Design of a recombinant immunotoxin against the human granulocyte-colony stimulating factor receptor.

Immunotoxin is a new strategy for protein therapy of cancer. This engineered protein contains two parts, the immune part which is an antibody or cytokine, directed against the cancer cell receptor, and the toxin part consisting of a plant or bacterial toxin leading to apoptosis by protein synthesis inhibition. The knowledge of cell-surface receptor overexpression in cancer cells can help scientists to construct new anti-cancer agents. The granulocyte colony stimulating factor (G-CSF) receptor is expressed on the cell surface of some blood cancers such as acute myeloid leukemia (AML). Therefore, this receptor can be used as an immunotoxin for treatment of some cancers. The aim of this work was to design and produce DT-GCSF immunotoxin using truncated DT fused to G-CSF. For fusion protein construction, DT389 and G-CSF fragments, were amplified by PCR using specific primers. A flexible linker SerGly4SerMet (SG4SM) was used to fuse the PCR products by SOEing PCR procedure to achieve an appropriate fusion protein, and the fused fragment was subcloned into pET21b. The new construction (pET-DT389GCSF) was transformed into E. coli strain BL21 (DE3) and the expression of the construction was confirmed by SDS-PAGE and Western blotting techniques. The data demonstrated the expression and purity rates of DT389GCSF about 25% and 90%, respectively. This chimeric protein construction can be used as a new anti-AML drug, but its in vitro and in vivo biological activity should be analyzed.

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia.

Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life.

Estudio comparativo sobre la utilidad de la profilaxis antibacteriana con levofloxacino en pacientes receptores de un trasplante de progenitores hematopoyéticos / Comparative study on the usefulness of antibacterial prophylaxis with levofloxacin in patients submitted to hematopoietic stem cell transplantation

Fundamento: La infección bacteriana continúa siendo una complicación frecuente en pacientes receptores de un trasplante de progenitores hematopoyéticos (TPH). No obstante, el impacto de la profilaxis antibacteriana en la mortalidad de estos pacientes es controvertido. Pacientes y métodos: Comparación retrospectiva de 2 grupos consecutivos de receptores de TPH según recibieran (n = 132) o no (n = 107) profilaxis antibacteriana con levofloxacino. Resultados: En el 41% de los procedimientos de TPH en los que se administró profilaxis con levofloxacino se constató infección microbiológicamente documentada (IMD) con bacteriemia, frente a un 40% de los que no recibieron levofloxacino. La frecuencia de bacteriemia por bacilos gramnegativos fue del 11 y del 38%, la resistencia a levofloxacino fue del 39 y del 14%, y hubo un 8 y 7% de muertes, respectivamente. Conclusiones: En nuestra experiencia, el uso de levofloxacino se asoció a una menor frecuencia de bacteriemia por microorganismos gramnegativos, pero no se asoció a disminución en la tasa de IMD ni influyó en su evolución. En cambio, hubo un aumento de la resistencia a quinolonas en los pacientes tratados con levofloxacino (AU)

Background: Bacterial infection remains a frequent complication in patients receiving a hematopoietic stem cell transplantation (HSCT). However, the impact of the antibacterial prophylaxis mortality in these patients is controversial. Patients and methods: Retrospective comparison of 2 consecutive groups of patients undergoing HSCT receiving (n = 132) or not (n = 107) antibacterial prophylaxis with levofloxacin. Results: 41% of patients receiving prophylaxis with levofloxacin had microbiologically documented infection (MDI) with bacteremia, compared with 40% of those not receiving levofloxacin. The frequency of gram-negative bacteremia was 11 and 38%, the resistance to levofloxacin was 39 and 14%, and the mortality was 8 and 7%, respectively. Conclusions; In our experience, the use of levofloxacin as prophylaxis in HSCT was associated with a lower frequency of gram-negative bacteremia but was not associated with a decreased rate of MDI and did not influence their outcome. In contrast, there was an increase in quinolone resistance in patients treated with levofloxacin (AU

M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

Toll-like receptor-mediated airway IL-17C enhances epithelial host defense in an autocrine/paracrine manner.

IL-17A, IL-17F, and IL-25 belong to the IL-17 family of cytokines, and are well known to play important roles in the host defense against infection and inflammatory diseases. IL-17C, also a member of the IL-17 family, is highly expressed in the epithelium; however, the function and regulatory mechanism of IL-17C in airway epithelium remain poorly understood. In this study, we demonstrate that polyinosinic-polycytidylic acid (polyI:C), the ligand to Toll-like receptor 3, is a potent inducer of IL-17C mRNA and protein expression in primary normal human bronchial epithelial (NHBE) cells. IL-17C induction by polyI:C was both time dependent and dose dependent, and was attenuated by inhibitors of the Toll-IL-1 receptor domain-containing adaptor-inducing INF-ß (TRIF)-NF-κB pathway, Pepinh-TRIF, BAY11, NF-κB inhibitor III, and NF-κB p65 small interfering RNA, suggesting that IL-17C expression is induced by polyI:C via the Toll-like receptor 3-TRIF-NF-κB pathway. Both IL-17C and polyI:C increased the expression of antimicrobial peptides and proinflammatory cytokines, such as human ß-defensin (hBD) 2, colony-stimulating factor 3 (CSF3), and S100A12 in NHBE cells. Knockdown of IL-17 receptor (IL-17R) E, the specific receptor for IL-17C, using IL-17RE small interfering RNA, attenuated polyI:C-induced hBD2, CSF3, and S100A12 expression, without any reduction of polyI:C-induced IL-17C expression, which suggest that IL-17C enhances hBD2, CSF, and S100A12 expression in an autocrine/paracrine manner in NHBE cells. Knockdown of IL-17C also decreased polyI:C-induced hBD2, CSF3, and S100A12 expression. Thus, our data demonstrate that IL-17C is an essential epithelial cell-derived cytokine that enhances mucosal host defense responses in a unique autocrine/paracrine manner in the airway epithelium.

A highly phagocytic cell line TO from Atlantic salmon is CD83 positive and M-CSFR negative, indicating a dendritic-like cell type.

Leucocyte cell lines are valuable tools for immunological studies. In this study the TO cell line, originating from Atlantic salmon head kidney leucocytes, is described with respect to enzyme cytochemistry, functional studies, reactivity with leucocyte specific antibodies and immune gene expression. Pronounced characteristics of the TO cell line are the rapid adherence to the plastic growth surface, high phagocytic capacity and bactericidal functions. No respiratory burst activity, and little or no NO production were detected under the experimental conditions tested, and thus the TO cells appear to have other effective killing mechanisms. The cells are reactive with a leucocyte specific monoclonal antibody (MAb), but does not bind a neutrophil specific MAb or stain for myeloperoxidase. Real-time RT-PCR showed the expression in TO cells of several immune genes, some of which were significantly regulated following LPS stimulation. The expression of CD83 might indicate a dendritic cell (DC) origin of the TO cells, as this marker is considered a hallmark for DC. Expression of TCR-alpha or the macrophage marker M-CSFR was not detected. Based on the present analyses the TO cells display a mixture of known characteristics for macrophages and DCs. At the same time the TO cells lack some central functions of phagocytic/myeloid cells. As the TO cells are developed to a long-term culture one cannot exclude that some functions might have been lost in this process. Nevertheless, the features of the TO cells indicate their potential as a model system for immunological studies of salmon phagocytic cells.

Colony-stimulating factors in inflammation and autoimmunity.

Although they were originally defined as haematopoietic-cell growth factors, colony-stimulating factors (CSFs) have been shown to have additional functions by acting directly on mature myeloid cells. Recent data from animal models indicate that the depletion of CSFs has therapeutic benefit in many inflammatory and/or autoimmune conditions and as a result, early-phase clinical trials targeting granulocyte/macrophage colony-stimulating factor and macrophage colony-stimulating factor have now commenced. The distinct biological features of CSFs offer opportunities for specific targeting, but with some associated risks. Here, I describe these biological features, discuss the probable specific outcomes of targeting CSFs in vivo and highlight outstanding questions that need to be addressed.

Effects of monoclonal anti-c-kit antibody (ACK2) on melanocytes in newborn mice.

Previous studies indicate that c-Kit is required for postnatal melanocyte development. To understand the precise mechanisms of c-Kit dependence, we studied melanocyte development in newborn C57BL/6 mice by means of peritoneal injection of a monoclonal anti-c-Kit antibody (ACK2), which blocks c-Kit functions. The mice were injected once or more with ACK2 at various intervals after birth. In experiment 1, skin samples were examined on day 10 post-partum and in experiment 2 they were examined daily until day 10 post-partum. We studied melanocytes in the hair follicles, epidermis, and dermis by light and electron microscopy with dopa reactions and immunohistochemistry. Epidermal melanocytes in untreated mice were dopa negative and c-Kit positive on day 0 post-partum but became dopa positive soon thereafter. In ACK2-treated mice, the earlier the mice received ACK2 injections after birth, the fewer melanocytes they had, not only in the epidermis, but also in follicles. In these mice, melanocytes that had undergone apoptosis in the dermis and the follicles were detected ultrastructurally. Some appeared to have produced tyrosinase, because they had dopa-positive melanosomes. These results suggest that melanocytes in newborn mice are c-Kit dependent and undergo apoptosis when c-Kit receptors are blocked by ACK2 in the early days after birth. During this c-Kit-dependent period, melanocytes differentiate from dopa negative to positive and migrate from the epidermis to hair follicles.

Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/c-kit.

Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with CD117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.

c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a network similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.

Effect of stem cell factor on myelopoiesis potential in human Dexter-type culture systems.

Hematopoiesis is influenced by the presence of the hematopoietic microenvironment, and Dexter-type liquid culture systems represent an in vitro representation of some aspects of the microenvironment that are optimal for the propagation of myeloid progenitors. Marrow stromal layers, which constitute part of these culture systems, produce growth factors, including stem cell factor (SCF), a ligand for the c-kit proto-oncogene that has been found to increase detection of myeloid, erythroid, and megakaryocytic progenitors in short-term marrow colony assays. In this work, the role of SCF in Dexter-type culture systems was examined to better define its contribution to steady-state myelopoiesis. When cultured in the continued presence of 100 ng/mL SCF, both primary and recharged cultures demonstrated significantly greater CFU-GM output, with quantitative differences noted throughout culture duration (up to 6 weeks). This increase in CFU-GM could be inhibited specifically with the addition of 1:1500 SR-1, a neutralizing anti-c-kit monoclonal antibody (MAb) that neutralizes the biological effects of SCF, and the increase was noted both with recharged light-density marrow cells and purified CD34+ progenitor cells. On the other hand, when primary or recharged marrow cultures were established in the absence of exogenous SCF, but in the continuous presence of SR-1, no inhibition of CFU-GM output was observed. When light-density marrow cells were purged of pre-existing CFU-GM by 4-hydroperoxycyclophosphamide (4-HC) and were seeded over irradiated stromal layers, exogenous SCF resulted in detection of CFU-GM from 4-HC-treated cells as early as 1 week of culture, as compared to the lack of significant emergence of CFU-GMs at 4 weeks in the control cultures. This SCF effect was also inhibited by SR-1. Purified CD34+ progenitor cells did not adhere to SCF immobilized to tissue culture plates, and the adhesion of such progenitors to murine Steel lines transfected with membrane-bound SCF was not greater than to the parent nontransfected Steel line, suggesting that the effect of SCF was not on CD34+ cell adhesion. These studies confirm the action of SCF at a pre-CFU level, and they demonstrate the ability of SCF to stimulate increased production of myeloid progenitors in long-term liquid culture systems.

Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML.

In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117+ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients. Our results show that most of normal BM CD117+ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117+ cells are CD34+, these cells displaying a different FSC/SSC distribution when compared to the CD117+/CD34- cells. No CD117+/CD15+ and CD117+/CD10+ cells were detected and very few CD117+ cells (< 1 x 10(-3) expressing the HLADR-/CD34-, CD33+/HLADR- and CD34+/HLADR- phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117+/CD15+ blast cells and eight had the CD117+ phenotypes detected at low frequencies (< 1 x 10(-3)) in normal BM. In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117+/CD15+ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.

The fourth immunoglobulin domain of the stem cell factor receptor couples ligand binding to signal transduction.

Receptor dimerization is ubiquitous to the action of all receptor tyrosine kinases, and in the case of dimeric ligands, such as the stem cell factor (SCF), it was attributed to ligand bivalency. However, by using a dimerization-inhibitory monoclonal antibody to the SCF receptor, we confined a putative dimerization site to the nonstandard fourth immunoglobulin-like domain of the receptor. Deletion of this domain not only abolished ligand-induced dimerization and completely inhibited signal transduction, but also provided insights into the mechanism of the coupling of ligand binding to dimer formation. These results identify an intrinsic receptor dimerization site and suggest that similar sites may exist in other receptors.

Interactions between stem cell factor and c-Kit are required for intestinal immune system homeostasis.

Interactions between stem cell factor (SCF) and its receptor, c-Kit, are important for development of hematopoietic, melanocytes, and germ cells. T lymphocytes appeared normal in c-Kit (W/Wv) or SCF (SI/SId) mutant mice, except for those residing within the intestinal epithelium, the intraepithelial lymphocytes (IEL). Normally, IEL are composed of equal numbers of cells with alpha beta or gamma delta T cell receptors. In mutant mice, beginning at 6-8 weeks of age, the number of gamma delta IEL decreased, whereas alpha beta IEL increased. The latter was due largely to an increased CD4+ CD8+ TCR alpha beta subset, suggesting that these cells may be intermediates in the alpha beta IEL lineage. c-Kit or SCF was expressed by IEL or intestinal epithelial cells, respectively, indicating a potential for direct intercellular interaction. This possibility was supported by reconstitution studies that demonstrated that c-Kit mutations directly affected IEL. Thus, SCF-c-Kit interactions are important for homeostasis of the intestinal immune compartment.

Switching of mouse spermatogonial proliferation from the c-kit receptor-independent type to the receptor-dependent type during differentiation.

Testicular cells composed mostly of germ cells and immature Sertoli cells from neonatal mice 2 and 5 days old were cultured to investigate germ-cell proliferation mediated by the c-kit receptor. The addition of antibody to block the interaction of the c-kit receptor with its ligand inhibited the proliferation of cultured spermatogonia from 5-day-old mice in a dose-dependent manner, but not from that of 2-day-old mice. The addition of anti-c-kit ACK2 monoclonal antibody also inhibited the proliferation of spermatogonia from 5-day-old mutant Sld/Sld mice but not of 5-day-old mutant Wv/Wv mice. The results indicate that c-kit-positive type A spermatogonia in the testes of 5-day-old mice require steel factor (kit ligand) for their proliferation, whereas self-renewal and differentiation of c-kit-negative primitive type A spermatogonia in the testes of 2-day-old mice do not.

Estudio de la clinica, patologia, estadios, evolucion del melenoma y marcadores pronosticos (PCNA y EGFR) / Study of the clinic, pathology, stadiamevolution from melanoma and prognosis makers

Se estudiaron 29 melanomas,en forma prospectiva en relacion a la exprecion del antigeno de proliferacion nuclear(PCNA)indicador de proliferacion celular y del receptor del factor de crecimiento epidermico(EGFR) responsable de estimular la hiperplasia.La PCNA mostro valores elevados y se correlaciono con los parametros habituales.El EGFR con bajo nivel de expresion,presento en el seguimiento un llamativo incremento en los niveles de invasion mas agresivos(nivel V)y en los casos del grupo de"fallecidos".

Estudio de la clinica, patologia, estadios, evolucion del melenoma y marcadores pronosticos (PCNA y EGFR) / Study of the clinic, pathology, stadiamevolution from melanoma and prognosis makers

Se estudiaron 29 melanomas,en forma prospectiva en relacion a la exprecion del antigeno de proliferacion nuclear(PCNA)indicador de proliferacion celular y del receptor del factor de crecimiento epidermico(EGFR) responsable de estimular la hiperplasia.La PCNA mostro valores elevados y se correlaciono con los parametros habituales.El EGFR con bajo nivel de expresion,presento en el seguimiento un llamativo incremento en los niveles de invasion mas agresivos(nivel V)y en los casos del grupo de"fallecidos".AU

c-kit expression by B cell precursors in mouse bone marrow. Stimulation of B cell genesis by in vivo treatment with anti-c-kit antibody.

To examine the in vivo role of c-kit receptor in B lymphopoiesis we have evaluated precursor B cell populations expressing c-kit in mouse bone marrow and the effects on B cell genesis of administering a neutralizing anti-c-kit mAb, ACK2. Double immunofluorescence labeling and mitotic arrest were used to examine bone marrow cells from BALB/c mice. Almost one-half of TdT+ cells and one-quarter of B220+ cells coexpressed c-kit, mainly at low intensities, and were actively proliferating in vivo. c-kit+ cells that lacked B lineage markers expressed c-kit in high intensities and entered mitosis at an exceptionally rapid rate. In ACK2-treated mice, erythroid and granulocytic cells were almost completely absent from the bone marrow, whereas, in contrast, B lymphopoiesis was stimulated. Pre-B cells expressing cytoplasmic mu-chains; as well as TdT+B220+ cells before mu expression, were increased two- to fourfold in number and production rate. IgM-bearing B lymphocytes were increased in bone marrow and spleen. The results demonstrate that many early precursor B cells in mouse bone marrow constitutively express c-kit receptor. The failure of ACK2 treatment to block B lymphopoiesis, however, suggests that c-kit receptor function is not essential for precursor B cell development in vivo, but can be replaced by alternative signaling systems. The stimulation of B cell genesis by ACK2 treatment may reflect a conferred advantage in the competition for microenvironmental factors which underlies the balance between B lymphopoiesis and other hemopoietic lineages in vivo.

Epitope mapping and functional studies with three monoclonal antibodies to the c-kit receptor tyrosine kinase, YB5.B8, 17F11, and SR-1.

Three monoclonal antibodies (MAbs) to the human c-kit receptor tyrosine kinase (P145c-kit), derived in independent laboratories, have been extensively used in studies of c-kit expression and the role of its ligand, steel factor (SLF), in hemopoiesis and mast cell differentiation and function. In this study, the relationship between the epitopes they identify, and their effects on SLF binding, receptor internalization, and signal transduction are compared. Epitope mapping studies carried out on the high P145c-kit-expressing cell line HEL-DR showed that SR-1 identifies an epitope independent of those bound by YB5.B8 and 17F11, while the latter two antibodies bound to distinct but interacting epitopes. SR-1 potently blocked the binding of SLF to P145c-kit on these cells and also on cells of the factor-dependent line MO7e. In contrast, YB5.B8 and 17F11 had minimal effects on ligand binding. Conversely, SLF partially blocked the binding of SR-1 and YB5.B8 to cells, while binding of 17F11 was actually enhanced by SLf on some target cells. Preincubation of HEL-DR and MO7e cells with MAbs prior to exposure to SLF revealed that 17F11 itself brought about partial down-regulation of P145c-kit and did not inhibit SLF-mediated down-regulation. SR-1 caused minimal down-regulation and inhibited SLF-mediated receptor internalization. YB5.B8 had minimal effects on either cell line in this assay. To determine whether the antibodies had any agonist activity, they were compared with SLF for their ability to bring about receptor phosphorylation in intact MO7e cells. All three antibodies induced detectable tyrosine phosphorylation with 17F11 being the most effective, while YB5.B8 was the least effective. Finally, the ability of the antibodies to influence the proliferation of the MO7e cells was examined. As expected, SR-1 potently inhibited the proliferative response to SLF, while 17F11 weakly inhibited and YB5.B8 had negligible effect. In the absence of SLF both 17F11 and YB5.B8 displayed very weak but reproducible agonist activity.

Assessment of the anti-c-kit monoclonal antibody YB5.B8 in affinity magnetic enrichment of human lung mast cells.

The monoclonal antibody, YB5.B8 binds to the second domain of the c-kit proto-oncogene product on human mast cells, a receptor associated with tyrosine kinase activity. This molecule is involved with cell proliferation, maturation and viability as well as cell activation and its natural ligand is stem cell factor (SCF). We have used this antibody coupled to Dynabeads to perform positive affinity enrichment of human lung mast cells. This procedure results in enrichment of mast cells from 2.6 +/- 0.3% to 85.0 +/- 1.6% purity (n = 29) with yields of 41.9 +/- 3.7% (n = 29). As YB5.B8 interacts with the same receptor domain as does SCF, it is important to demonstrate that this procedure does not modify mast cell function. Incubation of mast cells with 1-5000 ng/ml YB5.B8 for 30 min neither induced histamine release nor modulated histamine release induced by anti-IgE. Furthermore, incubation with YB5.B8 did not alter prolonged culture with SCF. Examination of cells enriched using YB5.B8 showed that they had a normal histamine content (3.8 +/- 0.3 pg/cell compared with 3.9 +/- 0.7 pg/cell unpurified, n = 20) and had unchanged behaviour in both histamine secretion and cell survival studies. These studies indicate that YB5.B8 does not influence mast cell function and thus its use in magnetic affinity purification procedures offers a simple and effective method for enriching human mast cell preparations.