Hemolymph Node - An Immunomorphlogical Organ: Modeling the Hemolymph Node by Allografting Renal Tissue in the Rat.

There is no authoritative characterization of the attributes of the hemolymph node (HLN) since Gibbes' first description in 1884. Early reports showed that HLN are found near the kidney in human and animals with the feature of numerous erythrocytes in sinuses. Subsequent studies mainly focused on anatomy and histology, such as the source, distribution, and quantity of erythrocytes in sinuses. Recent articles mentioned that the emergence of HLN was related to immunity, but there was no strong evidence to support this hypothesis. Therefore, it is still uncertain whether the HLN is an organ of anatomy, histology, or immunology. It has been found that the development of HLN could be elicited in the parathymic area by stimuli such as Escherichia coli, allogeneic breast cancer cells, and renal tissue that were injected/transplanted into the tail of rats in our pilot studies. In this study, the model of the HLN was established by transferring allogeneic renal tissue in the rat. Intrasinusoidal erythrocytes of the node were the component for producing a red macroscopic appearance, while macrophage-erythrocyte-lymphocyte rosettes were the major immunomorphological changes, reflecting the immune activity against the invasion of the allogeneic tissue within the node. Therefore, the HLN is an immunomorphological organ.

circTADA2As suppress breast cancer progression and metastasis via targeting miR-203a-3p/SOCS3 axis.

More and more evidence indicates that circular RNAs (circRNAs) have important roles in several diseases, especially in cancers. However, their involvement remains to be investigated in breast cancer. Through screening circRNA profile, we identified 235 differentially expressed circRNAs in breast cancer. Subsequently, we explored the clinical significance of two circTADA2As in a large cohort of triple-negative breast cancer (TNBC), and performed functional analysis of circTADA2A-E6 in vitro and in vivo to support clinical findings. Finally, we evaluated the effect of circTADA2A-E6 on miR-203a-3p and its target gene SOCS3. We detected two circRNAs, circTADA2A-E6 and circTADA2A-E5/E6, which were among the top five differentially expressed circRNAs in breast cancer. They were consistently and significantly decreased in a large cohort of breast cancer patients, and their downregulation was associated with poor patient survival for TNBC. Especially, circTADA2A-E6 suppressed in vitro cell proliferation, migration, invasion, and clonogenicity and possessed tumor-suppressor capability. circTADA2A-E6 preferentially acted as a miR-203a-3p sponge to restore the expression of miRNA target gene SOCS3, resulting in a less aggressive oncogenic phenotype. circTADA2As as promising prognostic biomarkers in TNBC patients, and therapeutic targeting of circTADA2As/miRNA/mRNA network may be a potential strategy for the treatment of breast cancer.

Pigment Epithelium-Derived Factor (PEDF) Improves Ischemic Cardiac Functional Reserve Through Decreasing Hypoxic Cardiomyocyte Contractility Through PEDF Receptor (PEDF-R).

BACKGROUND: Pigment epithelium-derived factor (PEDF), which belongs to the noninhibitory serpin family, has shown the ability to stimulate several physiological processes, such as antiangiogenesis, anti-inflammation, and antioxidation. In the present study, the effects of PEDF on contractility and calcium handling of rat ventricular myocytes were investigated. METHODS AND RESULTS: Adult Sprague-Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF-lentivirus was delivered into the myocardium along and away from the infarction border to overexpress PEDF. Video edge detection was used to measure myocyte shortening in vitro. Intracellular Ca(2+) was measured in cells loaded with the Ca(2+) sensitive fluorescent indicator, Fura-2-acetoxymethyl ester. PEDF local overexpression enhanced cardiac functional reserve in AMI rats and reduced myocardial contracture bordering the infracted area. Exogenous PEDF treatment (10 nmol/L) caused a significant decrease in amplitudes of isoproterenol-stimulated myocyte shortening, Ca(2+) transients, and caffeine-evoked Ca(2+) transients in vitro. We then tested a potential role for PEDF receptor-mediated effects on upregulation of protein kinase C (PKC) and found evidence of signaling through the diacylglycerol/PKCα pathway. We also confirmed that pretreatment of cardiomyocytes with PEDF exhibited dephosphorylation of phospholamban at Ser(16), which could be attenuated with PKC inhibition. CONCLUSIONS: The results suggest that PEDF depresses myocyte contractility by suppressing phosphorylation of phospholamban and Ca(2+) transients in a PKCα-dependent manner through its receptor, PEDF receptor, therefore improving cardiac functional reserve during AMI.

PEDF improves cardiac function in rats with acute myocardial infarction via inhibiting vascular permeability and cardiomyocyte apoptosis.

Pigment epithelium-derived factor (PEDF) is a pleiotropic gene with anti-inflammatory, antioxidant and anti-angiogenic properties. However, recent reports about the effects of PEDF on cardiomyocytes are controversial, and it is not known whether and how PEDF acts to inhibit hypoxic or ischemic endothelial injury in the heart. In the present study, adult Sprague-Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF-small interfering RNA (siRNA)-lentivirus (PEDF-RNAi-LV) or PEDF-LV was delivered into the myocardium along the infarct border to knockdown or overexpress PEDF, respectively. Vascular permeability, cardiomyocyte apoptosis, myocardial infarct size and animal cardiac function were analyzed. We also evaluated PEDF's effect on the suppression of the endothelial permeability and cardiomyocyte apoptosis under hypoxia in vitro. The results indicated that PEDF significantly suppressed the vascular permeability and inhibited hypoxia-induced endothelial permeability through PPARγ-dependent tight junction (TJ) production. PEDF protected cardiomyocytes against ischemia or hypoxia-induced cell apoptosis both in vivo and in vitro via preventing the activation of caspase-3. We also found that PEDF significantly reduced myocardial infarct size and enhanced cardiac function in rats with AMI. These data suggest that PEDF could protect cardiac function from ischemic injury, at least by means of reducing vascular permeability, cardiomyocyte apoptosis and myocardial infarct size.

Mangiferin regulates interleukin-6 and cystathionine-b-synthase in lipopolysaccharide-induced brain injury.

Mangiferin has been extensively applied in different fields due to its anti-inflammatory properties. However, the precise mechanism used by mangiferin on lipopolysaccharide (LPS)-induced inflammation has not been elucidated. Here, we discuss the potential mechanism of mangiferin during a LPS-induced brain injury. Brain injury was induced in ICR mice via intraperitoneal LPS injection (5 mg/kg). Open- and closed-field tests were used to detect the behaviors of mice, while immunoblotting was performed to measure the expression of interleukin-6 (IL-6) and cystathionine-b-synthase (CBS) in the hippocampus after mangiferin was orally administered (p.o.). Mangiferin relieved LPS-induced sickness 6 and 24 h after LPS injection; in addition, this compound suppressed LPS-induced IL-6 production after 24 h of LPS induction as well as the downregulation of LPS-induced CBS expression after 6 and 24 h of LPS treatment in the hippocampus. Therefore, mangiferin attenuated sickness behavior by regulating the expression of IL-6 and CBS.

[Ex vivo expansion and clonal variation of CD34(+)CD59(+) cells from bone marrow in children with paroxysmal nocturnal hemoglobinuria].

OBJECTIVE: To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH. METHODS: CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted. RESULTS: The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01). CONCLUSIONS: The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.

Toxic effects of cypermethrin on the male reproductive system: with emphasis on the androgen receptor.

The 15-day intact adult male assay was used to investigate the reproductive toxicity of cypermethrin. We also evaluated the contributions of the androgen receptor (AR) to cypermethrin-induced reproductive impairments. Sixty adult male Sprague-Dawley rats were randomly divided into five groups and treated with different doses of cypermethrin (0, 6.25, 12.5, 25 and 50 mg kg(-1) per day) by oral gavage for 15 days. After the rats were sacrificed, the testes, epididymides, seminal vesicles and prostates were excised and weighed. One testis was frozen to be used for daily sperm production. Another testis was processed for AR immunohistochemical analysis and electron microscopic observation. We found that the weights of prostates were significantly decreased in cypermethrin treatment at doses of 25 and 50 mg kg(-1) per day. Rats treated with cypermethrin at 50 mg kg(-1) per day exhibited a significant reduction in testicular daily sperm production. Seminiferous tubule changes were noted, including atrophy and distorted seminiferous tubules, reduction and deformation of spermatogonia, spermatocyte and disordered arrangement of spermatoblasts. Ultrastructural changes were found in cypermethrin-treated groups with disrupted cellular junctions, abnormal morphology of the nucleus, necrosis of spermatogonia spermatocytes and Sertoli cells. To clarify the possible mechanism, AR expression and the serum levels of testosterone were assayed. AR levels were significantly reduced in the rats treated with cypermethrin and the serum levels of testosterone were reduced in cypermethrin treatment at a dose of 50 mg kg(-1) per day. These data suggested that cypermethrin can induce impairments of the structure of seminiferous tubules and spermatogenesis in the male rats. The impairments can be attributed to the reduced AR expression.

Effects of isoflurane inhalation on the male reproductive system in rats.

The 15-day intact adult male assay was used to evaluate effects of isoflurane on the testes and sexual hormones. Forty adult male Sprague-Dawley rats were divided into five groups exposed to air containing 0, 50, 300, 1800 or 10,800 ppm isoflurane. After the treatments, serum was collected for the hormones assay. The right testis was to be used for daily sperm production. The left testis was processed for histopathology and electron microscopy observation. Daily sperm productions were significantly decreased at doses of 300, 1800 and 10,800 ppm. Impaired seminiferous tubules were noted at doses of 300, 1800 and 10,800 ppm. Ultrastructural changes included nucleus agglutination of spermatocytes, big lipid drops and autophagosome in cytoplasm. The serum follicle-stimulating hormone and testosterone concentrations reduced significantly at doses of 1800 and 10,800 ppm. Isoflurane induced impairments of seminiferous tubules and spermatogenesis. The testicular damages caused by isoflurane can be related to the imbalances in the sexual hormones.

Xanthogranulomatous inflammation of the female genital tract: report of three cases.

PURPOSE AND METHODS: This is a series of three cases diagnosed with xanthogranulomatous inflammation of the female genital with emphasis on the etiology, clinical-pathologic features and biological behavior. Clinical, pathologic, radiologic and follow up data are reported. RESULTS: The three cases of Xanthogranulomatous inflammation of the female genital tract are the followings: 1) one case affecting the endometrium, 2) one case affecting the fallopian tube, and 3) one case confined to the ovary. The patient's age was 37, 22 and 62 year-old, respectively. Histologic examination revealed extensive infiltration of foamy histiocytes admixed with variable amount of inflammatory cells. The later include plasma cells, lymphocytes, and occasional multinucleated giant cells. Immunohistochemistry showed positive staining for CD68, a histiocytic marker, in foamy histiocytes, CD3, a T cell marker, and CD20, a B cell marker, in the background lymphocytes. The plasma cells were polyclonal with expression of both κ and λ light chains. CONCLUSION: Xanthogranulomatous inflammation of the female genital tract is an unusual lesion, and clinically forms mass- like lesion in the pelvic cavity that invades the surrounding tissues, which may mimic the tumor clinically and by imaging.

Mobilization of bone marrow-derived Nkx2-5(+) cardiac progenitor cells under condition of acute myocardial ischemia.

The present study aimed to observe the morphological distribution of bone marrow (BM)-derived Nkx2-5(+) cardiac progenitor cells (CPCs) in bone marrow niche and evaluate the effect of acute myocardial ischemia (AMI) on the mobilizion of BM-derived Nkx2-5(+) CPCs. Animal models of BALB/c mouse AMI, cerebral and hind-limb ischemia were established. Nanogold labeling method, immunofluorescence and Western blot were used to identify the distribution of BM-derived Nkx2-5(+) CPCs and the expressions of Nkx2-5 protein in peripheral blood and BM after AMI. Meanwhile, in different ischemia organ models and after AMD3100 (SDF-1/CXCR4 antagonist) pretreatment in AMI model, Nkx2-5 protein expressions in peripheral blood were also assayed. Nkx2-5(+) CPCs were found to locate in cavitas medullaris. The percentage of Nkx2-5(+) CPCs in blood increased immediately after AMI. Nkx2-5 protein expression in peripheral blood was also upregulated at the timepoint of 24 h post-AMI (P<0.01) and kept stable without further enhancement from day 1 to day 7 post-AMI. In BM, Nkx2-5 protein expression was upregulated immediately after AMI and downregulated afterwards (P<0.01). After AMD3100 pretreatment in AMI group, Nkx2-5 protein expression was significantly inhibited in peripheral blood (P<0.05). In cerebral and hind-limb ischemia models, Nkx2-5 protein expressions were significantly lower than that in AMI group (P<0.01), but with no significant difference to control group. These results suggest that Nkx2-5(+) CPCs are physiologically resident in BM and AMI initiates mobilization of BM-derived Nkx2-5(+) CPCs in a predominant organ-specific manner. In the procedure of mobilization, SDF-1 may play a critical role in a chemoattracted manner.

[High power microwave radiation damages blood-testis barrier in rats].

OBJECTIVE: To determine the effect of high power microwave (HPM) radiation on the structure and function of blood-testis barrier (BTB) in rats. METHODS: One hundred and sixty-six male Wistar rats were treated by heart perfusion of lanthanum-glutaraldehyde solution and tail vein injection of evans blue (EB) at 6 h, 1, 3, 7 and 14 d after exposed to 0, 10, 30 and 100 mW/cm2 HPM radiation for 5 minutes, the structural change of BTB and distribution of lanthanum or EB observed through the light microscope, electron microscope and laser scanning confocal microscopy (LSCM). RESULTS: Testicular interstitial edema, vascular congestion or hyperemia with accumulation of plasma proteins and red blood cells in the inner compartment of seminiferous tubules were observed after exposure to HPM. The above-mentioned pathological changes were aggravated at 1-7 d and relieved at 14 d after radiation, obviously more severe in the 30 and 100 mW/cm2 exposure groups than in the 10 mW/cm2. Both lanthanum precipitation and EB were deposited in the inner compartment. CONCLUSION: HPM radiation may damage the structure and increase the permeability of BTB.

[Delayed disappearance of h-VEGF165 mRNA and protein under regulation of hypoxic response element].

Transfer of vascular endothelial growth factor (VEGF) gene to ischemic myocardium may provide a useful approach for angiogenesis and improve cardiac performance. However, uncontrolled expression of VEGF in vivo may result in certain side effects, such as hemangioma formation, retinopathy, and tumor development. We investigated the feasibility of using the nine copies of hypoxic response element (HRE) to control the expression of human VEGF(165) (h-VEGF(165)) under anoxic condition at cell level and also observed the synchron of h-VEGF(165) mRNA and protein expressions. Recombinant adeno-associated viral (rAAV) vector was prepared by using the three-plasmid system and cotransfected to human embryo kidney 293T cells by the calcium phosphate precipitates method. The rAAV vector was purified by chloroform-PEG8000/NaCl-chloroform and added to cultured myocardiocytes. Myocardiocytes of Sprague-Dawley rat were cultured in serum-free medium and then randomly divided into eight groups. Group I: cultured under normoxic conditions (21% O2) for 8 h as control; Group II: cultured under anoxic conditions (1% O2) for 8 h; Group III: cultured under normoxic conditions (21% O2) for 8 h with gene transfer; Group IV: cultured under anoxic conditions (1% O2) for 8 h with gene transfer; Group V, VI, VII: cultured under anoxic conditions (1% O2) for 8 h with gene transfer and then tured to normoxic conditions (21% O2) for 4, 8 or 12 h, respectively; Group VIII: cultured under anoxic conditions (1% O2) for 20 h with gene transfer. After completion of cell culture, the amount of h-VEGF(165) protein in culture supernatant was quantified by using enzyme linked immunosorbent assay (ELISA). Expression of h-VEGF(165) protein in cultured cardiacmyocytes was also evaluated by immunofluorescence. RT-PCR was employed to detect the expression of h-VEGF(165) mRNA. The results revealed that there were no expressions of h-VEGF(165) mRNA and protein in groups I, II, III, VI and VII. After gene transfer, the expressions of h-VEGF(165) protein and mRNA were significantly higher in groups IV and VIII than those in other groups (P<0.01); Immunofluorescence positive cells were observed in groups IV, V and VIII. RT-PCR revealed that a 484-bp strip can be found in groups IV and VIII, but unavailable in other groups. We conclude that HRE is a promising regulator for h-VEGF(165) gene expression following the changes of oxygen environment. HRE can induce the expression of h-VEGF(165) gene after hypoxia, but in normal oxygen condition, the expression of h-VEGF(165) was inhibited. Although expression of h-VEGF(165) mRNA ceased in normal oxygen condition under the control of HRE, expression of h-VEGF(165) protein was hysteretic to h-VEGF(165) mRNA expression.

Effects of endothelin-1 on differentiation of cardiac myocyte induced from rabbit bone marrow stromal cells.

BACKGROUND: Cardiomyocyte transplantation for the therapy of myocardial ischaemia is being paid close attention. However, how the microenvironment controls the differentiation of transplanted bone marrow stromal cells (BMSCs) is unknown. Endothelin-1 (ET-1), a cytokine, increases during myocardial infarction, but it is not known whether ET-1 is responsible for the fate of transplanted BMSCs. In the present study, we investigated the effects of ET-1 on differentiation and maturation of induced rabbit BMSCs, in vitro, to elucidate the cellular biological mechanisms. METHODS: The proliferation of BMSCs isolated from femur of rabbits was induced by ET-1 only, by 5-azacytidine (5-aza) or ET-1 combined with 5-aza. After 4 weeks of induced culturing, the differentiation rate and the diameter of induced myocyte like cells were estimated and the expressions of GATA-4 protein and phosphorylation level were assayed by Western-blot, RT-PCR analysis of beta-myosin heavy chain (MHC). mRNA expression, levels of troponin-I by immunohistochemical staining and ultrastructure of induce-cultured BMSCs were also determined. RESULTS: By induction with ET-1 and 5-aza, mean cell diameter of induced BMSCs was larger than induced with 5-aza [(6.26 +/- 0.22) microm cf (5.29 +/- 0.19) microm] (P < 0.001). There was no difference in rate of differentiation of myocyte like cells between the groups induced with 5-aza and ET-1 combined with 5-aza [(29.82 +/- 0.23)% cf (29.94 +/- 0.18)%] (P > 0.05). The expressions of GATA-4 protein and phosphorylation were enhanced significantly in groups induced with ET-1 combined with 5-aza (P < 0.05). In the group induced with ET-1 combined with 5-aza, expression of beta-MHC mRNA was higher than control [(0.122 +/- 0.008) cf (0.022 +/- 0.003)] (P < 0.01), and more troponin-I positive cells were also detected in this group. Differentiated BMSCs showed formations of myofilaments and primitive sarcomere, i.e., morphological characteristics of myocyte like cells. CONCLUSIONS: This study suggests that induced culturing of BMSCs by ET-1 combined with 5-aza can express cardiomyocytic characteristics whereas ET-1 alone could not induce BMSCs to differentiate to myocyte like cells. ET-1 upregulates the expression of GATA-4 protein and phosphorylation level of induced BMSCs, and rapidly promotes the differentiation and maturation of myocyte like cells from BMSCs.

Myocardioprotective effects of the combination of ischemic preconditioning with hypothermia and crystalloid cardioplegia in immature rabbits.

This study was undertaken to explore the myocardioprotective effects of the combination of ischemic preconditioning (IP) with hypothermia and St.II Thomas crystalloid cardioplegic solution (CCS) on immature hearts in the rabbit. Isolated immature rabbit hearts were perfused with Krebs-Henseleit bicarbonate buffer on Langendorff apparatus. In experiment 1, 24 hearts were divided into 4 groups (n=6 in each group): Con, IP1, IP2 and IP3 group. Hearts of the four groups underwent 0, 1, 2 or 3 cycles of IP respectively. Then all the hearts were subjected to a sustained ischemia period of 2 h at 20 degrees C and a postischemic reperfusion period of 30 min at 37 degrees C. In experiment 2, 48 hearts were divided into 6 groups (n=8 in each group): SCon1, SIP1, SCon2, SIP2, SCon3 and SIP3 group, according to hypothermia and the duration of sustained ischemia (30 min at 32 degrees C; 90 min at 25 degrees C, 2 h at 20 degrees C). The SIP1, SIP2 and SIP3 groups were preconditioned twice before the sustained hypothermic ischemia, while the SCon1, SCon2 and SCon3 groups were not preconditioned. CCS was applied during sustained ischemia, all the hearts were reperfused for 30 min at 37 degrees C. Heart rate (HR), left ventricular developed pressure (LVDP) and peak rate of increase or decrease of left ventricular pressure (+/-dp/dt(max)) were recorded. Tissue concentration of adenosine triphosphate (ATP), malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured. At the end of reperfusion, values of product of LVDP and HR, +/-dp/dt(max) in IP2 group were 96%+/-21%, 101%+/-19% and 84% +/-15% of the baseline values respectively, which were significantly higher than those of Con group and IP3 group (P<0.01, P<0.05); also, the ATP content of IP2 group was higher than that of the Con group (P<0.01). When CCS was applied during sustained period of hypothermic ischemia at 32 degrees C or 25 degrees C, recovery rates of RPP (rate product, =LVDPxHR) and +dp/dt(max) in SIP1 group were 87% +/-14% or 99% +/-26% of the baseline values respectively (P<0.05, vs SCon1 group), the values in SIP2 group changed to 87% +/-16% or 102% +/-20% respectively (P<0.05, vs SCon2 group). Contents of ATP in SIP1 and SIP2 groups were significantly higher than those of SCon1 or SCon2 groups respectively (P<0.05), but MDA content of the two groups were significantly lower than those of SCon1 or SCon2 groups (P<0.05) respectively. The study indicates that IP attenuates hypothermic ischemia/reperfusion injury to immature rabbit hearts under 20 degrees C ischemia, two cycles of IP showing better myocardioprotective effects than 1 or 3 cycles of IP. When IP was combined with CCS which were applied during hypothermic ischemia period, the beneficial effects of IP were weakened as the temperature during the hypothermic period was elevated.

[Study of biological characteristics of the CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients' bone marrow].

OBJECTIVE: To explore the characteristics of CD(34)(+) CD(59)(+) cells from paroxysmal nocturnal hemoglobinuria(PNH) patients' bone marrow and the possible reasons of hematopoietic clonal dominance of PNH clones. METHODS: CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients and CD(34)(+) cells from normal control were selected from the bone marrow mononuclear cells by means of immunomagnetic microbead-flow cytometry two step sorting method undergone ex vivo expansion in liquid culture for two weeks and performed semisolid cultures before and after expansion. RESULTS: (1) Cultivation for seven days was the optimum for ex vivo expansion of PNH CD(34)(+) CD(59)(+) cells and normal CD(34)(+) cells, both cell populations remained CD(59) positive after expansion. (2) Normal CD(34)(+) cells had higher capacities of proliferation and expansion, and stronger potential to survival than that of both PNH CD(34)(+) CD(59)(+) and PHN CD(34)(+) CD(59)(-) cells. (3) In terms of semisolid culture, there was no significant difference in the yields of CFU formation between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells. (4) In liquid culture with combinations of hematopoietic factors SCF + IL-3 + IL-6 + FL + Tpo or SCF + IL-3 + IL-6 + FL + Tpo + Epo, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells; but with combination of SCF + IL-3 + IL-6 + FL + Tpo + Epo + GM-CSF, CD(34)(+) CD(59)(-) cells had better proliferation and expansion capacities and stronger potential to survival than that of CD(34)(+) CD(59)(+) cells. CONCLUSIONS: (1) Normal CD(34)(+) cells had better proliferation, expansion capacities and stronger potential to survival than that of PNH CD(34)(+) CD(59)(+)cells. (2) In semisolid and liquid culture with hematopoietic factor combinations, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells. It was suggested that CD(34)(+) CD(59)(-) cells had no clonal hemotopoiesis dominance. GM-CSF might be one of the reasons for PHN clones to possess clonal hematopoiesis dominance.

[Application of double immunomagnetic positive sorting to ex vivo expansion of marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria].

Since flow cytometry was not feasible for sorting a huge amount of cells for clinical use, the method of double immunomagnetic positive sorting was used for selection of CD34(+)CD59(+) cells from bone marrow mononuclear cells in patients with paroxysmal nocturnal hemoglobinuria (PNH), which laid the groundwork for clinical ABMT/APBSCT of patients with PNH. Immunomagnetic positive selection was used for two times, the microbeads were removed from the CD34(+) cells selected firstly by means of overnight culture, then the sufficient CD34(+)CD59(+) cells were used for ex vivo expansion. The results showed that the survival, proliferation and colony-forming units of the selected CD34(+)CD59(+) cells by double immunomagnetic positive sorting had no significant difference as compared with that of CD34(+)CD59(+) cells selected by flow cytometry technique. It is suggested that the double immunomagnetic positive sorting promotes the use for separation and purification hematopoietic stem/progenitor cells and other cells with double or multiple markers cells for autologous hematopoietic stem cell transplantation in PNH patients.

Gene expression profile in human leukocytes.

Leukocytes are classified as myelocytic or lymphocytic, and each class of leukocytes consists of several types of cells that have different phenotypes and different roles. To define the gene expression in these cells, we have performed serial analysis of gene expression (SAGE) using human leukocytes and have provided the gene database for these cells not only at the resting stage but also at the activated stage. A total of 709,990 tags from 17 libraries were analyzed for the manifestation of gene expression profiles in various types of human leukocytes. Types of leukocytes analyzed were as follows: peripheral blood monocytes, colony-stimulating factor-induced macrophages, monocyte-derived immature dendritic cells, mature/activated dendritic cells, granulocytes, natural killer (NK) cells, resting B cells, activated B cells, naive T cells, CCR4(-) memory T cells (resting T(H)1 cells), CCR4(+) memory T cells (resting T(H)2 cells), activated T(H)1 cells, and activated T(H)2 cells. Among 38,961 distinct tags that appeared more than once in the combined total libraries, 27,323 tags were found to represent unique genes in certain type(s) of leukocytes. Using probability (P) and hierarchical clustering analysis, we identified the genes selectively expressed in each type of leukocytes. Identification of the genes specifically expressed in different types of leukocytes provides not only a novel molecular signature to define different subsets of resting and activated cells but also contributes to further understanding of the biologic function of leukocytes in the host defense system.

Molecular analysis of lipid-depleting factor in a colon-26-inoculated cancer cachexia model.

Cachexia in cancer is characterized by progressive emaciation involving depletion of host adipose tissue stores, the molecular mechanism of which remains largely unknown. In this study, we have attempted to clarify the biologic characteristics of lipid-depleting factor in a mouse cachexia model. Utilizing differentiated 3T3-L1 adipocytes, we established an assay method quantifying the lipid-depleting activity in plasma derived from colon-26-inoculated mice and then analyzed the associated molecular mechanism. Injection (s.c.) of a mouse colon adenocarcinoma cell line, colon-26 clone 20, induced cachexia, as evidenced by progressive weight loss. Addition of clone 20-derived cachexigenic, but not clone 5-derived noncachexigenic, plasma to the culture medium of differentiated 3T3-L1 adipocytes reduced the TG content in cultured cells. The ability of the introduced plasma to induce TG loss in 3T3-L1 cells paralleled the body weight changes of tumor-inoculated host mice. Clone 20 plasma, but not clone 5 plasma or recombinant IL-6, elicited lipolytic activity, which induced glycerol release from 3T3-L1 cells. Addition of clone 20 plasma to cultured 3T3-L1 adipocytes reduced TG synthesis from [(14)C]-glucose compared to clone 5 plasma, indicating that the lipid-depleting activity resulting from addition of clone 20 plasma depended not only on induction of lipolysis but also on inhibition of lipogenesis. Addition of clone 20 plasma to cultured 3T3-L1 adipocytes reduced the quantity of mature SREBP-1 in the nucleus of 3T3-L1 cells without affecting PPAR-gamma and C/EBP-alpha. Although TNF-alpha induced apoptosis in 3T3-L1 cells, clone 20 plasma did not. These results suggest that the lipid-depleting factor in clone 20 plasma is different from either IL-6 or TNF-alpha, and that this factor interfered with not only lipolysis but also lipogenesis through SREBP-1 of 3T3-L1 adipocytes.

WISP-2 is a secreted protein and can be a marker of estrogen exposure in MCF-7 cells.

As many structurally diverse chemicals have been reported to function as estrogens, evaluations for estrogenicity of compounds are of widespread concern. Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in human breast cancer cells. In this study, we examined whether WISP-2 could be utilized as a marker for screening environmentally relevant compounds for estrogenicity. In MCF-7 cells, progesterone, dexamethasone, tri-iodothyronine, and 2,3,7,8-tetrachlorodibenzo-p-dioxin did not regulate the expression of WISP-2, indicating that its induction is highly specific for hormones that interact with the estrogen receptor. Western blot analysis detected WISP-2 protein induced by 17-beta-estradiol (E2), not only in the cell lysates but also in the culture supernatant of exposed cells, indicating that WISP-2 was a secreted protein. The induction of WISP-2 protein by E2 in the culture supernatant was dose-dependent with estimated EC(50) levels between 10 and 100 pM. Our results demonstrated the capacity to screen environmental compounds for estrogenicity via WISP-2 induction.