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Biomedical subjects

S Perkins

Publications and source records attributed to S Perkins.

At least 73 records · Page 4Linked to original sources
PubMed1990

Efficient hybridization of mouse-human cell lines by means of hypo-osmolar electrofusion.

The fusion of a mouse-human heteromyeloma with a mouse hybridoma is used as a model to define parameters to generate human hybridomas. Electrofusion of these cells in 300 mosM and 75 mosM solutions showed that strong hypo-osmolar conditions resulted in a dramatic increase in the efficiency of hybridoma formation. In contrast to iso-osmolar electrofusion, a high hybrid yield could be obtained by injection of only a single field pulse. The field strength was adjusted in proportion to the increased size of the cells in hypo-osmolar solutions. Hypo-osmolar electrofusion allowed the generation of approximately 0.45% hybrids at a suspension density of 1.75 X 10(5) mouse-human cells/ml corresponding to an input number of 3.5 X 10(4) mouse-human cells. A further increase in the efficiency of hybridoma formation to about 0.6% was achieved by cell alignment in an alternating field of modulated field strength. Experiments in which the total cell number per fusion chamber was decreased at constant optimum suspension density showed that a further increase in the efficiency of hybridoma formation in hypo-osmolar solution was not possible because of the increasing influence of the heterogeneity of the cell lines with decreasing cell number. The results allow the conclusion that hypo-osmolar electrofusion is a potential tool to enhance successful immortalisation of human B lymphocytes.

Animals↗
PubMed1990

Stromal cell progeny of murine bone marrow fibroblast colony-forming units are clonal endothelial-like cells that express collagen IV and laminin.

Studies of human and murine bone marrow explants have demonstrated the existence of stromal cell precursors that give rise to colonies of adherent cells in short-term cultures. Because previous data suggested that these colonies were composed of fibroblasts, the precursor cells were termed fibroblast colony-forming units (CFU-F). However, we have recently shown that the stromal cells which support hematopoiesis in murine long-term bone marrow cultures (LTBC) express collagen IV and laminin, markers associated with an endothelial cell lineage, but are negative for collagen I and III, markers associated with a fibroblast cell lineage. Because these conflicting results suggest major functional differences between the stromal cells observed in long-term cultures and the short-term assay, we re-examined the lineage of CFU-F-derived stromal cells. Using two-color immunofluorescence, we characterized virtually all of the cells comprising individual "CFU-F" colonies derived from mouse radiation chimeras. Identification of donor (hematopoietic) or host (stromal) origin was based on surface staining for strain-specific H-2 surface antigens, and, for endothelial or fibroblast properties, on cytoplasmic staining for laminin and collagen IV, or collagens I and III, respectively. The results demonstrate that a large proportion of the cells in CFU-F colonies are donor-derived and fail to stain with any of the antisera specific for nonhematopoietic cells. In addition, these donor-derived cells exhibit marked phagocytic capacity and stain positively with monoclonal antibodies characteristic of the monocyte-macrophage hematopoietic cell lineage (anti-T200, anti-Mac-1, F4/80). However, the remainder of the cells are host-derived cells that stain positively with antisera to collagen IV and laminin. In contrast, stains for collagen types I and III were negative under conditions that allowed for strong staining of control skin fibroblasts. In separate studies, using mixtures of two genetically distinct bone marrows, the cells expressing collagen IV were further shown to be clonal in origin within individual colonies, directly demonstrating that the CFU-F assay provides a quantitative measure of the numbers of marrow stromal cell precursors. Thus, the current studies establish a remarkable similarity between the hematopoietic microenvironment in the short-term CFU-F assay and the long-term culture system: the majority of adherent cells are hematopoietic cells of the monocyte-macrophage lineage, while the remainder are stromal cells whose precise lineage remains uncertain, but whose pattern of collagen expression is more consistent with an endothelial rather than a fibroblast cell origin.

Animals↗
PubMed1989

Electric field-induced cell fusion and human monoclonal antibodies.

A persistent problem in the generation of antigen-specific human monoclonal antibodies is the rarity of appropriate B cells in human blood or splenic tissues. In order to immortalize the rare antigen-specific cells that are available, an electric field-induced cell fusion technique has been shown to markedly increase the fusion efficiency in comparison to polyethylene glycol-induced cell fusion using Epstein-Barr virus (EBV) or pokeweed mitogen activated B cells. Fusion efficiency of 10(-3)-10(-4) has been achieved by this process with as low as 1 X 10(6) input EBV-activated B cells. A panel of human monoclonal antibodies to human cytomegalovirus has subsequently been produced using this technique. This improvement should enable wider therapeutic and diagnostic applications of human monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1989

Human monoclonal antibodies to human cytomegalovirus.

Human monoclonal antibodies (HMAbs) to human cytomegalovirus (HCMV) have been developed by using electric field-induced cell fusion of human B lymphocytes to the human-mouse cell line SBC-H20. By this procedure, multiple hybridomas have been produced that secrete IgG 1 HMAbs with distinct patterns of indirect immunofluorescence on HCMV-infected cells. HMAbs Z01 and X20 immunoprecipitated a major protein at 64 kDa. HMAb Z02 immunoprecipitated a major protein of 48-50 kDa. HMAb Z10 identified a single protein at 65 kDa and HMAb X16 identified proteins at 100, 65, and 36-38 kDa. The HMAbs demonstrated varying degrees of virus-neutralizing activity. The production of HMAbs to HCMV provides an important approach to studying the human host response to HCMV by elucidating biologically relevant antigens and epitopes. In addition, HMAbs are a potentially unlimited source of relevant human antibodies for treating life-threatening HCMV infection.

Antibodies, Monoclonal↗
PubMed1988

Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice.

Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells.

Adipose Tissue↗
PubMed1987

Relationships between B-lineage lymphocytes and stromal cells in long-term bone marrow cultures.

In long-term culture of mouse bone marrow, the growth and differentiation of B-lineage lymphocytes depends on interaction with adherent cells or their products. The objectives of these studies were to characterize the types of cells present in the supporting adherent layer as well as the physical relationships of these cells with lymphocytes. With an extensive panel of antibodies against hemopoietic and lymphocyte antigens, two discrete nonlymphoid populations were identified: macrophages and undefined, large cells which we termed "stromal cells". Lymphocyte clusters grew in actual contact with the latter cells only. Stromal cells lacked expression of most hemopoietic antigens, including the common leukocyte antigen, J11d, heat stable antigen (M1/69), Thy-1 and BP 1. Antigens expressed by stromal cells were detected by AA4.1, our 94.2 antibody, and antibody to the Forsmann antigen, but the most distinguishing characteristics of the lymphocyte-binding stromal cells were production of basement membrane components, laminin and collagen IV, and the extremely low uptake of acetylated low density lipoprotein (LDL). Using acetylated LDL uptake as a sorting criterion, the lymphocyte-binding stromal cells were separated from the macrophages, recultured and shown to support lymphocyte proliferation. We found the binding between stromal cells and lymphocytes to be highly selective and dependent on divalent cations; hence, specialized adhesion mechanisms may have a role in B cell development. Moreover, our studies suggest that phosphatidylinositol-anchored cell surface molecules may be involved in this adhesion. Our findings demonstrate the possibility that a single cell type provides physical support and proliferation stimuli for early B-lineage cells. This accessory cell is not a macrophage; rather, it has features of an endothelial or epithelial cell.

Animals↗
PubMed1986

A human monoclonal antibody to rhG.

Hybridomas secreting a human monoclonal IgG1 antibody against a variant of the rhG antigen were produced from B lymphocytes of an Rh-negative donor with serum antibodies to D and G [correction of C] antigens. The antibody reacts by indirect hemagglutination with nearly all C- or D-positive cells, confirming the strong association in the expression of G with D or C antigens. The lack of antibody reactivity to C-negative Du cells suggests a particular epitope on the D complex associated with the G antigen.

Antibodies, Monoclonal↗
PubMed1985

Human monoclonal antibodies neutralizing varicella-zoster virus.

Hybridomas secreting human monoclonal antibodies to varicella-zoster virus were produced by fusing B cells of a patient recovering from acute varicella infection with a human-mouse cell line. Two hybrid lines have continued to secrete IgG1, one with kappa and the other with lambda chains, for at least 12 months. Each antibody neutralizes virus infectivity between 1-5 micrograms of partially purified immunoglobulin/ml, each shows a different pattern of immunofluorescent staining of virus-infected cells, and one identifies three viral proteins with molecular weights of 60,000, 95,000, and 97,000.

Animals↗
PubMed1985

Treatment of neutropenic infection: a randomized trial comparing latamoxef (moxalactam) with cephradine plus tobramycin.

Sixty neutropenic patients with infection were studied in a randomized trial comparing treatment with latamoxef (moxalactam) alone or with cephradine and tobramycin. The two treatment groups were comparable in respect to their clinical sites of infection, degree of neutropenia underlying malignancy, and organisms. Forty-two bacterial isolates were obtained from various clinically infected sites. All but two of these isolates were sensitive to latamoxef (30 highly sensitive, ten moderately sensitive). In contrast 29 of the isolates were resistant to cephradine and eight were resistant to both cephradine and tobramycin. Control of infection was achieved in 72% of patients treated with latamoxef and 55% treated with cephradine plus tobramycin. Latamoxef appears to be an effective antibiotic for the treatment of neutropenic patients with infection.

Adult↗
PubMed1985

A new approach to the diagnosis of active Rh0(D) immunization in passively immunized pregnant women.

An assay has been developed to distinguish active from passive Rh0(D) immunization in a patient who had recently received hyperimmune anti-Rh0(D) immunoglobulin therapy. Isolated peripheral B lymphocytes from a pregnant woman at 32 weeks gestation were co-cultured with Epstein-Barr virus in a liquid growth medium. After 7 days, anti-Rh0(D) antibodies produced in vitro by the transformed lymphocytes were detected in culture supernatants, thereby proving active immunization and indicating the potential of hemolytic disease of the newborn in the current pregnancy. This assay was also performed with peripheral B lymphocytes from three groups of individuals: mothers known to be Rh0(D) immunized and who recently delivered Rh-positive infants, women with longstanding Rh0(D) immunization, and women who were treated with anti-Rh0(D) globulin. In the first group, anti-Rh0(D) antibodies were again detected after in vitro viral stimulation. In the latter two groups, essentially no anti-Rh0(D) activity was detected.

Adult↗
PubMed1984

Rescue of human monoclonal antibody production from an EBV-transformed B cell line by fusion to a human-mouse hybridoma.

A human-mouse cell line that is hypoxanthine-aminopterin-thymidine sensitive and ouabain resistant was derived from a fusion between human B lymphocytes and a mouse myeloma line. This new mutant, when fused to a relatively unstable EBV-transformed B cell secreting a human monoclonal anti-A (red blood cell antigen) antibody, resulted in stable hybridomas capable of long term production of the specific human monoclonal antibody. Furthermore, some of the hybrid clones secreted antibody in far greater titer than the original EBV cell line. We conclude that fusion to this human-mouse line is an efficient approach to the production of human monoclonal alloantibodies and an effective method of 'rescuing' secretion of desired antibody from EBV cell lines.

ABO Blood-Group System↗
PubMed1984

Canine gastrocnemius disuse atrophy: resistance to paralysis by dimethyl tubocurarine.

Ten dogs developed unilateral gastrocnemius disuse atrophy after unilateral hindlimb immobilization in a cast for 25 days. Dose-response curves to dimethyl tubocurarine (MTC) were determined during anesthesia with pentobarbital sodium-N2O. Bolus and continuous infusion increments of MTC every 30 min provided steady-state blood levels at each stage of paralysis. Both gastrocnemius tendons were sectioned and attached to transducers. Both sciatic nerves were stimulated every 30 min: 2 Hz for 2 s, a 15-s pause, 50 Hz for 2 s. Dose-response curves, computer calculated by nonlinear regression using a sigmoid maximal effect model of the Hill equation, were parallel for the data relating blocking of tetanus to dose of MTC. The 50% paralyzing dose (tetanus) for control vs. casted gastrocnemius muscle was 64 vs. 813 mg/kg; corresponding plasma concentrations were 0.12 vs. 2.0 micrograms/ml. Thus in vivo simultaneous tension measurements of both gastrocnemius muscles, one casted and one uncasted, demonstrated resistance to paralysis by MTC in muscle with disuse atrophy.

Animals↗
PubMed1983

Motor neuron disease associated with carcinoma. A report of 2 cases.

Paraneoplastic complications are obscure and difficult to understand. The association of motor neuron disease and carcinoma may sometimes be more than coincidental, and 2 cases are described. One patient had motor neuron disease, limbic encephalitis (a recognized paraneoplastic disorder) and carcinoma of the oesophagus; the other had motor neuron disease and adenocarcinoma of the rectum. In the elderly male with motor neuron disease simple screening tests to exclude lymphoma and carcinoma of the lung, bowel and genito-urinary tract are advocated.

Adenocarcinoma↗