Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells.

CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.

Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-gamma production, and expansion of natural killer cells.

We investigated the in vitro effects of combining interleukin-18 (IL-18) and IL-2 on human lymphocytes. The combined use of these two cytokines synergistically enhanced the proliferation, cytolytic activity, and interferon-gamma production of peripheral blood mononuclear cells. Phenotypic analysis revealed a preferential expansion of CD56+CD3- cells and an up-regulation of IL-2 receptor-alpha expression on natural killer cells. Isolated natural killer cells showed a substantial increase in proliferation and cytotoxicity compared with CD4+ and CD8+ T cells. The combined use of IL-18 and IL-2 should be considered a viable strategy to induce an antitumor response in vivo.

Identification of unique truncated KC/GRO beta chemokines with potent hematopoietic and anti-infective activities.

SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GRO beta. In comparison to their full-length forms, truncated KC and truncated GRO beta were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GRO beta. To our knowledge, this represents the first example where any form of KC or GRO beta were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.

Immunogenic and tolerogenic properties of monomethoxypoly(ethylene glycol) conjugated proteins.

An immunogenic and tolerogenic characterisation of monomethoxypoly(ethylene glycol) conjugated proteins was carried out using, as immunogen models, an anti-malaria chimera monoclonal antibody (PfChMab) and a macrophage colony stimulating factor (M-CSF). Two conjugates of PfChMab were prepared by polymer derivatisation of 19 and 33% protein amino groups and one conjugate of M-CSF was obtained by modification of 24% amino groups. In mice M-CSF was found to elicit rapidly high IgG and IgM levels whereas the monomethoxypoly(ethylene glycol) derivatised M-CSF stimulated a significantly lower immunoresponse. Native PfChMab was found to induce a delayed immunoresponse with high IgM levels but low production of IgG. Furthermore, similar immunogenic profiles were obtained with the native and modified protein forms. The pre-administration of polymer conjugated M-CSF to mice subsequently treated with the native protein was found to suppress up to 75% of anti-native M-CSF IgG, while IgM production was not affected. On the other hand the pre-administration of monomethoxypoly(ethylene glycol) derivatised PfChMab was found to reduce significantly the generation of anti-native PfChMab IgM. Such suppression depended on the degree of modification: the conjugate with the higher number of polymer chains was more effective in suppressing the immunoresponse.

Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus.

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Anti-human RP105 sera induces lymphocyte proliferation.

Cellular environment dictates whether antigen binding to the B lymphocyte receptor together with co-stimulatory molecules will result in proliferation, anergy, or apoptosis. Murine RP105 is a member of the leucine-rich repeat family of proteins, which is specifically expressed on mature B cells. Monoclonal antibodies to the murine RP105 induce proliferation and protect B cells from apoptosis, suggesting an important regulatory role in murine B lymphocyte function. We identified a human RP105 homolog and mapped the gene to chromosome 5q12.3-13.1. Tissue distribution analysis shows that the transcript is found predominately in lymphoid tissues including spleen, tonsils, appendix, and peripheral blood leukocytes. Polymerase chain reaction analysis of isolated primary human cell populations confirms that mRNA exists in spleen B lymphocytes and monocytes but not T lymphocytes. Western blot analysis demonstrates specific expression of human RP105 in human B lymphocytes. Murine anti-human RP105 sera was generated using DNA immunization. The antisera contained antibodies that recognized and bound to human B lymphocytes from both spleen and peripheral blood as assessed by flow cytometry. Assessment of biological function showed that human peripheral blood leukocytes incubated with anti-RP105 sera were induced to proliferate as measured by tritiated thymidine incorporation. Moreover, anti-CD40 and interleukin-4-treated cells but not those exposed to anti-RP105 sera produced soluble CD23, suggesting distinct functional roles. This is the first demonstration of both the existence of RP105 protein on human B lymphocytes and its role in the regulation of B lymphocyte activation.

A novel human bone marrow stroma-derived cell line TF274 is highly osteogenic in vitro and in vivo.

A novel, immortalized, human bone marrow stroma-derived cell line TF274 is described which has the ability to form bone both in vitro and in vivo. Under basal conditions these cells expressed alkaline phosphatase (ALP) and type I collagen genes which are characteristic of the osteoblast phenotype. ALP levels were upregulated in the presence of osteotropic agents such as parathyroid hormone (PTH), transforming growth factor beta (TGF-beta), and BMP-2. In addition, PTH also increased cAMP levels in these cells. The capacity of these cells to form bone in vitro was evaluated by culturing them in the presence of L-ascorbic acid and beta-glycerophosphate. Matrix mineralization in these cultures was assessed by Alizarin Red staining and increased 45Ca uptake. Under these conditions mineralized nodule formation was observed in less than 2 weeks. Northern analysis of TF274 cells at various times during the mineralization process indicated a temporal expression of the osteocalcin gene that is typically associated with differentiating osteoblasts. The osteogenic nature of TF274 cells was confirmed in vivo using the severe combined immunodeficient (SCID) mouse model. Antibodies to human leukocyte antigens (HLA), class I antigens, and human OKa blood group antigen were used to demonstrate that the lesions formed were of human origin. By 21 days, the lesion consisted of a homogeneous focus of ALP-positive cells containing areas of mineralized bone lined with tartarate-resistant acid phosphatase (TRAP) positive osteoclasts. Thus, the TF274 cells exhibit osteogenic potential both in vitro and in vivo. This immortalized cell line represents a consistent source of cells that can be used to study human osteoblast differentiation both in vitro and in vivo.

Osteopontin and its integrin receptor alpha(v)beta3 are upregulated during formation of the glial scar after focal stroke.

BACKGROUND AND PURPOSE: Microglia and astrocytes in the peri-infarct region are activated in response to focal stroke. A critical function of activated glia is formation of a protective barrier that ultimately forms a new glial-limiting membrane. Osteopontin, a provisional matrix protein expressed during wound healing, is induced after focal stroke. The present study was performed to determine the spatial and temporal expression of osteopontin and its integrin receptor alpha(v)beta3 during formation of the peri-infarct gliotic barrier and subsequent formation of a new glial-limiting membrane. METHODS: Spontaneously hypertensive rats (n = 19) were subjected to permanent occlusion of the middle cerebral artery and killed 3, 6, and 24 hours and 2, 5, and 15 days after occlusion. The spatial and temporal expression of osteopontin mRNA was determined by in situ hybridization, and that of osteopontin ligand and its integrin receptor alpha(v)beta3 was determined by immunohistochemistry. RESULTS: Osteopontin mRNA was expressed de novo in the peri-infarct region from 3 to 48 hours; by 5 days osteopontin mRNA expression was restricted to the infarct. Osteopontin protein was expressed by peri-infarct microglia beginning at 24 hours and by microglia/macrophages at 48 hours in the infarct. Integrin receptor alpha(v)beta3 was expressed in peri-infarct astrocytes at 5 and 15 days. CONCLUSIONS: Early microglial/macrophage expression of osteopontin mRNA defines the borders and final infarct area at 24 hours. At 5 days osteopontin ligand is at a distance from the peri-infarct astrocytes expressing integrin receptor alpha(v)beta3. By 15 days astrocytes expressing integrin receptor alpha(v)beta3 are localized in an osteopontin-rich region concomitant with formation of the new glial-limiting membrane. The de novo expression and interaction of osteopontin ligand with its receptor integrin alpha(v)beta3 suggest a role in wound healing after focal stroke.

Successful in vitro antigen-dependent activation of 24-hour-old peripheral blood lymphocytes.

We describe a simple, rapid and reproducible in vitro culture system in which human peripheral blood lymphocytes (PBLs), donated 24 h prior to initiation of culture can be stimulated to produce antigen-specific antibodies. Peripheral blood lymphocytes purified by Ficoll-Hypaque centrifugation were passed over a G10 Sephadex column and then activated in vitro in the presence of 0.003% staphylococcus Cowan A, 2.8 x 10(-6) M indomethacin and appropriate concentrations of tetanus toxoid antigen. After the first 24 h in culture, a five-fold concentrated supernatant from an allogeneic mixed lymphocyte culture was added. The cell surface phenotypes of the PBLs were analyzed by flow cytometry at the initiation and termination of culture, in order to provide a comprehensive characterization of the cellular composition of a successful in vitro stimulation system. Our results clearly show that the majority of peripheral blood B cells can be induced to an activated stage (blast transformation) and interleukin 2 (IL-2) receptor expression, following very simple manipulations of the lymphoid population. Tetanus toxoid-specific antibody production can be readily generated in this cell population. In contrast, T cells were not activated to express IL-2 receptors and reach blast transformation, and did not show appreciable proliferation. Our system provides a population of B cells producing antibodies of desired specificity which could be utilized for the generation of human hybridomas or could serve as a donor population for antibody engineering via the combinatorial library approach. Careful light scattering and cell surface phenotypic analyses of the cells entering, proliferating and differentiating in these cultures enabled several novel observations to be made.

Reperfusion following focal stroke hastens inflammation and resolution of ischemic injured tissue.

Previously, we described cellular changes following Permanent Middle Cerebral Artery Occlusion (PMCAO) in spontaneously hypertensive rats. Ischemic changes following PMCAO included a time-related focal pan necrosis, inflammatory cell infiltration, gliosis, and eventual loss of necrotic tissue post PMCAO. We have now characterized changes which occur after Temporary Middle Cerebral Artery Occlusion (TMCAO; 80 or 160 min) followed by reperfusion and compared these changes to those which occur following PMCAO. TMCAO with reperfusion results in cortical infarcts which vary in size in an occlusion-time-dependent manner. After 1 h of reperfusion, ischemic changes were observed histologically, including microhemorrhages and the beginning of a slight inflammatory infiltration in and around the meningeal vasculature. This infiltrate consisted primarily of neutrophils, which by 6 h of reperfusion was significant with infiltration from deep blood vessels into brain tissue, including the presence of some monocytes adhering within blood vessels. Neutrophil infiltration occurred sooner and to a greater extent in reperfused tissues than in permanently occluded tissues, where it only began at 12 h post PMCAO. As occurred following PMCAO, increased Glial Fibrillary Acidic Protein (GFAP) immunoreactivity indicating astrogliosis was first observed at 12 h postTMCAO. Over 1-3 days of reperfusion, a heavy macrophage infiltrate was observed in the reperfused tissues in addition to a continued influx of neutrophils. Following 5 days of reperfusion, the lesion was completely replaced with inflammatory cells, of which macrophages predominated. Unlike PMCAO, which resulted in focal spots of neutrophil accumulation, neutrophils were more distributed throughout the infarcted cortex following TMCAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120.

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.

Development of tissue damage, inflammation and resolution following stroke: an immunohistochemical and quantitative planimetric study.

Development and resolution of the lesion produced by occlusion of the middle cerebral artery (MCAO) was studied through quantitative planimetry and histologic/immunohistochemical techniques. MCAO, performed in spontaneously hypertensive rats (SHR), initially (1-3 days) produced large, consistent cerebral cortical infarctions and an increase in ipsilateral hemispheric size (i.e., swelling) quantitated by planimetry on 2,3,5-triphenyltetrazolium chloride (TTC)-stained gross tissue sections. These initial changes correlated well with changes identified from 2 h to 3 days using hematoxylin and eosin stained histologic tissue sections and immunohistochemical techniques including: the progressive development of a cortical area of pan necrosis, infiltration of neutrophils into infarcted tissues, and activation of astroglia. During the initial 2 days following MCAO, glial fibrillary acidic protein immunoreactive cells increased in number and became larger and more intensely fluorescent medial to the cortical infarct. At 5 to 15 days, both the infarct and the ipsilateral hemisphere decreased in size. These changes correlated with the presence of abundant macrophages, and cavitation of the lesion along its medial border. Also during this period, a loose connective tissue matrix formed along the superficial aspect of the infarct. This connective tissue contained fibroblasts, extracellular matrix immunoreactive for laminin and collagen, capillary buds indicating neovascularization, and abundant macrophages. By the final timepoint (30 days), necrotic tissue could no longer be detected in either gross or histologic tissue sections, the inflammatory infiltrate had resolved, and the connective tissue was removed.(ABSTRACT TRUNCATED AT 250 WORDS)

A human lymphoid recombinant cell line with functional human immunodeficiency virus type 1 envelope.

Our goal has been to develop a safe and effective system that would allow us to explore the functions of the human immunodeficiency virus (HIV) envelope. We have generated a human lymphoid cell line (TF228.1.16) that stably expresses functional HIV envelope proteins on its cell surface, and therefore closely mimics the viral envelope and virus-infected cells. The TF228.1.16 line forms syncytia with human cells of the CD4+ phenotype and provides a facile virus-free cell-based assay for examining the mechanism of syncytia formation and for evaluating novel agents that may disrupt this process. The TF228.1.16 cells also provide an opportunity to present the HIV envelope proteins to the immune system in cellular form. In vitro immunization of human peripheral blood mononuclear cells (PBMC) and in vivo immunization of rhesus monkeys with this reagent results in the production of antibodies with neutralizing (anti-syncytia) activities. When the HIV envelope is expressed against the background of human lymphoid cells, it may exhibit immune protection with unique properties that have not yet been explored. Our results indicate that a virus-free cell system can play an important role in exploring the biology and function of HIV-envelope proteins without the interference of other viral components present in infected cells. This paper discusses these results, and examines the potential use of TF228.1.16 as a vaccine.

Manipulation of human B cells to confer immortality.

Electroporation was used to deliver genomic DNA from a lymphoid tumor to activated/stimulated human peripheral blood lymphocytes to create immortalized lymphoid cell lines. Activation of the recipient lymphocytes was essential for efficient immortalization. A panel of human B cell transfectant clones, each phenotypically representing specific stages of differentiation, resulted from the transfection. Monoclonal antibody production was measured, and the level produced depended on the phenotype of the cells, with the more mature B cell transfectants secreting up to 10 micrograms/mL of immunoglobulin. The transfectants were stable with respect to their morphological appearance, growth rate, and antibody production. Chromosome analysis indicated that the transfectants displayed a normal karyotype, devoid of abnormalities. We have shown that electroporation is an effective method of immortalizing human lymphocytes at different stages of differentiation. The transfectants provide a panel of cells that can readily be studied with respect to their phenotypic/karyotypic stability, regulation, and production of immunoglobulin, lymphokines, and growth factors. These data demonstrate the feasibility of generating immortalized human B cells to provide an important resource for the study of B cell differentiation and immortalization.

Gene transfer from targeted liposomes to specific lymphoid cells by electroporation.

Large unilamellar liposomes, coated with protein A and encapsulating the gene that confers resistance to mycophenolic acid, were used as a model system to demonstrate gene transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2a antibodies, was coupled to liposomes to target them specifically to defined cell types coated with IgG2a antibody. Protein A-coated liposomes bound human B lymphoblastoid cells preincubated with a mouse IgG2a anti-HLA monoclonal antibody but failed to adhere to cells challenged with an irrelevant (anti-H-2) antibody of the same isotype or to cells incubated in the absence of antibody. Transfection of target cells bound to protein A-coated liposomes was achieved by electroporation. This step was essential since only electroporated cells survived in a selective medium containing mycophenolic acid. Transfection efficiency with electroporation and targeted liposomes was as efficient as conventional procedures that used unencapsulated plasmids free in solution but, in the latter case, cell selectivity is not possible. This technique provides a methodology for introducing defined biological macromolecules into specific cell types.

Structure-activity studies of human IL-1 beta with mature and truncated proteins expressed in Escherichia coli.

IL-1 beta is synthesized as an inactive 31-kDa intracellular protein, which is then processed upon secretion to an active 17-kDa carboxyl-terminal fragment. To identify the minimal portion of IL-1 beta required for activity, we constructed several deletion mutants of mature IL-1 beta. These included three amino-terminal deletions of 10, 16, and 81 amino acids, two carboxyl-terminal deletions of 17 and 72 amino acids, and one internal fragment between amino acids 17 and 81. Expression of the mutants was monitored by Western blots and immunoprecipitation. With one exception, all of these mutants and the full length 17-kDa IL-1 beta were expressed as soluble protein in Escherichia coli and could be assayed for activity and receptor binding in lysates without further purification. Whereas the intact 17-kDa IL-1 beta retained full biologic activity (greater than 10(7) U/ml of lysate) and competed for binding with 125I-labeled IL-1 beta, none of the lysates containing IL-1 beta deletion mutant proteins had activity or competed for binding to receptor at significantly higher concentrations. The loss of function in the smallest C-terminal deletion mutant does not appear to be due to the direct involvement of these C-terminal residues in receptor binding because both monoclonal and polyclonal antisera directed to this region bind to IL-1 beta but do not neutralize its activity. Therefore, this region is probably indirectly involved in sustaining the structure of the receptor-binding site.

Production of continuous mouse plasma cell lines by transfection with human leukemia DNA.

Spleen cells from mice immunized with human cells were transfected with DNA from the human leukemia cell line, Reh. A calcium phosphate-DNA coprecipitate was introduced into the stimulated spleen cells by treatment with a polyethylene glycol-DMSO mixture. The cells which grew out from the transfected population could be passaged continuously in culture and cloned in semisolid agarose. The cell lines contain 40 acrocentric chromosomes, and Southern blot analysis with the cloned human Alu sequence indicates that human DNA is present. The transfected cell lines exhibit markers expressed on plasmacytoma cells and produce immunoglobulin in amounts equivalent to those produced by plasmacytoma cell lines. Five of nine cell lines tested produce antibodies that react with the human cells used to immunize the mice. These cell lines have been in culture for more than a year, and one of the lines has maintained a diploid karyotype and production of the specific antibody even after being passaged through a BALB/c mouse. Preliminary experiments indicate that these cells may be a useful model system for analysis of the early proliferative phase of leukocyte transformation.

Chronic idiopathic intestinal pseudo-obstruction caused by a degenerative disorder of the myenteric plexus: the use of Smith's method to define the neuropathology.

In this paper we report the pathologic basis of chronic idiopathic intestinal pseudo-obstruction in a patient who had a subtotal colectomy and ileorectal anastomosis for severe obstipation. Conventional light microscopy of the resected intestine showed an increased thickness of the longitudinal muscle, minimal amounts of smooth muscle fibrosis, and normal smooth muscle cells. The morphology of the myenteric plexus was difficult to interpret with this technique, but quantification of colonic neurons revealed a significantly decreased number compared with controls. Silver stains of the myenteric plexus by Smith's method showed: (a) patchy loss of nerve tracts with replacement by Schwann cells, (b) degeneration and decreased numbers of both argryophilic and argyrophobic neurons, (c) fragmentation and dropout of many axons, and (d) increased thickness and disorganized spatial arrangement of other axons. The pathology of this intestinal neuropathy could be missed by conventional light microscopy and may be apparent only when a silver technique is used to visualize the myenteric plexus.

Detection of neuroblastoma cells in human bone marrow using a combination of monoclonal antibodies.

Two different types of monoclonal antibodies, antineuroblastoma (PI153/3), and antilymphocyte (P3B1-C3) were used to identify and classify tumor cells in the bone marrow of patients with neuroblastoma and with other types of cancer. Cells expressing the antigens were detected with peroxidase-coupled anti-Ig. The cell-surface labeling is manifested as a dense black precipitate at the membrane visualized by light microscopy. The combination of the two antibodies gives specific staining patterns for each cell type. PI153/3+, P3B1-C3- is specifically associated with neuroblastoma cells. PI153/3+,P3B1-C3+ is expressed on blast cells from some types of acute lymphoblastic leukemia and a small subpopulation of normal lymphocytes. These monoclonal antibodies thus allow specific visual detection of single neuroblastoma cells in bone marrow samples. The results demonstrate how combinations of monoclonal antibodies can be effectively used to identify specific cell types by their expression of and lack of specific marker determinants. Application of this principle is particularly relevant for dissecting populations of related cells and/or molecules.