Ficoll-Hypaque Density Gradient Research Articles

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling

Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML chronic phase samples, about 70% of CML accelerated phase samples, and >80% CML blast crisis phase samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p < 0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p < 0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, and CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p < 0.05), implying cell-autonomous activation of Shh signaling play a predominant role in progenitor cells. Coactivation of Shh and β-catenin signaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh-neutralizing antibody or Wnt3a-neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis; however, Wnt3a peptide could salvage cell apoptosis, while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, and P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G(2)/M cell cycle arrest of progenitor cells. Our results demonstrate that autoactivated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, suggesting a novel therapeutic approach in CML.

Intracellular concentrations of micafungin in different cellular compartments of the peripheral blood

Whilst micafungin serum concentrations are well studied, little is known about its concentrations within cellular compartments of the peripheral blood. Hence, in this study blood samples were collected from patients receiving micafungin (n=26). These samples were separated by double-discontinuous Ficoll-Hypaque density gradient centrifugation. Intracellular concentrations within the obtained cells, i.e. peripheral blood mononuclear cells (PBMCs), polymorphonuclear leukocytes (PMNs) and red blood cells (RBCs), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Within PBMCs and PMNs, the intracellular micafungin concentration was significantly increased compared with the concentration in plasma (P<0.001). The intracellular concentration within RBCs did not significantly differ from the plasma concentration. Micafungin reaches high concentrations in human PBMCs and PMNs and is present in RBCs. In vitro data showed that intracellular uptake of micafungin by PBMCs depends on the albumin concentration of the surrounding medium, but only at non-physiological protein concentrations.

Function of regulatory T-cells improved by dexamethasone in Graves' disease

Intrathyroid injection of dexamethasone (DEX) has been used to treat Graves' disease (GD); however, the mechanism of this treatment remains poorly understood. The objective of this study was to investigate the effects of DEX on the function of regulatory T (Treg) cells (CD4(+)CD25(+)T cells) in patients with GD. Peripheral blood was obtained from 20 patients with GD, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient separation. CD4(+)CD25(-)/CD4(+)CD25(+)T cells were isolated by immunomagnetic selection and DEX was co-cultured with PBMCs or isolated T-cells for 72 h. Treg cell function was analyzed using the proliferation rate of CD4(+)CD25(-)T cells. The proportion of Treg cells and the transcription factor forkhead box P3 (FOXP3) mRNA expression in PBMCs decreased in GD patients compared with healthy subjects, and Treg cell function was impaired in patients with GD. Although the proportion of Treg cells and FOXP3 mRNA expression in PBMCs did not increase, the function of Treg cells improved after the treatment with DEX. Moreover, the proportion of T-helper 2 (Th2) cells was decreased by the DEX treatment. DEX could effectively improve the function of Treg cells and set up a new balance of Th1/Th2 in GD patients. This study might help to further understand the immune mechanism of the intrathyroid injection of DEX in the treatment of GD and facilitate the potential use of this therapy.

Expression of inhibitory receptors on CD4+ and CD8+ T cells from healthy Colombian donors

&lt;p&gt;T cell activation involves positive cellular signals that promote effector functions and negative signals that contribute to the regulation of these responses. These regulatory signals are generated upon activation of receptors on T cells that include CD160, 2B4, Programmed Death-1 and CTLA-4. &lt;strong&gt;Objective&lt;/strong&gt;. To evaluate the expression of inhibitory receptors like CD160, 2B4, Programmed Death-1 and CTLA-4 on CD4+ and CD8+ T cells from healthy Colombian donors. &lt;strong&gt;Materials and methods&lt;/strong&gt;. Peripheral blood mononuclear cells from 30 healthy donors from Bogotá (Colombia) were obtained via Ficoll-Hypaque density gradient and cells were stained with specific conjugated antibodies previously titrated. &lt;strong&gt;Results&lt;/strong&gt;. The CD160, 2B4, and Programmed Death-1 inhibitory markers were detected on CD4+ T cells&lt;br /&gt;with expression levels of 0.35%, 1.04%, and 1.35%, respectively. On CD8+ T cells, these markers were expressed at higher levels: 16%, 8.97%, and 4.3%, respectively. In contrast to the other receptors, CTLA-4 frequency of expression showed no significant difference between CD4+ (1.56%) and CD8+ (1.53%) T cells. Frequency of CD160/2B4 and CTLA-4/ Programmed Death-1 coexpression was 0.18% and 0.09% on CD4+ cells, and 4.02% and 0.2% on CD8+ T cells. &lt;strong&gt;Conclusions&lt;/strong&gt;. This is the first report showing the frequency of inhibitory receptors such as CD160, 2B4, Programmed Death-1, and CTLA-4 on CD4+ and CD8+ T cells from healthy Colombian donors. Our findings serve as a baseline for the analysis and comparison of these receptors in Colombian populations with different disease conditions.&lt;/p&gt;&lt;p&gt;&lt;br /&gt;&lt;strong&gt;Key words&lt;/strong&gt;: inhibitory receptors, T cells&lt;/p&gt;

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma

To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment. One hundred and two paraffin blocks and 33 fresh samples of CRC tissues were subject to this study. Immunohistochemistry was performed for B7-H1 and CD3 staining in CRC tissues. Ficoll-Hypaque density gradient centrifugation was used to isolate peripheral blood mononuclear cells of fresh CRC tissues; flow cytometry and immunofluorescence staining were used for detection of regulatory T cells. Data was analyzed with statistical software. Costimulatory molecule B7-H1 was found strongly expressed in CRC tissues, localized in tumor cell membrane and cytoplasm, while weak or none expression of B7-H1 was detected in pared normal colorectal tissues. Meanwhile, CD3 positive T cells were found congregated in CRC tumor nest and stroma. Statistic analysis showed that B7-H1 expression level was negatively correlated to the total T cell density in tumor nest (P < 0.0001) and tumor stroma (P = 0.0200) of 102 cases of CRC tissues. Among the total T cells, a variable amount of regulatory T cells with a clear Foxp3⁺ (forkhead box P3) staining could be detected in CRC tissues and patients' blood. Interestingly, in the 33 samples (15 cases of B7-H1(high) CRC tissues and 18 cases of B7-H1(low) CRC tissues) of freshly isolated mononuclear cells from CRC tissues, the percentages of CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ regulatory T cells were found remarkably higher in B7-H1(high) CRC tissues than in B7-H1(low) CRC tissues (P = 0.0024, P = 0.0182), indicating that B7-H1 expression was involved in proliferation of regulatory T cell. No significant difference was found in CRC peripheral blood (P = 0.0863, P = 0.0678). PD-1 is the specific ligand for B7-H1 pathway transferring inhibitory signal to T cell, which is expressed by activated T cell. Our further analysis of PD-1 expression on T cells in CRC tissues showed that conventional T cells (CD4⁺Foxp3⁻/CD8⁺Foxp3⁻), which was thought to contribute to the anti-tumor immune response, highly expressed PD-1; while regulatory T cells (CD4⁺Foxp3⁺/CD8⁺Foxp3⁻) almost failed to express PD-1. The average percentage of PD-1 expression on regulatory T cells was significantly higher than the percentage of PD-1 on conventional T cells (CD4⁺Foxp3⁻ T cell, P < 0.0001; CD8⁺Foxp3⁻ T cell, P < 0.0001). The diverse expression of PD-1 might lead to different fate of T cell subsets in B7-H1 over-expression CRC tumor microenvironment. B7-H1 expression in tumor cells can inhibit the conventional T cell proliferation in tumor microenvironment through the PD-1 expression on conventional T cells.

Genetically Engineered Natural Killer (NK) Cell Immunotherapy for Poor Risk B-Cell (CD20+) Leukemia and Lymphoma

Abstract 1003 Background:Patients with CD20+ Leukemia and Lymphoma (L/L) at diagnosis and a majority of those at relapse have a chemotherapy resistant phenotype and a dismal prognosis. Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells from cord blood (CB) play an important role in tumor surveillance post allogeneic stem cell transplantation (Beziat V et al, Leukemia, 2009) but cell number and tumor recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Genetically Engineered expanded CBNK cells with anti-CD19 chimeric receptor has been previously reported (Li L et al, Cancer Gene Ther. 2010). Objective:We investigated the functional activities and cytolytic effect of chimeric anti-CD20 receptor (anti-CD20 CAR) engineered CBNK cells expanded with modified K562 (generously supplied by D. Camapana, MD, PhD) against CD20+ L/L both in vitro and in vivo. Methods:CBMNC were obtained by Ficoll-Hypaque density gradient centrifugation. CBMNC were cocultured with 100Gy irradiated MODK562 (K562-mbIL15-41BBL cells) or wildtype (WT) K562 in RPMI+ 10 IU/ml IL-2 for 7 days. Medium was changed every 2 days with fresh medium and IL-2. CD56 and CD3 expression were evaluated by flow cytometry. NK activation marker LAMP-1 and cytolytic mechanism markers, perforin and granzyme B were evaluated by flow cytometry in expanded CBNK. NK cytotoxicity was assessed by europium release assay at 20:1 E:T ratio against CD20+ Ramos. Luciferase expressing (ffLuc+) Ramos were generated by transfecting ffLUCZeo-pcDNA plasmid DNA (generously supplied by L. Cooper, MD, PhD) and selected by Zeocin. In vivo ffLuc+ Ramos engraftment in NOD/SCID mice was verified using a Xenogen IVIS-200 system (Caliper Life Sciences). 6 weeks old xenografted mice were treated for 5 weeks with PBS, Ramos, 5×106 WTK562 expanded (E) CBMNC, 5×106 or 5×107 MODK562E CBMNC IP. Tumor growth was monitored by volume using bioluminescent imaging and mice survival was observed for 10 weeks. Different retrovirus preps that express the anti-CD20 CAR or control GFP only (CAR−) were generated independently. The anti-CD20 CAR was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD, St. Jude Children’s Hospital, Memphis, Tenn). MSCV-anti-CD20BB-CD3-zeta-GFP or control empty vector (MSCV-GFP) were transfected to 293T cells with helper plasmids pEQ-PAM3(-E) and pRDF. Conditioned medium containing retrovirus was harvested 48 and 72 h after transfection. Jurkat cells were used to examine infection efficiency. Anti-CD20 CAR expression was examined by flow cytometry with staining cells with goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin (Jackson Immunoresearch, West Grove, PA, USA) followed by PE-conjugated streptavidin and the expression was further confirmed by western blot of whole cell lysates for CD3zeta. Results:CBNK cells were significantly increased compared to WTK562 and media alone (72 ± 4 {35-fold} vs 43 ± 6 vs 9 ± 2%, p<0.01) (Figure 1). Cytotoxicity against Ramos was increased compared to WTK562 CBMNC (p<0.01); along with increased expression of NK activation marker, CD107a (p<0.05), perforin and granzymeB (42 ± 1.5 vs 15 ± 0.5%,p<0.001; 22 ± 0.5 vs 11 ± 0.3%, p<0.0.001, respectively). At 5 weeks, tumor volume in mice receiving either dose of MODK562E CBMNC was significantly decreased vs WTK562E CBMNC (2 ± 0.6 and 0.4 ± .05 vs 3 ± 0.25/mm3, p=0.0086, p=0.001, respectively and luminescence p<0.01). Xenografted mice receiving MODK562E CBMNC showed increased survival vs WTK562E (p<0.001). CAR+ and CAR− retrovirus supernants infected Jurkat cells efficiently. 3 independent retrovirus preps infected Jurkat cells with 97.71% CAR−GFP+ vs 80.25% CAR+ GFP+, 40.25% CAR−GFP+ vs 74.03% CAR+ GFP+, and 88.57% CAR−GFP+ vs 62.64% CAR+ GFP+. The anti-CD20 CAR expression was further confirmed by western blots (Figure 2). Conclusion and Future Directions:CBMNC stimulated with MODK562 was associated with significantly increased NK LAMP-1, perforin and granzyme B expression, enhanced cytotoxicity in-vitro and in-vivo survival. Anti-CD20 CAR expressed was confirmed in infected cells. Future directions include infecting MODK562 expanded CBNK with retrovirus expressing anti-CD20 CAR and examining the cytotoxicity activity of engineered CBNK against L/L in-vitro and survival in xenogafted mice. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression of Kindlins in Acute Myeloid Leukemia

Abstract 4910The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells. 1. Materials and methodsK562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group.Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5’-CCGCTCGAGCTATGCGTATCCCCGTAG-3’; (b) Kindlin-2 antisense primer, 5’-CGACGCGTCTAGCGAGGGGTTGTC-3’; (c) Kindlin-3 sense primer, 5’-CCGCTCGAGCTATGCGTATCCCCGTAG-3’; (d) Kindlin-3 antisense primer, 5’-CGACGCGTCTAGCGAGGGGTTGTC-3’; (e) GAPDH sense primer, 5’-GAAGGTGAAGGTCGGAGTC-3’; (f) GAPDH antisense primer, 5’-GAAGATGGTGATGGGATTTC-3’. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients’ clinical data were analyzed.2. Results Expression of Kindlins in newly diagnosis AMLTable 1Characteristics of the AML patients and expression of KindlinsCharacteristicNo. of Patients (%)Kindlin-2 (RQ-In)Kindlin-3 (RQ-In)SexMale46(52.3%)0.307±1.6590.066±0.738Female42(47.7%)0.005±1.6760.117±0.942p-value0.3980.776WBC<30×109/L22(25.3%)0.409±1.534−0.013±0.753≥30×109/L65(74.7%)-0.547±1.8930.394±1.021p-value0.0200.049WHO typeAML with t(8;21)(q22;q22)16(18.2%)0.842±1.730−0.207±0.811AML with t(15;17)(q22;q12)14(15.9%)Acute myelomonocytic leukaemia5(5.7%)−0.240±1.4730.243±0.790Acute monoblastic and monocytic leukaemia31(35.2%)AML without maturation3(3.4%)−0.148±1.6300.267±0.869AML with maturation13(14.8%)Acute erythroid leukaemia6(6.8%)p-value0.0160.046NCCN risk status(APL excluded)Better-risk20(27.0%)0.446±1.8880.057±1.056Intermediate-risk41(55.4%)0.027±1.4690.17±0.741Poor-risk13(17.6%)-0.376±1.5000.347±0.794p-value0.3470.630Induction chemotherapy39Pretherapy390.216±1.602−0.114±0.628Postremission391.555±1.1020.372±0.633p-value<0.0010.004The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML. Disclosures:No relevant conflicts of interest to declare.

Characterization and in vitro immunomodulatory screening of fructo-oligosaccharides of Asparagus racemosus Willd.

Asparagus racemosus Linn. (Fam. Liliaceae) is an ethno-pharmacologically acclaimed Ayurvedic medicinal plant. In the present study, aqueous extract of A. racemosus (ARC) was fractionated and screened for the polysaccharide fraction (ARP). The characterization was done by enzymatic, Size Exclusion, gas chromatography with flame ionization detector (GC–FID), high pressure anion exchange chromatography (HPAEC) and thin layer chromatographic analyses. Phyto-chemical evaluation confirmed the presence of 26.7% of 2→1 linked fructo-oligosaccharides (FOS). They have a degree of polymerization (DP) of nearly 7–8. Cytotoxicity evaluation on P388 cell lines was consistent with low cytotoxicity of the extracts. In vitro Natural Killer (NK) cell activity was evaluated using human peripheral blood mononuclear cells (PBMC) isolated from whole blood on a ficoll-hypaque density gradient. K562 a myeloid leukemia cell line, were used as target cells. ARC, tested over the range 0.2–50μg/ml, showed a dose-related stimulation of NK cell activity with a peak increase of 16.9±4.4% at 5.6μg/ml. However, ARP demonstrated a higher stimulatory activity of 51.8±1.2% at 25μg/ml. The results indicate that the FOS from A. racemosus potentiates the NK cell activity and this could be an important mechanism underpinning the ‘Rasayana’ properties of this plant.

Histone H3 acetylation of tumor necrosis factor-alpha and cyclooxygenase-2 in patients with type 2 diabetes

To investigate the expression of tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) mRNA and evaluate the status of histone H3 acetylation at TNF-α and COX-2 promoter in peripheral blood mononuclear cells (PBMCs) from type 2 diabetics. The PBMCs from 12 type 2 diabetics and 12 healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation. The differential expression of TNF-α and COX-2 mRNA was measured by real-time PCR (polymerase chain reaction). Chromatin immunoprecipitation analysis was used to detect the status of H3 acetylation at TNF-α and COX-2 promoter region. TNF-α and COX-2 mRNA were overexpressed in PBMCs from Type 2 diabetics as compared with normal controls (2.28 ± 0.09 fold and 2.78 ± 0.26 fold).(P < 0.05). Compared with normal controls, H3 acetylation at the TNF-α (1.54 ± 0.43 vs 0.97 ± 0.39, P = 0.0094) and COX-2 (1.20 ± 0.58 vs 0.64 ± 0.21, P = 0.0161) gene promoter region was elevated in PBMCs from Type 2 diabetic patients. Increased H3 acetylation at TNF-α and COX-2 promoter in PBMCs from type 2 diabetics may contribute to the pathogenesis of type 2 diabetes through the elevated expressions of TNF-α and COX-2.

Abstract 2217: Primary tumor loss of heterozygosity proximal to M6P/IGF2R locus is predictive for the presence and persistence of disseminated tumor cells in the bone marrow of ovarian cancer patients

Abstract Introduction: Disseminated tumor cells (DTC) in the bone marrow (BM) are present in about 35% of all ovarian cancer patients before surgery and after platinum-based chemotherapy which has been associated with an adverse prognosis. Since invasive BM aspirations are less accepted and often not feasible, it would be highly desirable to unmask a molecular biomarker in the primary tumor predicting tumor cell spread and persistence. It was the purpose of the study to 1) investigate loss of heterozygosity (LOH) incidence at four ovarian cancer relevant chromosomal loci involved in inter alia apoptosis, platinum sensitivity or DNA repair in primary ovarian tumor tissue 2) assess the prognostic value of LOH after a median follow-up time of three years and 3) correlate LOH incidence with the occurrence of DTC before surgery and after platinum-based chemotherapy. Methods: In total, 88 ovarian cancer patients were recruited for this study. Primary tumor DNA was extracted from paraffin embedded tissue and analyzed for LOH status at four polymorphic microsatellite markers including D10S1765 (PTEN locus), D13S218 (BRCA1 locus), D17S855 (BRCA2 locus) and D6S1581 (proximal to M6P/IGF2R locus) using PCR-based fluorescence microsatellite analysis. BM aspirates were feasible in 53 patients before surgery and in 40 patients after chemotherapy. BM cells were isolated from heparinized BM by Ficoll-Hypaque density gradient centrifugation and analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Results: In our cohort of 88 patients, we recorded at least one LOH in 56/88 patients (63%), two LOH in 30/88 patients (34%), three LOH in 8/88 patients (9%) and LOH at all four markers in 3/88 patients (3%), respectively. The occurrence of LOH at the entire marker set significantly correlated with tumor grading (p=0.0001) and histology (p=0.004). The frequency of LOH at the marker D10S1765 correlated with FIGO stage (p=0.046) and grading (p=0.05) whereas LOH at D17S855 was associated with grading (p=0.023) and histology (p=0.012). The occurrence did not correlate with disease free as well as overall survival. Prior to surgery, DTC were detected in 26/53 patients (49%) with a median number of 6 cells (range 1-38 cells). After chemotherapy, 20/40 patients (50 %) revealed DTC in their BM with a median number of 10 cells, ranging from 1 to 35 cells. Interestingly, the LOH incidence proximal to D6S1581 significantly correlated with DTC presence before surgery (p=0.05) as well as DTC persistence after chemotherapy (p=0.022). Conclusion: In primary ovarian tumors, allelic loss proximal to D6S1581 might serve as a potential molecular biomarker for the presence and persistence of DTC in the BM. Further investigations have to prove whether such a molecular marker could replace invasive BM aspirations in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2217. doi:10.1158/1538-7445.AM2011-2217

P85α Regulates Osteoblast Differentiation by Cross-talking with the MAPK Pathway

Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.

Variability of BCP and Precore Region Among the Occult HBV Infected Voluntary Blood Donors from Eastern India

© 2011, INASL 2 PBMCs/well were stimulated with ORF-2 peptide (452–617 a.a) and subjected to MTT assay. Proliferation index (PI) at 570 nm was calculated. All the samples were subjected for anti-HEV IgM, IgG serology and HEV RNA detection by RT-PCR. Results: Patients with hepatitis were diagnosed as acute hepatitis E based on anti-HEV IgM/RNA positivity. PBMCs of acute hepatitis patients showed reactivity to the ORF-2 peptide in comparison to control subjects in terms of blast cell transformation. The proliferation indices of patient and control groups were 3.0398 ± 0.666 and 1.21 ± 0.077, respectively. Thus significant antigenic reactivity (p = 0.0126) was observed in hepatitis E patients. Anti-HEV IgM was positive in 15 (93.75%), IgG in 10 (62.5%) and RNA in 7 (43.75%) patients. None of the parameters were positive in control group. There was no significant difference in PI values between patient groups with and without HEV RNA positivity (2.99 ± 0.664, 3.077 ± 0.705; p = 0.359). Conclusion: HEV specific ORF 2 peptide (452–617 a.a) effectively stimulates the PBMCs in the patient group as compared to healthy controls. Thus, the tested peptide was found to be antigenic and specific for HEV and can be used in understanding the pathogenesis of HEV infection in vitro with regards to gene modulation and various cytokine profiles. Conflict of Interest: None Gene Expression Analysis in Peripheral Blood Mononuclear Cells in Patients with Acute Hepatitis E A Naik*, A Goel*, D Goel*, A Pandey**, S Rastogi†, R Aggarwal* *Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow **Department of Obstetrics and Gynecology, King George’s Medical College, Lucknow †Command Hospital, Lucknow Background: Hepatitis E is the commonest cause of acute viral hepatitis in India and several developing countries. Pathogenesis of liver injury and immune events during this disease are not well understood. To better define immune events during hepatitis E, we undertook a gene expression study of peripheral blood mononuclear cells in patients with this disease. Methods: PBMCs were obtained from 12 patients with acute hepatitis E (typical clinical and biochemical findings, and positive IgM anti-HEV [Genelabs, Singapore]), 4 patients with acute hepatitis B (HBsAg positive, IgM anti-HBc positive, IgM anti-HEV negative) and 10 healthy controls, using Ficoll-Hypaque density gradient centrifugation. Total RNA was isolated from PBMCs using Qiagen RNeasy Protect Mini Kit, and cDNA synthesis and microarray analysis were done using Illumina HumanWG-6 v3.0 Expression BeadChips. Genes showing differential expression in hepatitis E, but not in hepatitis B, were identified and downstream pathway analysis was done. Results: PBMCs from patients with hepatitis E showed differential expression of 170 genes (upregulated 140, downregulated 30). PBMCs from patients with hepatitis B showed differential expression of 80 genes (up 62, down 18). Of these, 24 differentially-expressed genes were common. Genes differentially expressed in PBMCs from patients with hepatitis E but not in hepatitis B belonged to immune pathways that included cell-mediated immune response, immune cell trafficking and antigen presentation. The genes that were overexpressed in PBMCs from patients included CDCA5, CCNA2, CCNB2, PCNA, IGLL1 and TNFRSF17; most of these genes are involved in cell division and cellular function. In comparison, most of the genes that were underexpressed in PBMCs from hepatitis E (CCL20, TNF, IL1RN, CCL4L1, CCL3L3, CCL3, CCL3L1, IL1RN and IL1B) belonged to chemokine and pro-inflammatory cytokine families. Conclusion: Downregulation of chemokines and pro-inflammatory cytokines in PBMCs from patients with hepatitis E may contribute to disease pathogenesis by allowing enhanced viral replication. Conflict of Interest: None Variability of BCP and Precore Region Among the Occult HBV Infected Voluntary Blood Donors from Eastern India A Biswas*, R Panigrahi*, A Pal*, M Bandopadhyay*, BK De**, S Chakrabarti†, R Chakravarty* *ICMR Virus Unit, Kolkata **Department of Medicine, Calcutta Medical College, Kolkata †National Institute of Cholera and Enteric Diseases, Kolkata Background: In India prevalence of occult hepatitis B virus (HBV) infection in the general population is widespread. Data about the variability of the basal core promoter region (BCP) and precore (PC) region of the occult HBV strains is lacking. Objective: Mutations in the BCP and PC regions are associated with persistent/intermittent HBV replication. We evaluated the variability BCP and PC region of HBV in asymptomatic carriers and aimed to characterize the variability with different viral strains. Methods: From voluntary blood donation camps initially 1400 blood samples were collected. The anti-HBc and HBsAg status of the samples were confirmed by ELISA. PCR amplification to test the presence of HBV DNA was done from anti-HBc positive but HBsAg 03_JCEH-Abstract.indd 2 3/18/2011 11:13:03 AM

Immunomodulatory Polysaccharide from Chlorophytum borivilianum Roots

Chlorophytum borivilianum Santapau & Fernandes (Liliaceae) is an ayurvedic Rasayana herb with immunostimulating properties. The polysaccharide fraction (CBP) derived from hot water extraction of C. borivilianum (CB), comprising of ∼31% inulin-type fructans and ∼25% acetylated mannans (of hot water-soluble extract), was evaluated for its effect on natural killer (NK) cell activity (in vitro). Human peripheral blood mononuclear cells (PBMCs), isolated from whole blood on a Ficoll-Hypaque density gradient, were tested in the presence or absence of varying concentrations of each C. borivilianum fraction for modulation of NK cell cytotoxic activity toward K562 cells. Preliminary cytotoxicity evaluation against P388 cells was performed to establish non-cytotoxic concentrations of the different fractions. Testing showed the observed significant stimulation of NK cell activity to be due to the CBP of C. borivilianum. Furthermore, in vivo evaluation carried out on Wistar strain albino rats for humoral response to sheep red blood cells (SRBCs) and immunoglobulin-level determination using enzyme-linked immunosorbent assay (ELISA), exhibited an effectiveness of C. borivilianum aqueous extract in improving immune function. Present results provide useful information for understanding the role of CBP in modulating immune function.

Lymphocyte activation test for diagnosis of seronegative Brucellosis in humans

Seronegative brucellosis is occasionally encountered in clinical practice especially in localized disease where diagnosis is reached mainly through positive blood culture. Cellular immune responses are pivotal for protection against intracellular bacteria such as Brucella. This study was performed to evaluate the expression of activation markers on peripheral blood mononuclear cells in response to in vitro stimulation by whole-cell suspension of Brucella melitensis for the diagnosis of brucellosis. Fifteen seronegative patients with positive blood cultures for Brucella and twenty-five unexposed healthy blood donors serving as controls were recruited for the study. Peripheral blood mononuclear (PBMC) cells were obtained by the method of Ficoll Hypaque density gradient and stimulated in vitro with Brucella antigen. Expression of activation markers was assessed by flow cytometry after staining of PBMC with mononuclear cells with relevant monoclonal antibodies. Incubation with mitogen induced expression of all the four markers was demonstrated in all blood samples. In contrast, samples from all patients of Brucellosis showed significant positive responses with the expression of activation markers (CD38, CD69, CD25, and CD71) on both CD4+ and CD8+ cells as compared with the control group (P < 0.001). It is inferred that there was a remarkable upregulation of activation markers on CD4+ and CD8+ in seronegative patients with Brucellosis. It is recommended that the method can be utilized as a novel diagnostic test for detection of brucellosis where serology is negative.

Analysis of acrolein-derived 1,N2-propanodeoxyguanosine adducts in human leukocyte DNA from smokers and nonsmokers.

Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pollutant and toxicant that is also formed endogenously through metabolism of amino acids and polyamines and lipid peroxidation. Acrolein reacts with DNA, producing two pairs of regioisomeric 1,N(2)-propanodeoxyguanosine adducts: (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). Previous studies indicate that these adducts might be involved in producing mutations in the p53 tumor suppressor gene, as observed in lung tumors in smokers, but there are only limited published data comparing acrolein-DNA adducts in smokers and nonsmokers. In this study, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to analyze Acr-dGuo adducts in human leukocyte DNA. The potential for artifactual formation was found in two steps of the assay: DNA isolation and DNA hydrolysis. This was eliminated by employing a Ficoll-Hypaque double density gradient to obtain leukocytes free of erythrocyte contamination and by adding glutathione to scavenge acrolein present in H(2)O. The accuracy and precision of the method were confirmed. Acr-dGuo adducts were analyzed in leukocyte DNA from 25 smokers and 25 nonsmokers. γ-OH-Acr-dGuo was the predominant isomer in all samples, while α-OH-Acr-dGuo was detected in only three subjects. There was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/10(9) nucleotides) and nonsmokers (9.8 ± 5.5 adducts/10(9) nucleotides). Although our study is limited in size, these results, together with the results of previous analyses of acrolein-derived mercapturic acids in the urine of smokers and nonsmokers, suggest that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus, Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

Intracellular Concentrations of Posaconazole in Different Compartments of Peripheral Blood

Therapeutic drug monitoring (TDM) of antifungal plasma concentrations is increasingly recommended. However, data on antifungal concentrations in the other compartments of the peripheral blood are limited. Hence, we collected 23 blood samples from 14 patients receiving posaconazole for prophylaxis of fungal infections. These samples were separated by double-discontinuous Ficoll-Hypaque density gradient centrifugation. The intracellular posaconazole concentrations of the obtained cells, i.e., the peripheral blood mononuclear cells (PBMCs), polymorphonuclear leukocytes (PMNs), and red blood cells (RBCs), were determined by liquid chromatography-tandem mass spectrometry. The intracellular concentrations of the PBMCs and PMNs were significantly higher than those of surrounding media (P < 0.001). The ratios between the intracellular and extracellular concentrations (C/E) were 22.5 +/- 21.2, 7.66 +/- 6.50, and 0.09 +/- 0.05 for the PBMCs, PMNs, and RBCs, respectively. Posaconazole reaches high concentrations within human PBMCs and PMNs and is, to a lesser extent, present in RBCs. The high intracellular concentrations might contribute to posaconazole efficacy and distribution.

Abstract 400: Establishment and characterization of a novel IL6-dependent HLA-A2+ human myeloma cell line LNT1

Abstract The novel multiple myeloma (MM) cell line LNT1 was established from peripheral blood of a 66-year-old female patient with recurrent plasma cell leukemia of IgG/kappa type. Peripheral blood mononuclear cells from the heparinized peripheral blood separated by Ficoll-Hypaque density gradient centrifugation were cultured in RPMI 1640 medium/20% FBS, 100 U/ml penicillin and 50 μg/ml streptomycin, without additional growth factors. Cells were cultured by replacing ½ of the volume of the culture with fresh medium every 3-4 days for 3 months. In the 4th month, a slow yet sustained growth of cultured cells was noted, and designated LNT1. These cells grow as single cell suspension; IL-6-induced proliferation in a dose-dependent manner with 2 ng/ml of IL-6 inducing maximal DNA synthesis (by [3H]thymidine uptake) and survival (by MTT). In contrast, IGF-1 had no effect. Subsequently, 2 ng/ml of IL-6 was added to cultures and growth now sustained at 7 months. LNT1 was negative for Epstein-Barr virus, as evidenced by PCR using primers specific for EBNA-1. May-Grunwald-Giemsa staining showed that LNT1 cells have a high nucleocytoplasmic ratio with excentrically located nuclei, prominent nucleoli, and a deeply basophilic and vacuolated cytoplasm. Some cells were bi- or multi-nucleated. Surface antigen analysis showed that LNT1 cells express CD138, CD38, HM1.24, CS1, and HLA-A2, but did not express CD19, CD20, CD3, CD16, and CD14. Chromosome analysis at 7 months revealed the karyotype: 43, X, -X, del(1)(p13p22), psu dic(6;1)(q13;p11), del(12)(p11.2), −13, add(19)(q13.4), −22[2]/43, idem, del(2)(q11.2q2?3), der(6)ins(6;?)(q21;?)[7]. DNA fingerprinting was performed by simultaneously amplifying eight short tandem repeat (STR) loci and the amelogenin gene in a multiplex PCR reaction using specific primers to confirm the authentication of cell line. The established LNT1 cell line and the primary tumor showed identical band patterns of these 9 markers and the same immunoglobulin H rearrangement. An unique and identical VH3/IgG fragment was amplified using the appropriate VH family-specific framework region primer in conjunction with CH isotype specific (IgG) primers in DNA samples of both LNT1 cell line and the original tumor. Moreover, identical DNA sequence of VH3/IgG PCR from cell line and primary tumor further confirms clonality. LNT1 cell line also bears a N-RAS G12D mutation, as in the original tumor. Finally, LNT1 cell line is responsive to conventional (Dex, Malphalan) and novel or emerging (bortezomib, lenalidomide, perifosine, AS703026) anti-MM therapies. Therefore, the novel LNT1 cell line will provide a useful model system to study genetics and biology of MM, as well as to evaluate novel therapeutic strategies. Since LNT1 cells highly express HLA-A2, this line also serves a useful model to validate novel tumor-associated antigens for immunotherapy in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 400.

Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.

Pegylated interferon α-2b up-regulates specific CD8+ T cells in patients with chronic hepatitis B

to investigate the effect of pegylated interferon (IFN) α-2b on specific CD8+ T lymphocytes in patients with chronic hepatitis B (CHB). twenty-one patients with CHB were treated with pegylated IFN α-2b. Periphery blood mononuclear cells were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation (density: 1.077 g/L, Pharmingen) at weeks 0, 4, 8, 12, and 24, respectively. Frequency of circulating hepatitis B virus (HBV) epitope-specific CD8 T cells was detected by flow cytometry. Cytokines were detected by cytometric bead assay. the frequency of circulating HBV core or env-specific CD8 T cells was higher (P < 0.05), the number of HBV core specific CD8 T cells was greater at week 24 (P < 0.05), the level of Th1-type cytokines [interleukin (IL)-12, tumor necrosis factor-α, and IFN-γ] was higher, while that of Th2-type cytokines (IL-4, IL-6, and IL-10) was lower in responders than in non-responders (P < 0.05) after pegylated IFN α-2b treatment. The IL-6 level was correlated with HBV DNA (r = 0.597, P = 0.04), while the inducible protein-10 (IP-10) level was correlated with serum alanine aminotransferase (ALT) (r = 0.545, P = 0.005). The IP-10 level at week 8 after pegylated IFN α-2b treatment could predict the normalization of ALT in CHB patients (positive predict value = 56%, negative predict value = 92%). pegylated IFN α-2b can enhance the immune response of CHB patients by increasing the frequency of HBV specific CD8+ T cells and regulating the Th1/Th2 cytokines.