The expression of T cell related costimulatory molecules in multiple myeloma.

Presentation of tumour antigen by malignant cells not expressing costimulatory molecules is considered to be a major cause of the failure of the host's immune response against tumours. This study has determined the expression of the B7 family of costimulatory molecules on malignant plasma cells and the expression of the counter receptor molecules, CD28 and CD152 (CTLA-4), on T cells of patients with multiple myeloma. CD28 expression was present on most CD4 cells but was lower on CD8 cells especially from those patients who also showed evidence of expanded T cell clones (median 40%.

The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover: pH sensitivity of F-actin binding by human ADF, but not of Acanthamoeba actophorin.

Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G-actin)-ATP, and both bind co-operatively to F-actin.

An alpha-actinin-profilin chimaera with two alternatively operating actin-binding sites.

Studying the mode of interaction between actin and actin-binding proteins, we constructed a chimaeric protein consisting of the sequence for bovine profilin I (P), to which the sequence for the actin-binding domain of Dictyostelium discoideum alpha-actinin (alphaA1-2) was fused N-terminally. The resulting hybrid clone was expressed in Escherichia coli, and the chimaeric protein, alphaA1-2P, purified by affinity chromatography on poly-(L-proline) (PLP) columns and identified using specific antibodies. High resolution electron microscopy demonstrated that this protein consists of two discrete subdomains.

Probing the effects of calcium on gelsolin.

Gelsolin is a calcium-regulated actin severing and capping protein that binds two calcium ions and has three sites for actin; two recognize monomeric actin and one attaches to the sides of filaments. It contains six repeating sequence segments (G1-6). Here, we have analyzed the effects of calcium ions on (i) limited proteolysis of bacterially expressed human gelsolin by plasmin and (ii) dynamic light scattering and circular dichroism of gelsolin and various of its subdomains.

The functional phenotype of the primitive plasma cell in patients with multiple myeloma correlates with the clinical state.

The malignant plasma cells from patients with multiple myeloma display considerable phenotypic heterogeneity. All plasma cells express high intensity CD38 (CD38++), cytoplasmic immunoglobulin and either kappa or lambda light chains. Subpopulations of mature (CD45-), immature (CD45+) and primitive (CD45++, CD19+) plasma cells can be defined but little is known about the functional differences and clinical significance of these subpopulations.

ARVD4, a new locus for arrhythmogenic right ventricular cardiomyopathy, maps to chromosome 2 long arm.

Autosomal dominant arrhythmogenic right ventricular dysplasia (ARVD; MIM 107970) is a genetically heterogeneous cardiomyopathy, which often causes sudden death in juveniles and athletes. Two disease loci were previously mapped respectively to 14q23-q24 (ARVD1) and to 1q42-q43 (ARVD2). A third possible locus was assigned to 14q12-q22.

Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function.

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits.

Plasma cells in peripheral blood stem cell harvests from patients with multiple myeloma are predominantly polyclonal.

A flow cytometric technique has been developed to detect individual plasma cells in PBSC harvests and to establish light chain restriction as a surrogate marker of their clonality. Plasma cells were identified by high intensity CD38 (CD38++) and cytoplasmic immunoglobulin (cIg) expression. The ratio of cytoplasmic kappa to lambda expression was used to detect light chain restriction.

Disease progression in patients with multiple myeloma is associated with a concurrent alteration in the expression of both oncogenes and tumour suppressor genes and can be monitored by the oncoprotein phenotype.

The dysregulation of specific oncogenes due to either mutation or activation has previously been reported in a small number of patients with myeloma but the extent of oncogene dysregulation during the course of the disease is not known. The oncoprotein phenotype of plasma cells in 146 bone marrow samples from 81 patients with multiple myeloma was determined by dual colour flow cytometry using a predetermined panel of 8 monoclonal antibodies. High intensity CD38 expression was used to distinguish the plasma cell population and the cells were permeabilised to detect intracellular antigen expression.

The labelling index of primitive plasma cells determines the clinical behaviour of patients with myelomatosis.

For patients with multiple myeloma the most important laboratory correlate of prognosis and disease activity is the bromodeoxyuridine (BrdUrd) plasma cell labelling index (LI). However, the traditional immunofluorescent microscope LI technique, like other manual enumeration assays, can suffer from poor precision and accuracy. In this study the LI of different subpopulations of plasma cells (CD38++) as determined by flow cytometry was correlated with disease state.

Nucleoside transporters, bcl-2 and apoptosis in CLL cells exposed to nucleoside analogues in vitro.

The purine nucleoside analogues fludarabine (F1) and chlorodeoxyadenosine (2-CdA) are considered to be cell cycle specific agents which require DNA synthesis for cytotoxicity. However, their efficacy in the treatment of CLL, an indolent lymphoid malignancy suggests additional mechanisms of action. Like cytosine arabinoside (AraC), F1 and 2-CdA gain access to the cell via a specific nucleoside transporter (NST) protein.

Tepoxalin blocks neutrophil migration into cutaneous inflammatory sites by inhibiting Mac-1 and E-selectin expression.

Inflammation is characterized by the migration of polymorphonuclear leukocytes from the vasculature into the tissue causing profound injury. Adhesion and migration of neutrophils across the vascular bed are governed by a series of complex events including cytokine/chemokine production which in turn orchestrates the temporal expression of a cohort of adhesion molecules mediating the migration. Many of these adhesion molecules and their inducers are under the control of inflammatory response transcriptional factors such as NF kappa B and AP-1.

Selective modulation of elements of the immune system by low molecular weight nucleosides.

To optimally modulate a system as complex as the immune system, one must ultimately control its elements individually. Up to this time, use of polyclonal immune stimulants has necessarily involved modulation of a block of immune functions, frequently including undesired activities as well as the activity of interest. We now report selective modulation of individual elements of the immune system by low molecular weight nucleosides, within the context of a fully functional immune system.

Tepoxalin, a novel immunomodulatory compound, synergizes with CsA in suppression of graft-versus-host reaction and allogeneic skin graft rejection.

Tepoxalin, a dual 5-lipoxygenase and cyclooxygenase inhibitor with nonsteroidal antiinflammatory effects, has recently been shown to suppress NF kappa B transactivation and inhibit T cell proliferation via a mechanism very different from cyclosporine (CsA). In this report, we demonstrate that this novel immunosuppressive effect of tepoxalin is manifested in in vivo transplantation models. Tepoxalin suppressed murine spleen cell proliferation in a mixed lymphocyte reaction (MLR) with an IC50 of 1.

The immunostimulatory compound 7-allyl-8-oxoguanosine (loxoribine) induces a distinct subset of murine cytokines.

C8- and N7, C8-substituted guanine ribonucleosides comprise a class of molecules with potent immunostimulatory activity for a variety of humoral and cellular immune responses. Although it has been suggested that the immunostimulatory activity may be partially mediated by cytokine production, to date there has been no systematic evaluation of the spectrum of cytokines elicited by these nucleosides. In this study, we examine the cytokines produced by murine spleen cells in response to the di-substituted guanosine analog loxoribine (7-allyl-8-oxoguanosine).

Protein kinase C inhibitor chelerythrine disrupts memory formation in chicks.

Chelerythrine (CHELE), a specific, potent protein kinase C (PKC) inhibitor, disrupts memory formation for a one-trial peck-avoidance task. Three predictions were made about how CHELE, injected into chick brain near the time of training, would affect memory formation, based on previous work with two classes of protein kinase inhibitors (M. R.

Identification of the trapped calcium in the gelsolin segment 1-actin complex: implications for the role of calcium in the control of gelsolin activity.

The X-ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin-actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH.

Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium binding sites.

Gelsolin is composed of six repeating segments of sequence (G1-6) and contains three distinct actin binding sites, two that bind to G-actin and one that binds to filaments. The calcium-dependent actin monomer binding site present in the carboxyl-terminal half of the protein (G4-6) plays a critical role both in the cooperative binding of actin by gelsolin and in its nucleating activity. Here we have localized this actin binding site to segment 4 (G4) by expressing the segments G4, G4-5, G5, and G5-6 in Escherichia coli and analyzing their actin binding properties.

Enhancement of immunostimulatory activity by dual substitution of C8-substituted guanine ribonucleosides: correlation with increased cytokine secretion.

Guanine ribonucleosides with single substitutions at the C8 position (monosubstituted) or with dual substitutions at the C8 and N7 positions (disubstituted) up-regulate a spectrum of immunologic responses, including cytolytic responses to tumor cells. The current studies were undertaken to determine the effects of dual substitution on a number of nucleoside-inducible immunological parameters. To do so, two monosubstituted analogues, 8-bromoguanosine and 8-mercaptoguanosine, were directly compared with two disubstituted analogues, 7-methyl-8-oxoguanosine and 7-allyl-8-oxoguanosine (loxoribine).

Murine strain variation in the natural killer cell and proliferative responses to the immunostimulatory compound 7-allyl-8-oxoguanosine: role of cytokines.

7-Allyl-8-oxoguanosine (loxoribine) is a di-substituted guanine ribonucleoside which has been shown previously to enhance murine NK activity, B lymphocyte proliferation, and antibody synthesis. In this study we examined the relationship among enhancement of NK activity, proliferation, and cytokine synthesis in the responses of different strains of mice to loxoribine to provide insight into the role of cytokines in these biological activities. The NK response of mice was enhanced both in vitro and in vivo in all strains tested with the exception of the NK-deficient beige (BgBg) mouse.

The oncoprotein phenotype of plasma cells from patients with multiple myeloma.

The expression of 6 different oncoproteins and 2 tumour suppressor gene products in the plasma cells of 63 bone marrow samples was used to determine a profile of the oncogenic phenotype of patients with multiple myeloma. Dual label flow cytometry after periodatelysine paraformaldehyde fixation was used to detect cell surface phenotype and intracellular protein expression simultaneously. The normal range for both the incidence and intensity of expression was determined for each protein by analysing plasma cells (high CD38 intensity) in 22 normal bone marrow samples.

Small-molecule immunostimulants. Synthesis and activity of 7,8-disubstituted guanosines and structurally related compounds.

A series of 7,8-disubstituted guanosine derivatives was designed and prepared as potential B-cell-selective activators of the humoral immune response. These compounds were evaluated for their ability to act as B-cell mitogens and to augment the antibody response of B cells to sheep red blood cell (SRBC) challenge (adjuvanticity). In addition, they were tested for their ability to stimulate the natural killer (NK) cell response in murine in vitro cell assays.

Multiple myeloma: expression of nucleoside transporters on malignant plasma cells and their relationship to cellular proliferation.

The expression of nucleoside transporters is a limiting factor in the pharmacology of the nucleoside analogue, cytosine arabinoside (AraC) and is associated with cellular proliferation. We investigated the expression of nucleoside transporters on plasma cells from the bone marrow of 51 patients with multiple myeloma by 2-colour immunofluorescence flow cytometry, utilising 5-(SAENTA-x8)-fluorescein, a fluorescent ligand for the nucleoside transporter and anti-CD38 conjugated to phycoerythrin, as CD38 expression has unique characteristics on plasma cells. Mean nucleoside transporter expression on bone marrow plasma cells from patients with myeloma (1777 +/- 2181 transporters/plasma cell) was not significantly different from expression on plasma cells from normal bone marrow (997 +/- 1096 transporters/plasma cell).