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An exciting report by C.L. Hall et al. reported on a unique mechanism of cell transformation in which a matrix receptor linked by a GPI anchor to the external surface of the plasma membrane, called RHAMM (receptor for hyaluronic acid mediated motility), functioned as a dominant oncogene requires some comment. Experiments do not agree with major splicing-based variation in RHAMM gene products. Also, the RHAMM cDNA clone used in Hall's experiments encodes an N-terminally truncated nonnative RHAMM protein.

Interphase fluorescence in situ hybridisation (FISH) is a sensitive diagnostic tool used for the detection of alterations in the genome on cell-by-cell basis. However, the cost-per-test and the technical complexity of current FISH protocols have slowed its widespread utilisation in clinical settings. For many cancers, the lack of a cost-effective and informative diagnostic method has compromised the quality of life for patients. We present the first demonstration of a microchip-based FISH protocol, coupled with a novel method to immobilise peripheral blood mononuclear cells inside microfluidic channels. These first on-chip implementations of FISH allow several chromosomal abnormalities associated with multiple myeloma to be detected with a ten-fold higher throughput and 1/10-th the reagent consumption of the traditional slide-based method. Moreover, the chip test is performed within hours whereas the conventional protocol required days. In addition, two on-chip methods to enhance the hybridisation aspects of FISH have been examined: mechanical and electrokinetic pumping. Similar agitation methods have led to significant improvements in hybridisation efficiency with DNA microarray work, but with this cell-based method the benefits were moderate. On-chip FISH technology holds promise for sophisticated and cost-effective screening of cancer patients at every clinic visit. [ABSTRACT FROM AUTHOR]

The malignant clone in myeloma is not eradicated by chemotherapy. Cyclosporins inhibit drug transport mechanisms, particularly the multidrug transporter p-glycoprotein 170, leading to their use as chemosensitizers. In myeloma, clonotypic blood B cells represent the major drug-resistant subset. This study compares the ability of cyclosporin A analogues and metabolites to inhibit cellular transporter(s) in myeloma and normal B cells in vitro, and evaluates their potential role in vivo. Cyclosporin A (CsA), CsG, PSC 833 or SDZ 280-446, and primary CsA and CsG metabolites, were tested for their ability to inhibit drug transport mechanisms of ex vivo malignant B cells from 81 patients with multiple myeloma as compared to B cells from normal donors, as measured by the export of the dye rhodamine 123 (Rh123) using multiparameter flow cytometry. The majority of myeloma B and normal B cells had efficient transporter function as measured by their CsA-sensitive export of Rh123. CsA and CsA analogues mediated efficient inhibition of this transport. Inhibition of dye transport by normal B cells required an approximately six-fold greater concentration of the synthetic peptolide SDZ 280-446 than was needed to optimally inhibit transport by myeloma B cells. PSC 833 and CsG were inhibitory at concentrations approximately five-fold lower than were required for CsA. Assessment of inhibitory potency in vivo indicated that the in vivo chemosensitizer levels of CsA and PSC 833 exceeded the transporter inhibitory concentration by four- and 20-fold respectively. In vivo, cyclosporins are rapidly and almost completely converted to metabolites. AM1 and AM4N, primary metabolites of CsA, mediated inhibition of transport, as did CsG metabolites GM1, GM4N and GM9. AM1 and GM9 are known to reach steady-state in vivo levels that exceed the inhibitory concentration identified here by 1.1- to 1.9-fold. Thus, cyclosporin metabolites, which accumulate in the blood during infusion of CsA and other cyclosporins, are shown here to be effective chemosensitizers for normally drug-resistant myeloma cells in vitro. Cyclosporin metabolites are considered to be less toxic than the parent drugs, suggesting that novel chemosensitization strategies designed to minimize concentrations of parent drug and maximize accumulation of primary metabolites in vivo may optimize cytotoxicity to the malignant clone in myeloma. [ABSTRACT FROM AUTHOR]

Human T-cell development appears to be relatively resistant to cyclosporin A (CsA). Children exposed to CsA in utero as part of kidney transplant maintenance have few abnormalities. The objective of the study described here was to analyse the effects of CsA on the development in vitro of human multinegative (MN) (CD3-4-8-) thymocytes as a model system for thymic progenitor development in vivo. MN thymocytes, prepared by depletion methods, differentiated in vitro to acquire CD3 and undergo transitions in CD45 isoform expression analogous to those postulated to occur in vivo. In this work MN thymocytes were cultured with IL-2 and on thymic epithelial cells (TEC) with or without IL-2, either in the presence or absence of CsA. For many thymocyte preparations, differentiation in the presence of CsA resulted in almost complete inhibition of the acquisition of CD3 and of the low Mr isoform CD45R0. Expression of CD45RA and of total CD45 were reduced but not eliminated and the density of CD29 was unaffected. For others, neither CD3 nor CD45 expression was affected, but selective inhibition of TCRδ expression occurred. At all doses of CsA (0.1 100 µg/ml), MN thymocytes continued to cycle indicating a CsA-resistant generative compartment. Treatment of peripheral blood T cells with CsA had no effect on surface expression of CD3 or CD45 isoforms but did reduce the amount of de novo-synthesized CD45R0 mRNA. Culture of MN thymocytes on TEC rendered them virtually resistant to the negative effects of CsA. CD3 acquisition was unhindered and total CD45 remained high, but the transition from CD45RA to CD45R0 appeared to be delayed. In the absence of TEC, expression of both TCRαβ and of TCRδ was inhibited, but on TEC, TCRδ was actually up-regulated in some conditions. The effects of CsA on human thymocyte development appeared to be modulated by the physiological state of the donor and the growth conditions to which the cells were subjected. Conditions which most closely approximated those manifest in vivo rendered thymocytes most resistant to the negative effects of CsA. The amount of CsA required to affect differentiation in vitro was significantly higher than could be attained in vivo suggesting that the immunomodulatory effects of CsA in the maintenance of organ transplants may derive from an as yet uncharacterized mechanism. [ABSTRACT FROM AUTHOR]

The objectives of this study were firstly, to compare the immunophenotype of patients with monoclonal gammopathy of undetermined significance (MGUS) with that of patients with newly diagnosed, untreated multiple myeloma (Unt. MM). Our second objective was to determine which variables might distinguish patients with MGUS and early MM. The CD4/CD8 ratio in both patient groups differed significantly from normal as a result of a decrease in the proportion of CD4+ cells. Similarly, surface immunoglobulin-positive (Ig+) B cells were significantly reduced m both groups. Also, some impairment of Ig secretion was observed. An in vitro specificity study of B cells showed an enriched proportion of B cells specific for tetanus toxoid (which may be indicative of enrichment for memory B cells) in both MGUS and Unt. MM patients. Further to this, in MM patients but not in MGUS patients, there was an enriched proportion of B cells specific for determinants on the F(ab′)2 fragment of Ig. This suggests an anomalous auto-immune reactivity to polyclonal Ig molecules. In one of the two patients studied, who progressed from MGUS to MM, disease progression was accompanied by an increase in this anti-Ig reactivity. In both patients there was a decrease in CD4/CD8 ratio. [ABSTRACT FROM AUTHOR]

Although antigen-presenting cells (APC) appear to be able to princess minor histocompatibility antigens (MIHA) expressed on allogencic cells anti present them in association with intrinsic H-2 of the APC in vivo, this does not occur in vitro. This could be due to fundamentally different mechanisms of antigen handling by APE in vitro, or it could represent compromised APC function. In order to distinguish these possibilities we designed a system m which BALB/c spleen cells are transferred into an MIHA-disparate irradiated host and allowed to reside for 2–3 days: the spleen cells of the repopulated host are removed and used as stimulator cells for the CTI response of primed BAI B/c responder cells to DBA/? MIHA These cells are referred to as in vivo-pulsed APC (IVP-APC). Donor BALB/c cells arc able to pick up DBA/2 MIHA after a passage in DBA/2 hosts and efficiently present MIHA to primed CTL precursors to generate DBA/2-specific CTL. The donor cell type able to pick up and present MIHA is present in spleen but not thymus or bone marrow, is Thy-l.2 negative. Ia+, and nylon wool- adherent. Its stimulatory capacity is as efficient on a per cell basis as that of DBA/2 spleen cells. The generation of IVP-APC requires repopulation of the irradiated host, which must express MIHA foreign to the BALB/c donor cells. When we attempted to generate IVP-APC in H-2 incompatible hosts, we found that, although the IVP-APC could present H-2 antigens, they were unable to present MIHA in association with intrinsic APC H-2 antigens. Use of intra-H-2 recombinant strains as host mice indicated that compatibility of donor and host at the KI region of H-2 was essential for the generation of IVP-APC able to present apparently unprocessed MIHA to primed BALB/c responder cells. Thus, we were unable to reproduce the antigen- processing function of APe encountering antigen in situ using an adoptive transfer method of pulsing APC with MIHA in vivo. In addition, we suggest that our results may impose constraints on the formulation of models to account for the association of MIHA with H-2 antigens. [ABSTRACT FROM AUTHOR]

The generation of antibody diversity can be discussed at two levels: genotype and phenotype. The genetic question is, does the germ line genome contain all antibody genes, or are some created (by mutation) during the life of the individual? An equally important question at the level of phenotype is, when is immune competence expressed during development of an individual, and, particularly, does antigen stimulation increase the repertoire of competent cells? In this short article we will concentrate on the last question, after first listing reasons why we prefer somatic mutation to germ line theories at the genetic level. We will draw attention to some of the considerable body of indirect evidence that supports antigen-generated diversity and briefly discuss experiments that can directly test whether entirely new kinds of immunocompetent cells appear during the course of an immune response. [ABSTRACT FROM AUTHOR]