CD45 Antibody Research Articles

Positive Role of CCAAT/Enhancer-Binding Protein Homologous Protein, a Transcription Factor Involved in the Endoplasmic Reticulum Stress Response in the Development of Colitis

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the α-Emitting Radionuclides Bismuth-213 or Astatine-211

We previously investigated the potential of targeted radiotherapy using a bismuth-213 ((213)Bi)-labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. Although this approach allowed sustained marrow engraftment, limited availability, high cost, and short half-life of (213)Bi induced us to investigate an alternative alpha-emitting radionuclide, astatine-211 ((211)At), for the same application. Biodistribution and toxicity studies were conducted with conjugates of the anti-murine CD45 antibody 30F11 with either (213)Bi or (211)At. Mice were injected with 2 to 50 muCi on 10 microg or 20 muCi on 2 or 40 microg of 30F11 conjugate. Biodistribution studies showed that the spleen contained the highest concentration of radioactivity, ranging from 167 +/- 23% to 417 +/- 109% injected dose/gram (% ID/g) after injection of the (211)At conjugate and 45 +/- 9% to 166 +/- 11% ID/g after injection of the (213)Bi conjugate. The higher concentrations observed for (211)At-labeled 30F11 were due to its longer half-life, which permitted better localization of isotope to the spleen before decay. (211)At was more effective at producing myelosuppression for the same quantity of injected radioactivity. All mice injected with 20 or 50 muCi (211)At, but none with the same quantities of (213)Bi, had lethal myeloablation. Severe reversible acute hepatic toxicity occurred with 50 muCi (213)Bi, but not with lower doses of (213)Bi or with any dose of (211)At. No renal toxicity occurred with either radionuclide. The data suggest that smaller quantities of (211)At-labeled anti-CD45 antibody are sufficient to achieve myelosuppression and myeloablation with less nonhematologic toxicity compared with (213)Bi-labeled antibody.

Common wound healing signature in murine and human involuting breast, implications for pregnancy-associated breast cancer.

Abstract Abstract #1049 Full term pregnancy increases breast cancer risk for up to ten years after childbirth and breast cancer diagnosed during this time has been referred to as pregnancy associated breast cancer (PABC). PABC patients have high rates of metastasis, which is independent of known prognostic factors. Following parturition or lactation, during involution, the breast returns to its pre-pregnant state and in the rat model, we found mammary gland involution employs tissue remodeling programs activated during wound healing and inflammation including high matrix metalloproteinase -2, -3, and -9 activities, release of bioactive fragments of FN and LN, deposition of fibrillar collagen, and increased cytokine levels, including TGFβ1, TGFβ3 and CTGF. We hypothesize that this physiologically normal, but pro-inflammatory remodeling of the gland accounts for the high rate of metastases seen in PABC.
 To further define involution features common to wound healing that may account for the metastasis promoting attributes of PABC, the question of whether macrophages are recruited to the involuting gland was investigated in both rat mammary and human breast tissue. Using the macrophage marker CD68, a lysosomal associated protein, the number of CD68 positive cells in rat mammary glands increased more than 6 fold during involution. Involution-associated macrophages were identified as subtype M2-like by arginase-1 expression and lack of subtype M1-marker iNOS expression. To address the question of whether breast involution in women also is characterized by a wound-healing signature, formalin fixed breast biopsy tissues were obtained for women who were either pregnant, lactating, or whose breast tissue was actively involuting at the time of biopsy. Using a novel computer assisted imaging program, over 200 images of human breast tissue have been quantitated for CD68. CD68 staining in the human breast followed the same trend as in rat, with 5 times more staining in involuting compared to pregnant or lactating tissue. Total leukocyte number, as assessed by CD45 antibody, and oncofetal ECM protein tenascin-C staining were also increased in involuting human breast tissue. With respect to these three stromal markers, involuting human breast tissue was more similar to breast cancer stroma (N=50 images) than stroma from breasts of nulliparous women (N=30 images). These data demonstrate for the first time that human mammary gland involution is characterized by a pro-inflammatory microenvironment, implicating physiologic tissue inflammation in the poor prognosis of PABC.
 In a newly developed animal model for PABC, MCF10DCIS cells were injected orthotopically into SCID mice whose mammary glands were either quiescent (virgin mice) or actively remodeling following weaning (involuting mice). Tumor incidence was 78% in involuting and 50% in virgin mice. Further, tumor multiplicity, size and DCIS conversion to locally invasive lesions were significantly higher in the involuting mice than control virgin mice. This model is being utilized for mechanistic and chemopreventive studies of PABC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1049.

Antibodies to glycosylphosphatidyl-inositol anchored proteins (GPI-AP) in antithymocyte and antilymphocyte globulin: possible role for the expansion of GPI-AP deficient cells in aplastic anemia

Antithymocyte globulin (ATG) and antilymphocyte globulin (ALG) are currently used successfully for immunosuppressive treatment of aplastic anemia. In this study we have investigated whether commercial ATG/ALG preparations contain antibodies against glycosylphosphatidyl-inositol anchored proteins (GPI-AP), which could be responsible for emergence of GPI-deficient populations in aplastic anemia after ATG/ALG therapy. We analyzed four commercial ATG/ALG preparations by competitive binding assays using flow cytometry. Quantification was achieved by calculating the concentration of ATG/ALG required to give 50% inhibition of binding the specific fluorochrome-labeled monoclonal antibody (EC50). High concentrations of antibodies against the GPI-anchored protein CD52 were found in all preparations (Lymphoglobulin Genzyme, Thymoglobulin Genzyme, ATGAM. Pharmacia & Upjohn, and ATG-Fresenius S Fresenius). Antibodies against the GPI-anchored protein CD48 are present in significant concentrations except in the preparation ATGAM. CD16 antibodies were found in lower concentrations. We could not detect significant concentrations of antibodies against the GPI-anchored proteins CD157 and CD14. Campath-1H, a monoclonal antibody against the GPI-anchored protein CD52, has been used as immunosuppressive tool for T-cell depletion. CD52 antibodies in ATG/ALG preparations might contribute in the same way to the immunosuppressive effects in treatment of aplastic anemia. It is known that in a substantial proportion of patients with aplastic anemia GPI-deficient cells are present in a low level at diagnosis or emerge after immunosuppressive therapy. GPI-anchored antibodies in ATG/ALG preparations might lead to a relative advantage for pre-existing GPI-deficient cells caused by an escape from the antibody-mediated attack.

Reticulated platelets and thrombopoietin in schistosomiasis patients

Schistosomiasis mansoni is a non-cirrhotic liver disease. In cirrhosis patients with portal hypertension, a decreased number of reticulated platelets associated with increased thrombopoietin serum levels were reported. We previously reported a 120/nl platelet cutoff level as a marker of clinically significant portal hypertension in schistosomiasis patients. To evaluate reticulated platelet counts and thrombopoietin serum levels (TPO) in schistosomiasis patients and correlate them with portal hypertension markers. Thirty-three schistosomiasis patients without co-morbidities were endoscopically classified as those with (n = 19) or without (n = 14) clinically significant portal hypertension. Flow cytometric determination of reticulated platelets was performed using CD41 antibody and thiazol orange. Ultrasonographic examinations were performed according to the Niamey protocol. TPO and hyaluronic acid serum levels were determined in duplicate using ELISA methods. The platelet number of 120/nl discriminates the two groups with 100% accuracy and 100% positive and negative predictive values, and correlates with spleen length and portal and splenic vein diameters. Differences in reticulated platelets and hyaluronic acid serum levels between both groups were significant (P = 0.025 and 0.012, respectively), but thrombopoietin serum levels were not (P = 0.769). Schistosomiasis patients with portal hypertension have increased reticulated platelets associated with normal TPO serum levels.

Role of LPS/CD14/TLR4-mediated inflammation in necrotizing enterocolitis: Pathogenesis and therapeutic implications

To establish the roles of lipopolysaccharide (LPS)/CD14/toll-like receptor 4 (TLR4)-mediated inflammation in a rat model of human necrotizing enterocolitis (NEC). Six pairs of intestinal samples from human NEC were collected before and after recovery for histological and molecular analysis of inflammatory cytokines and signaling components. In the rat NEC model, we isolated 10-cm jejunum segments and divided them into six groups (n = 6) for sham operation, treatment with LPS, bowel distension, combined bowel distension and LPS stimulation, and two therapeutic groups. The potential efficacy of a recombinant CD18 peptide and a monoclonal CD14 antibody was evaluated in the latter two groups. The serum and tissue levels of several inflammatory mediators were quantified by real-time polymerase chain reaction, ELISA and immunoblotting. Human acute phase NEC tissues displayed significant increases (P < 0.05) in levels of TLR4, CD14, myeloid differentiation protein (MD)-2, tumor necrosis factor (TNF)-alpha and nuclear factor-kappaB when compared to those after recovery. The histological and inflammatory picture of human NEC was reproduced in rats that were treated with combined bowel distension and LPS, but not in the sham-operated and other control rats. Serum levels of interleukin-6 and TNF-alpha were also elevated. The NEC pathology was attenuated by treating the NEC rats with a monoclonal CD14 antibody or an LPS-neutralizing peptide. LPS and distension are required to produce the histological and inflammatory features of NEC. A potential treatment option is blocking LPS activation and leukocyte infiltration.

Pretargeted radioimmunotherapy using anti-CD45 monoclonal antibodies to deliver radiation to murine hematolymphoid tissues and human myeloid leukemia.

Radioimmunotherapy (RIT) for treatment of hematologic malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted (P)RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by a biotinylated clearing agent and radiolabeled 1,4,7,10-tetraazacylodode cane N,N',N",N'''-tetraacetic (DOTA)-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the PRIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a large amount of labeled Ab for a prolonged time. Therapy experiments showed that (90)Y-DOTA-biotin significantly prolonged survival of mice treated with pretargeted anti-hCD45 Ab-SA compared with mice treated with conventional RIT using (90)Y-labeled anti-hCD45 Ab at 200 muCi. Because human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and PRIT using an anti-murine (m)CD45 Ab where the target antigen is present on normal hematopoietic tissues. After 24 h, 27.3% +/- 2.8% of the injected dose of activity was delivered per gram (% ID/g) of lymph node using (131)I-A20-Ab compared with 40.0 +/- 5.4% ID/g for pretargeted (111)In-DOTA-biotin. These data suggest that pretargeted methods for delivering RIT may be superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.

Interdependency of CEACAM-1, -3, -6, and -8 induced human neutrophil adhesion to endothelial cells

Members of the carcinoembryonic antigen family (CEACAMs) are widely expressed, and, depending on the tissue, capable of regulating diverse functions including tumor promotion, tumor suppression, angiogenesis, and neutrophil activation. Four members of this family, CEACAM1, CEACAM8, CEACAM6, and CEACAM3 (recognized by CD66a, CD66b, CD66c, and CD66d mAbs, respectively), are expressed on human neutrophils. CD66a, CD66b, CD66c, and CD66d antibodies each increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. This increase in neutrophil adhesion caused by CD66 antibodies is blocked by CD18 mAbs and is associated with upregulation of CD11/CD18 on the neutrophil surface. To examine potential interactions of CEACAMs in neutrophil signaling, the effects on neutrophil adhesion to human umbilical vein endothelial cells of a set of CD66 mAbs was tested following desensitization to stimulation by various combinations of these mAbs. Addition of a CD66 mAb in the absence of calcium results in desensitization of neutrophils to stimulation by that CD66 mAb. The current data show that desensitization of neutrophils to any two CEACAMs results in selective desensitization to those two CEACAMs, while the cells remain responsive to the other two neutrophil CEACAMs. In addition, cells desensitized to CEACAM-3, -6, and -8 were still responsive to stimulation of CEACAM1 by CD66a mAbs. In contrast, desensitization of cells to CEACAM1 and any two of the other CEACAMs left the cells unresponsive to all CD66 mAbs. Cells desensitized to any combination of CEACAMs remained responsive to the unrelated control protein CD63. Thus, while there is significant independence of the four neutrophil CEACAMs in signaling, CEACAM1 appears to play a unique role among the neutrophil CEACAMs. A model in which CEACAMs dimerize to form signaling complexes could accommodate the observations. Similar interactions may occur in other cells expressing CEACAMs.

Quantification of circulating endothelial cells in peripheral blood of systemic lupus erythematosus patients: a simple and reproducible method of assessing endothelial injury and repair

Quantification of circulating endothelial cells (CECs) in peripheral blood is developing as a novel and reproducible method of assessing endothelial damage/dysfunction. Accordingly, elevated levels of CECs may be a marker of vascular injury in systemic lupus erythaematosus (SLE). This study was undertaken to assess the blood level of CECs in SLE and to correlate its level with the activity of the disease and to find out the possibility that the presence of increased numbers of CECs can be used as a marker of immune-mediated vessel damage. The study included 33 patients with SLE and 20 healthy controls. They were subjected to clinical examination together with laboratory investigations including complete blood count (CBC), erythrocyte sedimentation rate (ESR), urine analysis, renal function test, C3, C4, ANA, anti-ds DNA antibody, antiphospholipid (IgM and IgG) antibodies and quantification of CECs in blood. CECs were calculated using flow cytometry after staining with a mouse anti-human CD45 antibody (pan-leukocyte marker), mouse anti-human CD146 antibody (endothelial cell marker) and 7-amino-actinomycin D (7-AAD) viability marker. CECs were defined as the live cells with 7-AAD negative, CD45 negative and CD146 positive. The number of CECs was significantly higher in patients with SLE compared with those in healthy control (mean +/- SD 38.6 +/- 21.2 versus 7.4 +/- 3.4). Furthermore, CECs were correlated positively with SLE disease activity index (SLEDAI) score, ESR and anti-ds DNA. CECs from patients with vasculitic skin lesions, renal and central nervous system (CNS) manifestation were significantly higher than patients free from the previous signs. An increased number of CECs observed in patients with SLE was associated with the active phase of the disease and may represent a marker of widespread endothelial injury.

CEACAM-1, -3, -6, and -8 Signaling in Human Neutrophils

Members of the carcinoembryonic antigen family (CEACAMs) are widely expressed, and, depending on the tissue, capable of regulating diverse functions including tumor promotion, tumor suppression, angiogenesis, and neutrophil activation. Four members of this family, CEACAM1, CEACAM8, CEACAM6, and CEACAM3 (recognized by CD66a, CD66b, CD66c, and CD66d monoclonal antibodies (mAbs), respectively), are expressed on human neutrophils. CD66a, CD66b, CD66c, and CD66d antibodies each increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. This increase in neutrophil adhesion caused by CD66 antibodies is blocked by CD18 mAbs and is associated with upregulation of CD11/CD18 on the neutrophil surface. To examine potential interactions of CEACAMs in neutrophil signaling, the effects of a set of CD66 mAbs on neutrophil adhesion to human umbilical vein endothelial cells was tested following desensitization to stimulation by various combinations of these mAbs. The current data show that desensitization of neutrophils to any two CEACAMs results in selective desensitization to those two CEACAMs, while the cells remain responsive to the other two neutrophil CEACAMs. In addition, cells desensitized to CEACAM-3, -6, and -8 were still responsive to stimulation of CEACAM1 by CD66a mAbs. In contrast, desensitization of cells to CEACAM1 and any two of the other CEACAMs left the cells unresponsive to all CD66 mAbs. Cells desensitized to any combination of CEACAMs remained responsive to the unrelated control protein CD63. Thus, while there is significant independence of the four neutrophil CEACAMs in signaling, CEACAM1 appears to play a unique role among the neutrophil CEACAMs. A model in which CEACAMs dimerize to form signaling complexes could accommodate the observations. Similar interactions may occur in other cells expressing CEACAMs.

Heterozygous Ala156Val Mutation in the GPIb Alpha (Heterozygous Bernard-Soulier Syndrome Type Bolzano) Induces Macrothrombocytopenia by Hampering Proplatelet Formation.

BACKGROUND. Pathogenesis of macrothrombocytopenia in Bernard-Soulier syndrome (BSS) is still obscure, although the normal amount of bone marrow megakaryocytes (MKs) and the normal platelet survival point towards defective platelet formation as the causative defect.PATIENTS AND METHODS. We analyzed in vitro megakaryocyte differentiation and maturation, as well as proplatelet formation (PPF) in 6 patients heterozygous for the Ala156Val mutation in the GPIb alpha (Bolzano mutation). All of them had mild to moderate thrombocytopenia and enlarged platelets. MKs were differentiated from cord blood CD34+ cells (1 patient) and peripheral blood mononuclear cells (5 patients) for 12 days. Mature MKs were grown in suspension or plated onto glass coverslips coated with collagen I, fibrinogen (FNG), or von Willebrand Factor (VWF). MK differentiationmaturation and PPF were evaluated by phase contrast and fluorescence microscopy upon cell staining with anti alpha-tubulin and CD41 antibodies. Controls were analyzed in parallel with each patient sample.RESULTS Immunomorphological evaluation of cultured cord blood progenitors at day 12 revealed that, the percentage of CD41+ cells with MK morphology was comparable in patient and relative control. Moreover, classification of MK morphology, according to standard criteria, revealed no significant differences in the maturation profiles between patient and control, indicating that the Bolzano mutation did not affect either differentiation or maturation of MKs. When reseeded in fresh medium, mature MKs derived from cord blood of both patient and control extended proplatelets. However, a time course analysis revealed a severe reduction of PPF by MKs derived from the patient: in fact, while 25% of control MKs were forming proplatelets after 48–72 hours of culture, the percentage of MKs with PPF from the patient's cord blood never exceeded 5%. Adhesion to FNG or VWF resulted in a similar impairment of PPF with respect to control. As in the control, type I collagen totally inhibited PPF by BSS cord blood derived MKs. PPF by MKs derived from the peripheral blood of 5 BSS type Bolzano patients was also analyzed. As for the cord blood we observed a significant reduction of PPF, both when MKs were grown in suspension and when MKs were adhering to fibrinogen or VWF. Thus, as in MKs from cord blood, the Bolzano mutation seemed to affect PPF also in MKs cultured from peripheral blood. In morphologic analysis proplatelets derived from both cord and peripheral BSS type Bolzano blood revealed an altered structure of the tips that presented altered alpha-tubulin distribution and an enlarged diameter with respect to controls. Interestingly, staining of patients' peripheral blood films, revealed that some of the circulating platelets had an altered alpha-tubulin distribution resembling that observed at proplatelets' tips.CONCLUSIONS. MKs from patients carrying the Bolzano mutation present both quantitative and qualitative abnormalities of in vitro PPF. This indicates a key role for GPIb in PPF and confirms the hypothesis that macrothrombocytopenia of BSS derives from defective platelet formation.

Culture of Mobilized Human CD34+ Cells in Hypoxic Conditions Improves Lentiviral Transduction Efficiency in SCID-Repopulating Cells

Under normal physiological conditions, hematopoietic stem cells (HSC) are sequestered in a hypoxic microenvironment in the bone marrow (BM), suggesting that low oxygen levels may play a fundamental role in the maintenance of normal stem cell function and protect these cells from the damaging effects of reactive oxygen species (ROS). In vitro culture of human BM CD34+ cells under hypoxic conditions has been shown to result in expansion of SCID-repopulating cells (SRC) as compared to culture under normoxic conditions (JCI 112 (1); 126, 2003). We investigated whether culture of human mobilized CD34+ cells under low oxygen conditions (5% O2) could improve lentiviral transduction efficiency in SRC compared with culture under atmospheric O2 conditions (21%). G-CSF mobilized CD34+ cells from 4 healthy volunteers were prestimulated for 48 hours in the presence of cytokines (SCF, Flt-3 ligand and thrombopoietin) and subsequently transduced in fibronectin coated plates for 24 hours with SIN-lentiviral vectors carrying the GFP gene under the control of an EF1α promoter. In 3 experiments, cells were used for in vitro assays, including ROS, phenotypic, cell cycle, clonogenic and apoptosis assays. In one experiment, cells were injected intravenously in the tail vein of sublethally irradiated NOD/SCID IL2rγ −/− mice after transduction. Intracellular ROS levels increased more significantly in human CD34+ cells cultured for 3 days in 21% O2 compared with cells cultured in 5% O2. When cultures were maintained more than 3 days, ROS levels were similar between the 2 conditions. The levels of expansion of CD34+ cells compared with baseline were similar in hypoxia (3.9-fold) and normoxia (3.5-fold) (p=0.47). In contrast, the expansion of CD34+CD38− cells, a subpopulation enriched in HSCs, was greater in hypoxia (3.8-fold) than in normoxia (2.2-fold) (p=0.02). After 3 days of culture, the total number of colony-forming cells (CFC) increased 1.1-fold and 1.3-fold under hypoxic and normoxic conditions, respectively (p=0.32) compared with freshly isolated CD34+ cells. The level of O2 had no significant effect on lineage commitment of the CFC. At baseline, the majority (59.5%) of the CD34+ cells were in the G0 phase of the cell cycle. After 3 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 5.5% and 3.5%, respectively (p=0.03). The differences in percentages of cells in the G1 and G2/S/M phases of the cell cycle were not statistically different. The percentages of CD34+ apoptotic cells were similar between hypoxic (32.8%) and normoxic (29.5%) conditions (p=0.18). The pO2 also had no impact on CD34+ cell death (12.2% at 5% O2 and 11.7% at 21% O2, p=0.9). When considering the bulk of CD34+ cells after transduction with GFP-lentiviral vectors, there was no statistically significant difference in the percentages of GFP+ cells under hypoxia (22.3%) or normoxia (21%) (p=0.88). In contrast, when CD34+ cells cultured under hypoxia were injected into NOD/SCID IL2rγ −/− mice at the end of the transduction period, improved human cell engraftment and lentiviral transduction efficiency were detected 2 months after transplantation compared with CD34+ cells cultured under normoxia. Human cell engraftment in the mouse BM, as determined by flow cytometry using a human specific CD45 antibody, was 84% in the hypoxic group (n=4) and 54% in the normoxic group (n=4) (p=0.04). The level of O2 had no significant impact on the lineage commitment of the SRC, with a majority of CD45+CD15+ granulocytes in both groups. The percentage of GFP+CD45+ cells was 54% (hypoxia) and 43% (normoxia) (p=0.02), indicating an improved transduction efficiency of SRC under hypoxic conditions. Overall, these data indicate that human CD34+ cells cultured under low oxygen conditions maintain a more primitive phenotype and have an increased susceptibility to lentiviral transduction compared with cells cultured in 21% O2 conditions. Improved engraftment and transduction efficiency do not appear to be related to decreased apoptosis in lower O2 concentrations; instead, increased ROS production in higher O2 concentrations could lead to increased cell signaling and differentiation. Use of low O2 levels for in vitro transduction of human CD34+ cells could have important clinical implications in gene therapy.

I-131-Anti-CD45 Radioimmunotherapy Effectively Targets and Treats T-Cell Non-Hodgkin's Lymphoma

T-cell non-Hodgkin lymphoma (T-NHL) exhibits inferior remission durations and survival rates compared to B-cell NHL. This may be at least partially due to the paucity of targeted therapies and the absence of approved radioimmunoconjugates available for T-NHL. The CD45 antigen is expressed on ~90% of T-NHL and is not readily internalized or shed, making it an appealing target for radioimmunotherapy (RIT). In order to test this hypothesis, we first demonstrated that CD45 was expressed in high copy numbers on the surface of human T-NHL samples and T-NHL cell lines with a median of 3.4×105 CD45 molecules/cell on T-NHL lines (CCRF-CEM, Karpas 299) and 2.25×105 CD45 molecules/cell on patient derived T-NHL specimens (T-LBL, CTCL, NK/T-NHL). We employed an athymic murine xenograft model with the above human T-NHL lines to show that targeting human CD45 could deliver more radioactivity to tumor nodules than to normal tissues. Mice with palpable human T-NHL xenografts were injected with 131I-labeled BC8 (a murine anti-human CD45 antibody [Ab]) and an 125I-labeled murine isotype matched nonbonding control Ab (BHV1). CCRF-CEM xenografts targeted with 131I-BC8 demonstrated 86% (p=.003) and 106% (p=.001) more radioiodine retention at 24h and 48h, respectively, compared to xenografts targeted with the control BHV1 Ab. More importantly, tumor sites exhibited 1.6 (p=.045), 2.4 (p=.008), and 2.7 (p=.007)-fold 131I retention compared to the lungs, liver, and kidneys, respectively (CCRF-CEM, 24h). Similar results were observed at later time points and with other T-NHL lines. We next hypothesized that this preferential tumor targeting would translate into improved tumor control and survival using therapeutic doses of 131I-BC8. Mice with palpable T-NHL xenografts (CCRF-CEM) were randomly assigned to receive 200μg of BC8 labeled with either 300 or 400μCi of 131I, 200μg of BHV-1 (control) labeled with 300μCi 131I, or no treatment. Tumor dimensions and survival were tracked. By day 9, complete remissions (CR) were attained in 90% of mice treated with 400 μCi of 131I-BC8 and 67% of mice that received 300 μCi of 131I-BC8 (Figure). In contrast, none of the untreated control mice or mice that received 300 μCi of 131I-BHV-1 achieved CR (p<0.0001). All 20 mice in the 2 control groups required euthanasia due to tumor growth, whereas only 4 of 20 of the mice in experimental arms needed to be sacrificed due to disease progression and unmaintained remissions persisted for over 72 days following therapy. Since CD45 is also expressed on most normal hematopoietic tissues, we evaluated the ability of anti-CD45 RIT to preferentially localize to tumor and hematolymphoid sites in a syngeneic model targeting murine CD45. Mice with palpable EL-4 (murine T-NHL) xenografts were treated with 200 μg of 131I-30F11 (anti-murine CD45) followed by harvest of tumor sites and normal organs. In this syngeneic model, the ratios of retained radioactivity in tumor sites to critical non-target sites were: lung 5.3 (p<.001), liver 5.4 (p<.001), and kidney 8.7 (p<.001). Comparable ratios were seen when other target sites (marrow, spleen) were compared to normal non-target organs. These data indicate that CD45 is highly expressed on T-NHL, allows reliable antibody targeting of tumor sites in both xenogeneic and syngeneic models, and translates into significantly improved control of T-NHL xenografts.These results support the hypothesis that CD45-targed RIT transplant conditioning regimens may improve outcomes for patients with relapsed/refractory T-NHL. [Display omitted]

Advanced Glycation End-Product of Low Density Lipoprotein Activates the Toll-Like 4 Receptor Pathway Implications for Diabetic Atherosclerosis

Diabetes is a major risk factor for coronary heart disease. Accumulation of advanced glycation end-products (AGEs) attributable to hyperglycemia in diabetics promotes the development of atherosclerosis. However, the underlying mechanisms remain unclear. The advanced glycation end-product of low-density-lipoprotein (AGE-LDL) induced proinflammatory cytokine production in human coronary artery endothelial cells and human- and mouse-macrophages. AGE-LDL stimulated cytokine synthesis was markedly reduced in mouse macrophages with a TLR4 loss-of-function mutation. Coimmunoprecipitation experiments indicated AGE-LDL interacts with TLR4, RAGE, and CD36. Incubation of cultured macrophages with TLR4, RAGE, or CD36 antibodies inhibited AGE-LDL stimulation of tumor necrosis factor (TNF)alpha production. A competitive binding inhibitor of TLR4 blocked AGE-LDL binding to the receptor. After transfection of a HEK293 cell system with wild-type TLR4, AGE-LDL activated a signaling pathway including p38 alpha, JNK, and ERK1 kinases and AP1, Elk1, and NF-kappaB transcription factors; the net result being increased cytokine production. These effects were absent when cells were transfected with empty plasmid. Two common polymorphisms in TLR4, D299G and T399I, reduced the response of TLR4 to lipopolysaccharide (LPS) but had no effect on AGE-LDL signaling. These results indicate that AGE-LDL activates a TLR4-mediated signaling pathway, thus inducing proinflammatory cytokine production. This mechanism may partly explain the increased risk of atherosclerosis observed in diabetics.

Vitamin D3 Induces Autophagy of Human Myeloid Leukemia Cells

Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.

Functional relevance of tetraspanin CD9 in vascular smooth muscle cell injury phenotypes: A novel target for the prevention of neointimal hyperplasia

Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in the development of neointima following vascular injury. In this study, we found that CD9 is constitutively expressed in the VSMC of the neointima of injured carotid arteries. The in vitro migration and proliferation of human coronary artery smooth muscle (hCASM) cells were reduced by 40% and 63%, respectively, by treatment with a CD9 specific monoclonal antibody mAb7 when compared to control antibody treatment. In a mouse carotid ligation injury model, a single application of a neutralizing anti-mouse CD9 antibody resulted in a 31% (day 14, n = 8, p < 0.05), and 32% (day 28, n = 5, p < 0.01) reduction in neointima formation. In support of these findings, exogenous expression of human CD9 by CD9-adenoviral transduction led to 43% increases in neointima ( p < 0.05, n = 6). Upon investigation of the mechanisms underlying CD9 mediated VSMC phenotypic events we found that integrin α5β1 was a constitutive partner of CD9 and that CD9 significantly augmented PI-3 kinase dependent Akt phosphorylation. Furthermore, enhanced Akt phosphorylation was attenuated by mAb7 binding. Cumulatively, a functional link between CD9, α5β1, PI3-K/Akt activity and enhanced VSMC migratory and proliferative phenotypes has been demonstrated. These studies suggest that agents that modulate CD9 mediated VSMC phenotypes may emerge as novel strategies for the treatment of abnormal vascular injury response.

Analysis of microvessels in pancreatic cancer: by light microscopy, confocal laser scan microscopy, and electron microscopy

The microvessel density (MVD) of most malignant tumors is considered to be strongly related to metastasis and prognosis. Weidner's "hot spot method" for determining MVD is in general use, but it is possible that cells other than endothelial cells will also be stained. In our previous study, no correlations were observed between MVD determined by the "hot spot method" and prognosis/metastasis. But, using the "lumen method," we found a correlation with the number of vessel structures only. In the present study, we analyzed the staining of microvessels in pancreatic cancer, using light microscopy, confocal laser scan microscopy (CLSM), and transmission electron microscopy (TEM). Microvessel staining of pancreatic cancer with CD34, factor VIII, and CD45 antibodies was examined in consecutive slices by light microscopy. For CLSM, freshly resected specimens were immunostained with factor VIII and fluorescein isothiocyanate. For TEM, specimens were fixed with 2.5% glutaraldehyde, treated with 1% osmium tetroxide, and embedded in epoxy resin. Staining of vessels with CD34 and factor VIII antibodies appeared similar under light microscopy. However, CD34-stained consecutive slices were judged not to reveal vessel structures, and some cells stained with CD45 antibody were similar in appearance to CD34-stained cells. Under CLSM, irregular arrangements of neovascularization, consisting of many branches, were observed, but many positively stained cells not identified as vessels were also seen. Microvessels were distinctly identified under TEM, but the types of individual cells could not be determined. An integrated, reproducible method for the measurement of MVD is vital. For pancreatic cancer, the "lumen method" is recommended.

Plant Sterols Cause Macrothrombocytopenia in a Mouse Model of Sitosterolemia

Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an inborn error of metabolism characterized by high plasma plant sterol concentrations. Recently, macrothrombocytopenia was described in a number of sitosterolemia patients, linking hematological dysfunction to disturbed sterol metabolism. Here, we demonstrate that macrothrombocytopenia is an intrinsic feature of murine sitosterolemia. Abcg5-deficient (Abcg5(-/-)) mice showed a 68% reduction in platelet count, and platelets were enlarged compared with wild-type controls. Macrothrombocytopenia was not due to decreased numbers of megakaryocytes or their progenitors, but defective megakaryocyte development with deterioration of the demarcation membrane system was evident. Lethally irradiated wild-type mice transplanted with bone marrow from Abcg5(-/-) mice displayed normal platelets, whereas Abcg5(-/-) mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. Treatment with the sterol absorption inhibitor ezetimibe rapidly reversed macrothrombocytopenia in Abcg5(-/-) mice concomitant with a strong decrease in plasma plant sterols. Thus, accumulation of plant sterols is responsible for development of macrothrombocytopenia in sitosterolemia, and blocking intestinal plant sterol absorption provides an effective means of treatment.

Use of an anti‐CD16 antibody for in vivo depletion of natural killer cells in rhesus macaques

Non-human primates serve as key animal models for a variety of viral infections. To evaluate the contribution of natural killer (NK) cells to the immune-mediated control of these viruses in macaque monkeys, we have described a method for depleting NK cells in vivo by administration of anti-human CD16 mouse monoclonal antibody. Using a fluorometric NK-cell cytotoxicity assay, we show that most NK-cell cytotoxicity in rhesus monkey peripheral blood mononuclear cells resides in the CD16(+) and/or CD159A(+) subset of lymphocytes. The anti-human CD16 antibody, 3G8, binds to subsets of rhesus monkey lymphocytes and monocytes but not to neutrophils. Intravenous administration of 10-50 mg/kg of 3G8 to normal rhesus monkeys resulted in anti-CD16 antibody persistence in the plasma for 1-3 weeks. This treatment also depleted 80-90% of CD3(-) CD159A(+) lymphocytes, putative NK cells, from blood for at least 1 week and was associated with the loss of NK-cell cytotoxicity when evaluated by in vitro assays. Using this method, transient depletion of NK cells from two rhesus monkeys chronically infected with simian immunodeficiency virus failed to cause changes in virus replication. These studies describe a non-human primate model for in vivo NK-cell depletion and suggest a limited role for cytotoxic CD16(+) NK cells in controlling AIDS virus replication during chronic infection.

The tripeptide feG inhibits leukocyte adhesion

BackgroundThe tripeptide feG (D-Phe-D-Glu-Gly) is a potent anti-inflammatory peptide that reduces the severity of type I immediate hypersensitivity reactions, and inhibits neutrophil chemotaxis and adhesion to tissues. feG also reduces the expression of β1-integrin on circulating neutrophils, but the counter ligands involved in the anti-adhesive actions of the peptide are not known. In this study the effects of feG on the adhesion of rat peritoneal leukocytes and extravasated neutrophils to several different integrin selective substrates were evaluated.ResultsThe adhesion of peritoneal leukocytes and extravasated neutrophils from rats to adhesive proteins coated to 96-well plates was dependent upon magnesium (Mg2+) ion, suggestive of integrin-mediated adhesion. feG inhibited leukocyte adhesion, but only if the cells were stimulated with PAF (10-9M), indicating that feG's actions in vitro require cell activation. In the dose range of 10-10M to 10-12M feG inhibited the adhesion of peritoneal leukocytes to fibrinogen and fibronectin, but not IgG, vitronectin or ICAM-1. feG inhibited the binding of extravasated neutrophils to heparin, IgG, fibronectin and CD16 antibody. Antigen-challenge of sensitized rats reduced the adhesion of peritoneal leukocytes to most substrates and abolished the inhibitory effects of feG. However, pretreating the animals with intraperitoneal feG (100 μg/kg) 18 h before collecting the cells from the antigen-challenged animal restored the inhibition of adhesion by in vitro feG of peritoneal leukocytes and extravasated neutrophils to fibronectin.ConclusionThe modulation of leukocyte adhesion by feG appears to involve actions on αMβ2 integrin, with a possible interaction with the low affinity FcγRIII receptor (CD16). The modulation of cell adhesion by feG is dual in nature. When administered in vivo, feG prevents inflammation-induced reductions in cell adhesion, as well as restoring its inhibitory effect in vitro. The mechanism by which in vivo treatment with feG exerts these effects remains to be elucidated.