Potential effects of Cu2+ stress on nitrogen removal performance, microbial characteristics, and metabolism pathways of biofilm reactor.

Sequencing batch biofilm reactors (SBBR) were utilized to investigate the impact of Cu2+ on nitrogen (N) removal and microbial characteristics. The result indicated that the low concentration of Cu2+ (0.5 mg L-1) facilitated the removal of ammonia nitrogen (NH4+-N), total nitrogen (TN), nitrate nitrogen (NO3--N), and chemical oxygen demand (COD). In comparison to the average effluent concentration of the control group, the average effluent concentrations of NH4+-N, NO3--N, COD, and TN were found to decrease by 40.53%, 17.02%, 10.73%, and 15.86%, respectively. Conversely, the high concentration of Cu2+ (5 mg L-1) resulted in an increase of 94.27%, 55.47%, 22.22%, and 14.23% in the aforementioned parameters, compared to the control group. Low concentrations of Cu2+ increased the abundance of nitrifying bacteria (Rhodanobacter, unclassified-o-Sacharimonadales), denitrifying bacteria (Thermomonas, Comamonas), denitrification-associated genes (hao, nosZ, norC, nffA, nirB, nick, and nifD), and heavy-metal-resistant genes related to Cu2+ (pcoB, cutM, cutC, pcoA, copZ) to promote nitrification and denitrification. Conversely, high concentration Cu2+ hindered the interspecies relationship among denitrifying bacteria genera, nitrifying bacteria genera, and other genera, reducing denitrification and nitrification efficiency. Cu2+ involved in the N and tricarboxylic acid (TCA) cycles, as evidenced by changes in the abundance of key enzymes, such as (EC:1.7.99.1), (EC:1.7.2.4), and (EC:1.1.1.42), which initially increased and then decreased with varying concentrations of Cu2+. Conversely, the abundance of EC1.7.2.1, associated with the accumulation of nitrite nitrogen (NO2--N), gradually declined. These findings provided insights into the impact of Cu2+ on biological N removal.

Application of calcium peroxide in promoting resource recovery from municipal sludge: A review.

The harmless disposal, resource recovery, and synergistic efficiency reduction of municipal sludge have been the research focuses for the last few years. Calcium peroxide (CaO2) is a multifunctional and safe peroxide that produces an alkaline oxidation environment to promote the fermentation of municipal sludge to produce hydrogen (H2) and volatile fatty acids (VFAs), thus realizing sludge resource recovery. This review outlines the research achievements of CaO2 in sludge resource recovery, improvement of sludge dewaterability, and removal of pollutants from sludge in recent years. Meanwhile, the mechanism of CaO2 and its influencing factors have also been comprehensively summarized. Finally, the future development direction of the application of CaO2 in municipal sludge is prospected. This review would provide theoretical reference for the potential engineering applications of CaO2 in improving sludge treatment in the future.

An injectable, in situ forming and NIR-responsive hydrogel persistently reshaping tumor microenvironment for efficient melanoma therapy.

BACKGROUND: Melanoma is a highly aggressive form of skin cancer with increasing incidence and mortality rates. Chemotherapy, the primary treatment for melanoma, is limited by hypoxia-induced drug resistance and suppressed immune response at the tumor site. Modulating the tumor microenvironment (TME) to alleviate hypoxia and enhance immune response has shown promise in improving chemotherapy outcomes. METHODS: In this study, a novel injectable and in situ forming hydrogel named MD@SA was developed using manganese dioxide (MnO2) nanosheets pre-loaded with the chemotherapy drug doxorubicin (DOX) and mixed with sodium alginate (SA). The sustainable drug delivery, oxygen generation ability, and photothermal property of MD@SA hydrogel were characterized. The therapeutic efficacy of hydrogel was studied in B16F10 in vitro and B16F10 tumor-bearing mice in vivo. The immune effects on macrophages were analyzed by flow cytometry, real-time quantitative reverse transcription PCR, and immunofluorescence analyses. RESULTS: The MD@SA hydrogel catalyzed the tumoral hydrogen peroxide (H2O2) into oxygen, reducing the hypoxic TME, down-regulating hypoxia-inducible factor-1 alpha (HIF-1α) and drug efflux pump P-glycoprotein (P-gp). The improved TME conditions enhanced the uptake of DOX by melanoma cells, enhancing its efficacy and facilitating the release of tumor antigens. Upon NIR irradiation, the photothermal effect of the hydrogel induced tumor apoptosis to expose more tumor antigens, thus re-educating the M2 type macrophage into the M1 phenotype. Consequently, the MD@SA hydrogel proposes an ability to constantly reverse the hypoxic and immune-inhibited TME, which eventually restrains cancer proliferation. CONCLUSION: The injectable and in situ forming MD@SA hydrogel represents a promising strategy for reshaping the TME in melanoma treatment. By elevating oxygen levels and activating the immune response, this hydrogel offers a synergistic approach for TME regulation nanomedicine.

Platinum nanoparticles (PtNPs)-based CRISPR/Cas12a platform for detection of nucleic acid and protein in clinical samples.

Early rapid screening diagnostic assay is essential for the identification, prevention, and evaluation of many contagious or refractory diseases. The optical density transducer created by platinum nanoparticles (PtNPs) (OD-CRISPR) is reported in the present research as a cheap and easy-to-execute CRISPR/Cas12a-based diagnostic platform. The OD-CRISPR uses PtNPs, with ultra-high peroxidase-mimicking activity, to increase the detection sensitivity, thereby enabling the reduction of detection time and cost. The OD-CRISPR can be utilized to identify nucleic acid or protein biomarkers within an incubation time of 30-40min in clinical specimens. In the case of taking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene as an instance, when compared to a quantitative reverse transcription-polymerase chain reaction (RT-qPCR), the OD-CRISPR test attains a sensitivity of 79.17% and a specificity of 100%. In terms of detecting prostate-specific antigen (PSA), aptamer-based OD-CRISPR assay achieves the least discoverable concentration of 0.01 ng mL-1. In general, the OD-CRISPR can detect nucleic acid and protein biomarkers, and is a potential strategy for early rapid screening diagnostic tools.

Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identification of SARS-CoV-2 from clinical samples.

The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).

Chromosomal microarray analysis of infertile men with azoospermia factor microdeletions.

Azoospermia factors, located in the long arm of the Y chromosome, are critical for spermatogenesis, the microdeletions of AZF are considered to be associated with male infertility. In addition to complete deletion, several AZFc partial deletions were also detected in infertile men with wide phenotypic heterogeneity. In this study, we investigated the relevance of Y chromosome deletions, Y-linked CNVs and variable phenotypes in infertile men. To clarify the relationship between phenotypic heterogeneity and Y chromosome deletion in male infertility, we performed chromosomal microarray analysis (CMA) capable of analyzing thousands of loci simultaneously to investigate Y-linked copy number variations (CNVs). Firstly, we reviewed the results of Y chromosome screening in 554 infertile patients and then compared the results of CMA to routine Y chromosome screening in 29 patients with Y chromosomal microdeletions. Then, the Y-linked CNVs associated with oligoasthenospermia were identified according to ACMG standards and guidelines. The results indicated that the prevalence of Yq microdeletions was 5.23% (29/554), with 93% (27/29) of the deletions in the AZFc region among 554 infertile men recruited in this study. The results of CMA and multiplex-PCR-based AZFc deletion analysis were generally concordant, but CMA provided more details about location, size and OMIM genes involved in deletion fragments of the AZF region. Of 29 clinically infertile phenotype-related CNVs detected by CMA, nine were pathogenic and the remaining 20 CNVs were OVUS. Except for a 15.69 Mb loss CNV in AZFa + b + c and an 8.25 Mb loss CNV in AZFb + c, others were located in the AZFc region. Based on a combination of the clinical symptoms and loss CNVs, we concluded that the CNV size and the involvement of spermatogenesis critical genes are two important factors that determine the relevance of a CNV in the AZFc region to the presence or absence of a clinically infertile phenotype.

Potential circulating biomarkers of circulating chemokines CCL5, MIP-1ß and HA as for early detection of cirrhosis related to chronic HBV (hepatitis B virus) infection.

BACKGROUND: Due to no clinical symptoms in the compensated stage of cirrhosis, it is usually diagnosed when decompensated complications occur. In this study, the noninvasive circulating biomarkers for early detection to compensated stage of cirrhosis in patients with chronic HBV (hepatitis B virus) infection was explored. METHODS: According to the Guideline of Prevention and Treatment of Chronic Hepatitis B (2015 Update), 78 patients with CHB (chronic hepatitis B) were divided into mild group, moderate-to-advanced group, while 73 patients with HBV-related cirrhosis were divided into compensated group and decompensated group. Nineteen cytokines and chemokines, four serum liver fibrosis markers were measured using chemiluminescence. The expression of CCL5 in liver tissue was determined with immunohistochemistry. RESULTS: The CCL5 expression level in serum increased in CHB patients with aggravated liver injury and significantly decreased in cirrhosis patients with advanced liver fibrosis. ROC analysis revealed that the serum levels of CCL5, HA and MIP-1ß were effective in distinguishing patients with cirrhosis from patients with CHB, especially for CCL5. Increasing serum level of CCL5 in CHB patients was severely associated with disease progression. CONCLUSIONS: The serum levels of CCL5, HA and MIP-1ß maybe used to distinguish cirrhosis from CHB patients, moreover, CCL5 was the most reliable marker. The increasing serum levels of CCL5 were significantly related to disease progression in CHB patients.

miR-494 induces EndMT and promotes the development of HCC (Hepatocellular Carcinoma) by targeting SIRT3/TGF-ß/SMAD signaling pathway.

EndMT has an important effect on metastasis and progression of tumor. This work will elucidate the effect of miR-494 on EndMT and development of HCC. Therefore, the differential miRNA expression among non-tumorous, para-tumorous and tumorous tissues was analyzed. Moreover, luciferase activities of SIRT3 3'UTR treated with miR-494 were determined. Then human hepatoma cell lines were dealt with mimics or inhibitors of miR-494, migration and proliferation ability were assessed. The expression of SIRT3 and markers of mesenchymal cell were analyzed. The influences of miR-494 on development of HCC through inducing EndMT by targeting SIRT3 and TGF-ß/SMAD signaling pathways in hepatoma cell lines were investigated. Xenograft mice were used to explore the potential roles of miR-494 on EndMT and development of HCC in vivo. Our results showed that, compared with non-tumorous tissues, 17 miRNAs were upregulated and 3 miRNAs were down-regulated in tumor tissues. In tumor tissues, the miR-494 expression level was much more than the expression of para-tumorous and non-tumorous tissues. MiR-494 suppressed SIRT3 expression, additionally enhanced expression of mesenchymal cell markers, while exerted effects on cell proliferation and migration of hepatoma cell lines. Moreover, the antagomir of miR-494 could protect against development process in xenogarft murine model. In conclusions, our work demonstrated that miR-494 targeted to SIRT3, and was a crucial mediator of EndMT and development of HCC through regulating SIRT3/TGF-ß/SMAD signaling pathway. It suggested that aim at SIRT3/TGF-ß/SMAD signaling pathway through suppressing the miR-494 expression level, was a feasible therapy strategy for HCC.

Spectral-optical-tweezer-assisted fluorescence multiplexing system for QDs-encoded bead-array bioassay.

As an efficient tool in the multiplexed detection of biomolecules, bead-array could achieve separation-free detection to multiple targets, making it suitable to analyze valuable and scarce samples like antigen and antibody from living organism. Herein, we propose a spectral-optical-tweezer-assisted fluorescence multiplexing system to analyze biomolecule-conjugated bead-array. Using optical tweezer, we trapped and locked beads at the focus to accept stimulation, offering a stable and optimized analysis condition. Moving the system focus and scanning the sample slide, we achieved emissions collection to QDs-encoded bead-array after the multiplexed detection. The emission spectra of fluorescence were collected and recorded by the spectrometer. By recognizing locations of decoding peaks and counting the intensities of label signals of emission spectra, we achieved qualitative and quantitative detection to targets. As proof-of-concept studies, we use this system to carry out multiplexed detection to various types of anti-IgG in the single sample and the detection limit reaches 1.52 pM with a linear range from 0.31 to 10 nM. Through further optimization of experimental conditions, we achieved specific detection to target IgG with sandwich method in human serum and the detection limit reaches as low as 0.23 pM with a linear range from 0.88 to 28 pM, validating the practical application of this method in real samples.

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice, and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro.

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross-talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G-protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G-protein activation, plays an important role in embryo implantation by interfering in the cross-talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17ß-estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17ß-estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17ß-estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.

MicroRNA-196b promotes cell proliferation and suppress cell differentiation in vitro.

MicroRNA-196b (miR-196b) is frequently amplified and aberrantly overexpressed in acute leukemias. To investigate the role of miR-196b in acute leukemias, it has been observed that forced expression of this miRNA increases proliferation and inhibits apoptosis in human cell lines. More importantly, we show that this miRNA can significantly increase the colony-forming capacity of mouse normal bone marrow progenitor cells alone, as well as partially blocking the cells from differentiation. Taken together, our studies suggest that miRNA-196b may play an essential role in the development of MLL-associated leukemias through inhibiting cell differentiation and apoptosis, while promoting cell proliferation.

Real-time fluorescence Loop-Mediated Isothermal Amplification (LAMP) for rapid and reliable diagnosis of pulmonary tuberculosis.

A reliable, simple and rapid diagnostic method that can be helpful in pulmonary tuberculosis diagnosis is urgently needed. Loop-mediated Isothermal Amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. In this study, real-time fluorescence LAMP was evaluated to rapidly detect Mycobacterium tuberculosis in sputum and was compared to the performance of real-time fluorescence quantitative PCR (Q-PCR). All the standard MTB strains were successfully detected and limit of detection (LOD) was 10(2)CFU/mL by real-time fluorescence LAMP within 20min. In light of MTB in sputum, the real-time fluorescence LAMP method yielded a sensitivity of 98.0% and a specificity of 78.3%, compared to Q-PCR assay, which yielded a sensitivity of 96.0% and a specificity of 82.6% for PTB diagnosis. There was an excellent overall agreement between LAMP and Q-PCR for PTB (κ=0.315) and non-PTB (κ=0.862). Therefore, the real-time fluorescence LAMP assay is a rapid, sensitive, and specific method to detect pulmonary tuberculosis.

[Enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol].

OBJECTIVE: To explore the enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol. METHODS: The value of 50% inhibition concentration (IC50) for KG-1a by resveratrol was analyzed using trypan blue staining. Peripheral blood mononuclear cells were separated, and then activated by interleukin (IL)-2 and IL-15. The sensitivity of KG-1a treated with and without resveratrol to activated immune cell-mediated cytolysis was assayed by lactate dehydrogenase (LDH) -releasing assay. The expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the surface of activated immune cells and its receptors (DR4/5 and DcR1/2) on the surface of KG-1a were detected by flow cytometry. RESULTS: Resveratrol could inhibit the proliferation of KG-1a and IC50 at 24 h was 25 mmol/L. At a ratio of 10:1 or 20:1 between effect and target, the cytolytic rates of treated KG-1a by activated immune cells were (55.80 ± 10.88)% and (72.31 ± 13.06)%, significantly higher than (24.96 ± 9.25)% and (37.93 ± 5.21)% of untreated KG-1a (P<0.05). The expression of DR5 on the surface of KG-1a treated with resveratrol was (9.05 ± 3.57)%, significantly higher than (3.11 ± 0.54)% of untreated KG-1a (P<0.05). Conversely, the expression of DcR1 on the surface of treated KG-1a was (13.23 ± 3.56)%, lower than (53.75 ± 10.51)% of KG-1a (P<0.05). When TRAIL pathway on the surface of activated immune cells was blocked, the cytolytic rates of treated KG-1a were (35.97 ± 6.36)% and (49.80 ± 10.68)%, significantly lower than (52.92 ± 6.98)% and (70.73 ± 9.79)% of untreated KG-1a (P<0.05) at the same ratio of effector and target. CONCLUSION: Resveratrol could enhance cytolytic sensitivity of KG-1a by activated immune cells through TRAIL pathway.

Eps8 vaccine exerts prophylactic antitumor effects in a murine model: a novel vaccine for breast carcinoma.

Cancer vaccines are an effective way to prevent the occurrence of cancer. Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel tumor-associated antigen, which is overexpressed in the majority of tumor types. In the present study, the Eps8 protein was cloned and characterized, and its feasibility as an antitumor agent in murine breast carcinoma was investigated. The results revealed that the Eps8 protein increased the secretion of interleukin (IL)-12 in the culture supernatant of dendritic cells (DCs). The Eps8 protein­pulsed DCs induced significant cytotoxic T lymphocyte (CTL) responses, T-cell proliferation and a higher level of interferon (IFN)-γ in the culture supernatant of the splenocytes ex vivo. Additionally, when the mice were immunized with the Eps8 vaccine, this resulted in a regression of 4T1 breast tumors and significantly prolonged survival time in the tumor­bearing mice compared with that in the phosphate-buffered saline (PBS) control group. The Eps8 vaccine induced higher CTL responses in the splenocytes of mice vaccinated against the 4T1 cells; the ratio of CD4+/CD8+ T cells was increased in the Eps8 group; and the percentage of CD4+CD25+ FoxP3+ regulatory T (Treg) cells in the Eps8 group was significantly lower compared with that of the PBS group. The results suggested that the Eps8 vaccine was able to stimulate antitumor effects against 4T1 breast cancer cells in vitro and in vivo, and it may provide a potential immunotherapeutic agent for the treatment of breast cancer.

[NVP-BEZ235 inhibits proliferation and colony-forming capability of CD34(+)CD38(-) human acute myeloid leukemia stem cells].

This study was aimed to explore the effect of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle and colony forming capability of CD34(+)CD38(-) human acute myeloid leukemia (AML) KG1a cells. Flow cytometry was used to detect expression of CD34 and CD38 on the surface of human AML KG1a cells; Trypan blue assay was used to analyze the effect of NVP-BEZ235 at various concentrations on proliferation of KG1a cells; flow cytometry was performed to examine the cell cycle of KG1a cells after NVP-BEZ235 treatment; Soft agar colony-forming experiment was used to detect the colony forming ability of KG1a cells treated with NVP-BEZ235 at various concentrations. The results indicated that the percentage of CD34(+)CD38(-) AML KG1a cells was (98.02 ± 0.72)%. NVP-BEZ235 (0.125 - 1 µmol/L) inhibited the proliferation of KG1a cells in a time-and dose-dependent manner (P < 0.05) and the 50% inhibition concentrations (IC50) at 24 h and 48 h were 0.597 µmol/L and 0.102 µmol/L, respectively. KG1a cells were arrested at G0/G1 phase after treating with 0.5 µmol/L NVP-BEZ235 for 24 h, it was significantly higher than that of control group (83.2 ± 3.80)% vs (43.47 ± 9.60)% (P < 0.05). KG1a cells treated with NVP-BEZ235 (0 - 1 µmol/L) for 14 d and 21 d, the number of colony decreased respectively from (375.67 ± 21.46) per 2500 KG1a cells and (706.33 ± 87.31) per 2500 KG1a cells to 0, with statistical significance (P < 0.05). It is concluded that NVP-BEZ235 can inhibit proliferation and colony-forming capability of CD34(+)CD38(-) human AML KG1a cells.

Resveratrol sensitized leukemia stem cell-like KG-1a cells to cytokine-induced killer cells-mediated cytolysis through NKG2D ligands and TRAIL receptors.

Human promyeloblastic leukemia KG-1a cells exhibit many characteristics similar to leukemia stem cells, which are resistant to chemotherapeutic drugs and hyposensitive to cytotoxic cells. Resveratrol (RES), as a member of plant polyphenols, has gained considerable attention due to its ability to prevent cancer from progressing. In this study, the potential of RES to sensitize KG-1a cells to cytolysis of cytokine-induced killer cells (CIKs) through NKG2D ligands and TNF-related apoptosis-inducing ligand (TRAIL) receptors were investigated. Twenty-five micromolars RES was found to inhibit approximately 50% of KG-1a cell growth and had the least growth-inhibition effect on peripheral blood mononuclear cells (PBMCs) after 24 h. Utilizing cytokines including interleukin-2 (IL-2) and interleukin-15 (IL-15) to activate PBMCs, we obtained substantial CD3 (+) CD56 (+) natural killer cell-like T lymphocytes that secreted cytokine interferon-γ (IFN-γ) and expressed NKG2D and TRAIL on their surfaces (i.e., cytokine-induced killer cells, CIKs). RES was shown to render KG-1a cells susceptible to CIK-mediated cytolysis estimated by LDH-release assay. This heightened sensitivity correlated with an increase in cell-surface expression of NKG2D ligands and death receptor 4 (DR4), coupled with a downregulation of cell-surface expression of decoy receptor 1 (DcR1) in KG-1a cells. Blocking NKG2D ligands or TRAIL with monoclonal antibodies could abrogate CIKs-mediated cytolysis. These results demonstrated that increased sensitivity of KG-1a cells, modulated by RES to alloreactive CIKs-mediated cytolysis is a phenomenon attributable to induced expression of NKG2D ligands and activation of TRAIL pathway. Thus, resveratrol combined with alloreactive CIKs merits clinical evaluation as a novel and effective immunotherapy strategy to eliminate residual leukemia stem cells.

Suppression of graft-versus-host disease after adoptive infusion of alloreactive NK cells induced by silencing Ly49C gene in mice.

BACKGROUND: To investigate suppression of graft-versus-host disease (GVHD) after adoptive infusion of alloreactive natural killer (NK) cells induced by silencing Ly49C gene. METHODS: Silencing efficiency was detected by quantitative real-time reverse transcriptase polymerase chain reaction and flow cytometric analysis. The recipient mice were randomly divided into three groups which were respectively treated with RNAi Ly49C alloreactive NK cells, NK cells alone and RPMI 1640 culture medium. The cumulative GVHD scores, pathological lesion of liver, spleen, gut and survival time in the recipients were observed. RESULTS: The valid one of four siRNA duplex we designed selectively suppressed Ly49C mRNA expression by 63% (Max 70%) when compared to control levels, while flow cytometric analysis indicated Ly49C protein expression on NK cells transfected with Ly49C-124 siRNA was significantly decreased in comparison with that of negative control or mock (P<0.05). The scores of GVHD in RNAi-Ly49C NK cells group, NK cells alone group and blank control group were (1.30+/-1.34), (3.10+/-2.33) and (5.70+/-1.77) during 30 days after transplantation, respectively. Pathologic manifestations of liver, spleen and gut in RNAi Ly49C alloreactive NK cells group were slighter to a certain extent than those in other two groups. Survival time(days) of simply irradiation group, RNAi Ly49C NK cells group, NK cells alone group and blank group were (8.70+/-1.49), (63.00+/-24.81), (36.00+/-17.38) and (24.40+/-7.44), respectively. CONCLUSIONS: Infusion adoptively of alloreactive NK cells induced by silencing Ly49C gene could reduce GVHD degree and prolong survival time in GVHD mice model.

[Expression of NKG2D ligands on dendritic cells at different development stages and its effect on cytotoxicity of NK cells].

AIM: To investigate the expression of NKG2D ligands on dendritic cells(DC) at different development stages and its effect on cytotoxicity of natural killer(NK) cells. METHODS: The monocytes were cultured into immature dendritic cells(iDC) and mature dendritic cells(mDC) with cytokines. NK cells were obtained from normal peripheral blood by CD56 antibody magnetic isolation.The expression of NKG2D ligands (MICA/B, ULBP1-3) was detected by flow cytometry. Cytotoxicity of NK cells and the NK cells blocked with anti-NKG2D mAbs against iDC and mDC was tested using LDH-releasing method. RESULTS: IDC and mDC were of typical morphology and phenotypes. MICA, MICB, ULBP1, and ULBP3 were expressed on iDC and their expression rate was (32.39+/-8.30)%, (17.75+/-3.40)%, (26.71+/-6.48)%, (38.37+/-6.89)%, respectively. MICA and ULBP3 were expressed on mDC and their expression rate was (7.81+/-3.33)% and (8.36+/-2.42)%, respectively, which was lower than that on mDC (P<0.01). At the each E:T ratio cytotoxicity of NK cells against iDC was stronger than that against mDC (P<0.01). cytotoxicity of NK cells blocked with anti-NKG2D mAb against iDC was decreased compared with that of NK cells unblocked (P<0.05) while cytotoxicity of NK cells blocked with anti-NKG2D mAb against mDC showed no decrease compared with that of NK cells unblocked (P>0.05). CONCLUSION: The expression of NKG2D ligands on iDC is higher than that on mDC, which plays an important role in the cytotoxic effect of NK cells against iDC, but has no effect on that against mDC. NKG2D-NKG2D ligands shows one of the molecular mechanisms that NK cells kill iDC selectively.

[Cytotoxicity of allogenetic natural killer cells against CD34+ acute myelogenous leukemia cells].

OBJECTIVE: To study the cytotoxic effect of allogenetic natural killer (NK) cells in vitro on human CD34+ acute myelogenous leukemia cells. METHODS: CD34 expression on acute myelogenous leukemia KG1a cells was detected by flow cytometry. KG1a cells were co-cultured at different effector-to-target (E:T) ratios with NK cells isolated from 5 healthy individuals using magnetic cell sorting. Lactate dehydrogenase (LDH) release assay was employed to examine the cytolysis of KG1a cells in the co-culture, and the inhibition rate of the KG1a cell colony formation in methylcellulose was determined with K562 cells sensitive to NK cells as the control. RESULTS: A expression rate as much as (98.0-/+1.1)% was detected for CD34 antigen on KG1a cells, and the isolated NK cells (CD3(-)CD16+CD56+ cells) had a purity of (93.2-/+3.7)% after magnetic cell sorting. Allogenetic NK cells exhibited obvious cytotoxicity and colony inhibition in vitro against KG1a cells at different E:T ratios, and the effects were significantly enhanced as the E:T ratios increased (P<0.05). At the same E:T ratio, the cytotoxicity and colony inhibition rate of allogenetic NK cells against KG1a cells was lower than those against K562 cells (P<0.05). CONCLUSION: Allogenetic NK cells exhibit obvious cytotoxicity and colony formation against CD34+ acute myelogenous leukemia cells.

LFA-1 and VLA-4 involved in human high proliferative potential-endothelial progenitor cells homing to ischemic tissue.

Cumulative evidences have revealed that endothelial progenitor cell (EPC) transplantation can promote the neovascularization in ischemic tissue, but the mechanism of EPCs homing to the site of ischemia is poorly understood. In this study, to investigate the mechanism of human umbilical cord blood-derived high proliferative potential-endothelial progenitor cells (HPP-EPCs) homing to ischemic tissue we evaluated the expression of lymphocyte function-associated antigen-1 (LFA-1, or CD11a/CD18) and very late antigen-4 (VLA-4, or CD49d/CD29) in EPCs and the changes of expression level of their ligands, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in ischemic tissue and performed the adhesion and migration assays to analyze the interaction between the receptors and ligands. Furthermore, we studied the roles of LFA-1 and VLA-4 in EPC homing in an ischemic model of mice. The results show that LFA-1 andVLA-4 were expressed in HPPEPCs and ICAM-1 and VCAM-1 were expressed in vessel endothelium in ischemic tissues. The pre-incubation of HPP-EPCs with neutralizing antibodies against CD11a or CD49d reduced adhesion and migration of HPP-EPCs in vitro and reduced recovery of hind-limb blood flow, capillary density and incorporation of HPP-EPC into ischemic tissues in vivo. Furthermore, the pre-incubation of HPP-EPCs with the combination of CD11a and CD49d antibodies led to synergistically negative effects on adhesion and transmigration of HPP-EPCs in vitro, and on the homing of HPP-EPCs to ischemic tissue and on neovascularization capacity in vivo. These results indicate that LFA-1 andVLA-4 are involved in HPP-EPC homing to ischemic tissues.