reverse hemodilution and improve cardiac function even with high flow rate or negative pressure. Prolonging the duration of MUF can filter out more inflammatory mediators, but the increased trend cannot be reversed in 15 minutes.

[Expression of retinoblastoma protein in child acute leukemia cells and its clinical significance].

The research was aimed to detect the expression levels of retinoblastoma protein (pRb) in child acute leukemia cells, and to explore its possible association with leukemia cells cycle, the risk of disease, minimal residual disease (MRD) monitoring and prognosis of B-ALL. Flow cytometry (FCM) was used to detect the expression of pRb in 89 cases of acute leukemia (including 25 AML, 10 T-ALL and 54 B-ALL) and bone marrows from 7 normal children (control group). Meanwhile the cell cycle in some cases was analyzed. The results showed that (1) the FCM could accurately detect the expression of pRb in acute leukemia cells; (2) the high level of pRb expression was frequent in all types of child acute leukemias. In the same case, the expression of pRb was significantly increased in leukemia cells when compared with non-leukemia cells. And no detectable pRb protein was found in partial cases of acute leukemia; (3) there was a close relation between expression of pRb and the cell cycle of leukemia cells, the number of G(1) phase cells in pRb positive case of B-ALL was more than that in pRb negative case (92% vs 77%); (4) in B-ALL, the level of pRb expression in MRD positive group was significantly lower than that in MRD negative group (P < 0.05), but pRb expression was stable in non-leukemia cells during therapy; (5) pRb expression was related to the early response to therapy in B-ALL, the expression of pRb was significantly increased in sensitive group when compared with insensitive group (P < 0.05). It is concluded that high level or absence of pRb expression can be found in child acute leukemia cells. The expression of pRb is positively related to cell cycle of leukemia cells, MRD monitoring and the early response to therapy. In short, the detection of pRb expression level can guide the therapy and the evaluation of prognosis in B-ALL.

[Prognostic significance of lymphocyte function associated anti-gen-3 (CD58) in childhood B cell-acute lymphocytic leukemia].

This study was aimed to investigate the value of CD58 in evaluation of early therapeutic effect on childhood B-ALL. The expression features of CD58 in 135 cases of childhood B-ALL were analyzed by four-color flow cytometry; MRD detection protocol for B-ALL using CD58/CD10/CD34/CD19 combination was established; the correlation between the expression features of CD58 and MRD detection was analyzed for the early therapeutic response in childhood B-ALL. The results showed that the mean value of CD58 MFI in 135 cases of B-ALL was 113.08 +/- 63.33, which was significantly higher than that in 15 cases of normal bone marrow controls (14.68 +/- 5.26, P < 0.01). In addition, CD58 was over expressed in 51.9% (70/135) of B-ALL patients, indicating that CD58 could be an effective marker in MRD detection. The CD58/CD10/CD34/CD19 was the second most effective combination next to TdT/CD10/CD34/CD19 in B-ALL MRD detection with flow cytometry. Meanwhile, the positive rate of MRD detection by flow cytometry was significantly lower in CD58 over expression group (P < 0.05). It is concluded that CD58 may be used as an indicator for detection of MRD in B-ALL patients, which would enrich the combination of MRD detection. The CD58 over expression may be considered as a marker of a favorable prognosis in childhood B-ALL.

[Expression of the transcription factor PAX5 in childhood acute leukemic cells].

To investigate transcription factor PAX5 expression characteristics in childhood acute leukemic cells, expression levels of PAX5 and CD19 mRNA in 6 hematological tumor cell lines and bone marrow cells of 6 normal children, 58 de novo patients and 4 relapse acute leukemic children, including 39 cases of B-ALL, 10 cases of T-ALL and 13 cases of AML, were detected by a real-time RT-PCR. The results showed that PAX5 and CD19 mRNA expression levels were 2.35% and 2.52% in Namalwa (B-cell lines) respectively, but almost not detectable in other T- and myeloid cell lines. Among clinical samples, expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML (P = 0.029 and P = 0.013 respectively). PAX5 expression was significantly lower in T-ALL and AML than that in normal controls. The difference of PAX5 mRNA expression levels between T-ALL and AML was not significant. Individual difference of PAX5 mRNA expression levels in children with B-ALL was great. Moreover, PAX5 mRNA expressions in de novo and relapse patients with B-ALL were significantly higher than those in remission (P = 0.011 and P = 0.006 respectively). As binding sites for B-cell specific activator protein have been identified in the promoter regions of CD19, the study found that in B-ALL, there was clear correlation between the expression levels of PAX5 and CD19, which was also studied by real-time RT-PCR. It is concluded that PAX5 transcripts are readily detectable and quantifiable in clinical materials with B-ALL by real-time RT-PCR. The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.

Ets1 as a marker of malignant potential in gastric carcinoma.

AIM: Ets1 proto-oncogene is a transcription factor involved in the activation of several genes of tumor invasion and metastasis. We aimed to determine the relationship between the extent and intensity of Ets1 expression and patients' clinicopathological factors in gastric carcinoma. METHODS: Immunohistochemical analysis was performed for gastric tumor paraffin-embedded sections, followed by image analysis. RESULTS: Ets1 was not expressed in the normal gastric epithelium and its surrounding cells. The percentage of Ets1 expressing cells detected increased significantly in both epithelial tumor and stromal cells from high T classification, lymph node metastasis positive, clinical advanced-stage groups (P<0.001). The level of Ets1 staining in epithelial tumor cells also reflected the degree of cell differentiation. The percentage of epithelial and stromal cells expressing Ets1 was significantly correlated with the presence of lymph node metastasis (P=0.014 and P<0.001 respectively). Ets1 expression was not observed in tissue samples from patients with benign gastric ulcers. CONCLUSION: Ets1 protein expression in epithelial tumor cells reflects the degree of differentiation, and the percentage of Ets1 positive tumor and stromal cells correlates with lymph node metastasis. Thus Ets1 is a valuable marker of malignant potential in terms of invasiveness and metastasis of gastric carcinoma. It is also possible that inhibition of Ets1 is a potential avenue for therapy in gastric cancer.

[Characterization of CD10 expression and its significance in minimal residual disease detection in childhood B-acute lymphoblastic leukemia].

To observe the expressions of CD10 in childhood B-acute lymphoblastic leukemia (B-ALL) and to define the role of CD10 in minimal residual disease (MRD) detection. 58 cases of childhood B-ALL were studied in this program. Four-color flow cytometry was used to analyze the characteristics of B-ALL phenotypes. The four-color fluorochrome labeled antibody combinations of CD10 with other markers were used to detect MRD. The results showed that CD10 overexpression (CD10(bright)) was detected in 65.5% (38/58) of B-ALL patients and a strong correlation between CD10(bright) and CD34 expression was also observed, i.e. CD10(bright) expression most frequently happened in B-ALL with high percentage of CD34 positive cells. In detection of MRD, CD10(bright), combined with other markers, could effectively distinguish normal cells with leukemic cells, even if there was no any other marker that can be used. It is concluded that CD10(bright) expression correlates with high expression of CD34 in B-ALL, it is a good marker for MRD detection. The combination of CD10 and other markers can be applied in B-ALL MRD detection with flow cytometry.

[Detection of minimal residual disease in childhood B-ALL by flow cytometry].

OBJECTIVE: To establish a flow cytometric method for detecting minimal residual disease (MRD) in children with B-ALL and evaluate its clinical application. METHODS: Fifty-eight childhood B-ALL cases entered this study and 30 MRD analyses were performed after remission induction therapy. Four-color fluorochrome labeled monoclonal antibodies were used to analyze the cell immunophenotypes. Cells from normal bone marrow were used as controls. The leukemic cell populations located in flow cytometry dot plots different from those of normal were considered to be the markers of interest in the first step screening, and then used to monitor MRD step after therapy. RESULTS: Fifty-eight cases of childhood B-ALL were screened for antibodies combinations of interest and were identified in 89.7% (52/58) of these cases. The four-color antibody combinations consisted of CD(10)/CD(34)/CD(19) plus another effective marker such as CD(38), CD(65), CD(66c), CD(21). The sensitivity of this method was 0.01%, much higher than microscopic inspection. In 8 cases whose bone marrow microscopically showed no residual leukemic cells, the percentage of leukemic cells were identified with this method of 0.028%, 1.430%, 3.050%, 0.015%, 5.660%, 2.700%, 0.027%, and 0.069%, respectively. CONCLUSION: The application of flow cytometry in MRD monitoring can significantly improve the detection sensitivity in childhood B-ALL, thus facilitate the further treatment decision and follow-up.