Abdominal obesity and low-grade systemic inflammation as markers of subclinical organ damage in type 2 diabetes.

AIM: This study aimed to explore the associations between abdominal obesity, inflammatory markers and subclinical organ damage in 740 middle-aged patients with type 2 diabetes. METHODS: Waist circumference (WC) and sagittal abdominal diameter (SAD) were measured, and blood samples were analyzed for C-reactive protein (CRP) and IL-6. Carotid intima-media thickness (IMT) was evaluated by ultrasonography, and aortic pulse wave velocity (PWV) measured with applanation tonometry. RESULTS: Abdominal obesity as determined by SAD and WC was significantly correlated with IL-6 (WC: r=0.27, P<0.001; SAD: r=031, P<0.001), CRP (WC: r=0.29, P<0.001; SAD: r=0.29, P<0.001), IMT (WC: r=0.09, P=0.013; SAD: r=0.11, P=0.003) and PWV (WC: r=0.18, P<0.001; SAD: r=0.21, P<0.001). In multiple linear regressions with IMT and PWV as dependent variables, and age, gender, statin use, systolic blood pressure (SBP), body mass index (BMI), CRP and HbA1c as independent variables, both SAD and WC remained associated with IMT and PWV. On stepwise linear regression and entering both SAD and WC, the association between SAD and PWV was stronger than the association between WC and PWV. CONCLUSION: Both SAD and WC are feasible measures of obesity, and both provide information on inflammation, atherosclerosis and arterial stiffness in type 2 diabetes, while SAD appears to be slightly more robustly associated with subclinical organ damage than WC.

The growth and metastasis of human, HER-2/neu-overexpressing tumor cell lines in male SCID mice.

HER-2/neu is overexpressed on a variety of human adenocarcinomas and overexpression has been associated with a poor prognosis. For this reason, HER-2 has become an attractive target for immunotherapy. To facilitate testing of anti-HER-2-monoclonal antibodies (MAbs) and immunotoxins (ITs), we have evaluated the in vivo growth and metastatic spread of three HER-2-overexpressing human breast cancer cell lines (BT474, MDA-MB-453 and HCC1954) and one ovarian cancer cell line (SKOV3.ip1) in pre-irradiated male SCID mice using subcutaneous (s.c.), intravenous (i.v.) and intraperitoneal (i.p.) routes of injection. All the cell lines tested grew as s.c. tumors and the growth of BT474 and MDA-MB-453 cells after s.c. injection was improved by co-inoculation with Matrigel. Metastases to the lungs were detectable by PCR or histopathology after s.c. injection of BT474 and to a much lesser extent after s.c. injection of HCC1954, MD-MB-453 and SKOV3.ip1 cells. I.p. injection of HCC1954 and SKOV3.ip1 cells produced fatal ascites while i.v. injection of SKOV3.ip1, but not BT474 or MDA-MB-453 cells, resulted in infiltration of lungs and death within 9-11 weeks.

The use of an anti-CD3 immunotoxin to prevent the development of lymphoproliferative disease in SCID/PBL mice.

Severe combined immunodeficient mice (SCID) reconstituted with normal PBLs (SCID/PBL) from Epstein-Barr virus-positive (EBV+) human donors often develop fatal human B lymphomas which resemble the EBV-induced lymphoproliferative disease (LPD) observed in immunosuppressed individuals. This phenomenon appears to be T cell dependent. In this study we used an immunotoxin (IT) prepared by conjugating the monoclonal anti-CD3 antibody, 64.1, to deglycosylated ricin A chain (dgRTA) to prevent LPD in SCID/PBL mice. We show that the incidence of LPD is greatly reduced by either a combination of in vitro treatment of PBLs followed by one in vivo treatment of the xenografted mice with 64.1-dgRTA immunotoxin or by repeated treatments in vivo with the immunotoxin. In contrast, in vitro treatment alone or in vivo treatment with only one injection of 64.1-dgRTA were less effective. As expected, this IT did not have any non-specific cytotoxic effects on already established EBV+ tumors from SCID/PBL mice. The use of this IT, therefore, represents a simple method to avoid LPD when injecting blood-containing tissues into SCID mice.

The development of monoclonal antibodies for the therapy of cancer.

Monoclonal antibodies (Mabs) were first described by Köhler and Milstein in 1975. Not only did this discovery lead to a Nobel prize, but it created an enormous scientific field that has now become a multimillion dollar industry. Mabs made the transition from laboratory reagents to clinical diagnostics very quickly. However, their development as therapeutic agents was, as predicted, more costly and time-consuming. Indeed, clinicians and scientists were required to learn a new set of rules for using these large, immunogenic, targeted agents in humans. Nevertheless, in 1997 the first Mab was licensed in the U.S. and several others will soon follow. In this review, we discuss Mab-based strategies for the treatment of cancer. We compare native, fragmented, recombinant and chimeric antibodies, bispecific antibodies, immunoconjugates, and immunoliposomes. The rationale for their development, their advantages, their in vitro and in vivo performance, and their clinical usefulness are discussed.

Immunotoxins: an update.

The use of immunotoxins (ITs) in the therapy of cancer, graft-vs-host disease (GvHD), autoimmune diseases, and AIDS has been ongoing for the past two decades. ITs contain a targeting moiety for delivery and a toxic moiety for cytotoxicity. Theoretically, one molecule of a toxin, routed to the appropriate cellular compartment, will be lethal to a cell. Newly developed MoAbs, toxins, and molecular biological technologies have enabled researchers to construct ITs that can effectively kill many different cell types. In fact, phase I/II clinical trials have given promising results. Although nonspecific toxicity and immunogenicity still limit the use of IT therapy, these agents hold enormous promise in an optimal setting to treat minimal disease.

IL-4-induced B cell migration involves transient interactions between beta 1 integrins and extracellular matrix components.

IL-4 has previously been shown to stimulate motile responses in murine B lymphocytes. This was studied as acquisition of motile morphology and migration through filters in microchemotaxis chambers. In this paper, we investigated IL-4-stimulated migration of B cells into gels of native collagen fibers, which may be a more physiologically relevant assay. When IL-4 was present in the gel and/or in the medium above, B cells were able to invade the collagen gel. Migration was dependent on the dose of IL-4 and was optimal after 45 h of incubation. It appeared that IL-4 acted by inducing both chemokinesis and chemotaxis. Fibronectin (FN) was found to be an important factor for B cell locomotion, since low concentrations of FCS or FN in the gel matrix greatly improved migration. B cell locomotion was inhibited by antibodies specific for beta 1, alpha 4 and alpha 5 integrins, indicating the presence of integrin-extracellular matrix (ECM) interactions in lymphocyte motility responses. Migration was not associated with an up-regulation of beta 1, alpha 4 or alpha 5 integrins. The adhesion between substrate and cells is likely to be of low affinity, since IL-4-stimulated, as well as non-stimulated B cells, did not adhere to ECM-coated culture wells. Our data suggest that transient interactions between integrins and the ECM matrix may favour B cell migration.

Interleukin-4 induces in vitro migration in naive B cells through direct mechanisms.

Interleukin-4 (IL-4) mediated locomotor responses by murine B cells in vitro were examined in this paper. The IL-4 induced migration was found to act directly on purified, splenic B cells, without involvement of secondary mediators. It appeared that only a subpopulation of B cells was able to respond in migration assays. Flowcytometric analysis showed that the migrating cells had the characteristics of naive B cells: I-Alo, J11dhi, IgDhi. They also displayed high expression of the adhesion molecule L-selectin and made predominantly IgM antibodies. This is contrary to what has previously been observed regarding motile responses to chemotactic factors by T cells, which mostly affect memory or activated T cells. However, this is in accordance with other studies indicating that IL-4 is a cytokine that exerts its effect mainly on resting B cells.

Interleukin-10 inhibits motility in murine and human B lymphocytes.

We here report the finding that the anti-inflammatory cytokine interleukin-10 (IL-10) inhibits motility of B lymphocytes. B cells were induced to display motile morphology and active migration by IL-4. IL-10 inhibited locomotor responses to IL-4, when B cells of both murine and human origin were used. The inhibitory effect of IL-10 was reversible, since washing of B cells preincubated in IL-10 restored the ability to respond to IL-4. Time-course experiments showed that IL-10 did not have to be present from the very onset of culture, but could be added as late as 5 hr after initiation. In addition, murine B cells stimulated with lipopolysaccharide (LPS) showed motile morphology, as well as cellular aggregation and proliferation. All these parameters were suppressed by IL-10. However, viability of B cells was not adversely affected by IL-10. Exposure to IL-10 did not result in any changes in the surface expression of molecules involved in adhesion, such as CD2, CD11a/CD18, CD44, CD54 or L-selectin, on B lymphocytes.

The effect of T cell-derived cytokines on B cell motility in vitro.

We have investigated the ability of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, and interferon-gamma (IFN-gamma) to induce motility in murine, splenic B lymphocytes. Two parameters of cellular locomotion were studied, namely acquisition of motile morphology (polarization) and in vitro migration through polycarbonate filters. Of the tested cytokines, only IL-4 gave a strong motile response among B cells. At the optimal concentration of 3 ng/ml, IL-4 induced polarization in 10-20% of the B cells within 30 min and in up to 40% after overnight exposure. The same amount IL-4 present in a microchemotaxis chamber stimulated migration through polycarbonate filters after overnight incubation in up to 15% of the input B cells. IFN-gamma could also induce some degree of polarization and migration, but only after 19 hr of exposure and to a much lesser extent than IL-4.

A comparative assessment of the roles of CD8 and CD2 in the functions of activated murine CD8+ T lymphocytes.

Both CD8 and CD2 are T cell surface receptors involved in physical cell interaction and in transmembrane signalling. The present paper addresses their role in the induction of two different functions of the cloned murine cytotoxic T cell C196: target cell lysis and IFN-gamma production. These functions were induced in C196 either by stimulation with the specific stimulator/target cell P815 or, bypassing specific recognition, by the aCD3 hybridoma 145-2C11 or by solid phase aTCR antibodies. These responses were tested for their susceptibility to inhibition/enhancement by a panel of aCD8 and aCD2 mAb. In addition, CD8 deficient and CD8/CD2 double-deficient variants of C196 were transfected with the CD8 and CD2 genes and the resulting cell lines were analysed for their functional capacities. The following results were obtained: (i) CD8 is primarily important in the specific recognition process of activated CTL; (ii) transmembrane signalling of activated CTL through the TCR does not require CD8, nor is it sensitive to modification through CD8; (iii) CTL can nevertheless be directly activated through CD8; however, this is restricted to induction of cytotoxicity but does not result in IFN-gamma production; (iv) CD2 does not seem to be important in any of these responses.

T and B cell collaboration: induction of motility in small, resting B cells by interleukin 4.

In this report we investigate if IL 4 can work as a chemoattractant factor by inducing locomotion in B cells. We found that murine recombinant IL 4 (rIL 4) induced motile morphology and migration through polycarbonate micropore filters of murine, splenic B cells at an optimal concentration of 3 ng/ml. Kinetic studies revealed optimal migration at 8-16 h, although a significant response could be detected already after 1 h. Flow cytometric studies confirmed that the migrated cells were indeed B cells. We also compared the activity of small, dense B cells and large, low-density B cells, based on Percoll gradient separation. We found no difference in IL 4-induced motility among the two groups. Furthermore, we looked at B cells activated in vitro by preculture in lipopolysaccharide (LPS) or IL 4. Our data indicate that both LPS and IL 4 can increase the general capacity for motility in B cells after preculture for 24 h. T and B cell collaboration requires close cell-cell contacts in order for T cell help to be administered to the B cell. One way of enhancing such cell contacts could be through directional cell migration induced by helper factors (chemotaxis). We suggest that IL 4 can play a role as a chemoattractant factor that enhances cell contacts between T helper cells and B cells.

The immunoglobulin receptors on B cells bind antigen, focus activation signals to them and initiate antigen-presentation.

We do not agree with the analysis of Langman and Cohn on the function of Ig receptors. We have reviewed the available literature regarding anti-Ig activation of B cells and found it contradictory and unconvincing. We have presented experimental evidence on the inability of Ig receptors on B cells to mediate activation or tolerogenic signals. We suggest that the Ig receptors serve to focus antigen to specific B cells so the B cells can be activated by TI antigens or helper T cells. The Ig molecules also bind foreign antigen and thereby initiate internalization and antigen processing. The processed peptides are exported to the membrane, where they associate with MHC class II antigens, thus transforming B cells into efficient antigen-presenting cells.

Forced contact between antigen-presenting cells and T cells: consequences for T-cell activation.

It is still not known how T cells are activated, which T-cell surface structures transmit activation signals, and if antigen-presenting cells possess activation structures for T cells. We have studied whether the T-cell receptor (TcR) must be engaged for T-cell activation to occur. By using membrane-incorporated monoclonal antibodies, we artificially forced T cells to bind to antigen-presenting cells in a mixed lymphocyte reaction system and thereby bypassed the need for TcR engagement and also made it possible for any surface molecule on antigen-presenting cells to deliver a stimulatory signal to the T cells. Theoretically, T cells would become polyclonally activated by this procedure. However, we found that they did not, even though they were intimately bound to the antigen-presenting cell, thus demonstrating that the TcR must participate in antigen/MHC binding in order for the T cells to become activated. This study does not exclude the possibility that antigen-presenting cells possess structures that can activate T cells.

Capping and co-capping of membrane immunoglobulin and lipid-conjugated immunoglobulin inserted in the cell membrane of B lymphocytes.

Human and mouse immunoglobulins (Ig) or F(ab')2 fragments of rabbit Ig were conjugated to lipids and the conjugates inserted into the membrane of mouse spleen cells. It was found that nearly all B cells, but not T cells, became decorated with lipid immunoglobulin. Both endogenous Ig receptors and inserted Ig capped after the addition of cross-linking F(ab')2 antibodies and in both cases capping required energy. Capping of endogenous mouse Ig led to co-capping of inserted human Ig, but the reverse was not true.

Recognition of major histocompatibility complex antigens on murine glial cells.

Recognition of autologous major histocompatibility complex (MHC) antigens by T cells is an essential step in the induction of an immunologic reaction to either endogenous or exogenous antigens. We investigated the ability of murine glial cells of different ages to stimulate clones of allospecific T lymphocytes. We also investigated the effects of supernatants from cultures of activated T cells on the immunologic recognition of MHC antigens on murine glial cells. Lymphocyte clones specific for Class I, Class II and non-MHC, background antigens were obtained from C57B1/6J-anti-DBA/2 mixed lymphocyte cultures. Glial cell cultures were prepared from newborn syngeneic (C57B1/6J) and allogeneic (DBA/2) mouse brains. Glial cultures 1-4 weeks of age were able to stimulate alpha-Class I-specific clones. No stimulation of alpha-Class II or alpha-background clones was noted. Incubation of glial cells with supernatants from cultures of alloantigen-activated spleen cells (C57B1/6J-anti-DBA/2) resulted in a decreased ability of glial cells to stimulate alpha-Class I responses. In contrast supernatant-treated cultures acquired the capacity to stimulate alpha-Class II-specific clones. No responses were noted in clones responsive to non-MHC antigens. The ability to stimulate alpha-Class II-specific clones was most prominent with one-week-old glial cultures and was lost by four weeks of culture. The increased susceptibility of younger glial cultures to the modulatory effects of lymphokines from activated T cells may be a factor in the increased susceptibility of the immature central nervous system to persistent viral infections and the development of autoimmune phenomena.