Marion C. Cohen

Investigation of promoter polymorphisms in the tumor necrosis factor-α and interleukin-10 genes in liver transplant patients

Background. Cytokines such as tumor necrosis factor (TNF)-&agr; and interleukin (IL)-10 play significant roles in the inflammatory and immune responses that mediate allograft rejection. The presence of a G→A polymorphism at position −308 in the promoter region of the TNF-&agr; gene increased its transcription 6- to 7-fold. A similar polymorphism at position −1082 of the IL-10 promoter results in decreased production of IL-10 protein. In this study we have determined whether the single nucleotide polymorphisms in the promoter regions of the TNF-&agr; and IL-10 genes can predict the outcome of the allograft in liver recipients. Methods. DNA was extracted from whole blood of liver recipients. The genotype of the patients was determined by polymerase chain reaction using sequence-specific primers. The level of TNF-&agr; and IL-10 protein was measured by ELISA after stimulation of peripheral blood mononuclear cells with concanavalin A. Results. There was significant correlation between acute cellular rejection and the presence of the −308A polymorphism (P <0.001), with 8 of 13 patients with the TNF-&agr; polymorphism having evidence of acute rejection. Cell stimulation studies revealed that the level of TNF-&agr; protein produced by patients with liver rejection was significantly higher than for patients without rejection (P =0.001). There were no strong associations between the presence of the IL-10 polymorphisms and rejection (P =0.71). Conclusions. This study adds to the understanding of the role of cytokine polymorphisms in liver transplants. The data suggest that cytokine promoter polymorphisms may be a risk factor associated with allograft rejection in the liver.

Correlation between agarose microdroplet and capillary tube procedures as assays for migration inhibition of target cells.

We have compared the capillary tube assay for migration inhibition studies with our modification of the agarose microdroplet technique, using several sources of factors with inhibitory activity (lymphokines, bacterial factors) and a variety of cell types (inflammatory exudate cells, tumor cells). In all circumstances both procedures gave quantitatively similar results, and high statistical correlation was found. These results suggest that the two procedures are measuring similar properties, and in any case may be used interchangeably as assays.

Serum migration inhibitory activity against macrophages and tumor cells

Abstract A factor that can inhibit the migration of tumor cells without cytotoxic effect (TMIF) is present in supernatants of activated lymphocytes. In the present experiments, we demonstrate that similar activity can be found in the serum of appropriately immunized animals. As is the case for macrophage migration inhibitory factor (MIF), TMIF activity appears only transiently after challenge with antigen. TMIF is also found in the serum of tumor-bearing animals. In this situation, activity persists from Day 7 postinoculation until the time of death. Although previous studies have demonstrated that TMIF and MIF are distinct, these results show that TMIF activity parallels serum migration inhibitory activity against macrophages. In the present experiments, the animals were given a lethal dose of tumor cells. However, it is likely that by virtue of its biologic properties, TMIF might have a protective effect under other experimental conditions.

Adherence of tumor cells to endothelial monolayers: inhibition by lymphokines

Tumor cells can adhere to endothelial cell monolayers in vitro. The kinetics of this reaction are rapid; 50% of maximal binding occurs by 30 min of incubation. In the case of the P815 mastocytoma, the maximal percentage of binding is approximately 70%, suggesting that there are both binding and nonbinding tumor cell populations. Binding is independent of tumor cell dose over a 200-fold range of cell concentrations. Lymphokine-containing preparations were found to markedly suppress the binding of either P815 mastocytoma or Ehrlich ascites cells to endothelium. This effect appeared to be due to both diminished attachment and enhanced dissociation. The activity is found in the same molecular weight range as tumor migration inhibition factor (TMIF), and is not found in preparations lacking TMIF activity. Thus, the factor may prove to be TMIF itself or a lymphokine related to it. Of equal interest is the possibility that it represents a previously undescribed factor.

In vitro migration of tumor cells from human neoplasms: inhibition by lymphokines

We have previously described a noncytotoxic lymphokine, TMIF, with the capacity of inhibiting the in vitro migration of a variety of serially passaged experimental animal tumors, but not non-neoplastic cells. In the present study, we describe conditions for the assay of human tumor cell movement utilizing agarose microdroplets. Using this procedure, we were able to demonstrate that TMIF is as effective in inhibiting the in vitro migration of suspensions of tumor cells obtained from spontaneous human neoplasms, as it is in inhibiting model tumor systems. This finding demonstrates that responsiveness to TMIF is not merely a property conferred on tumor cells by prior serial passage. Also, by demonstrating that tumors of human origin are responsive, the present study raises the possibility that studies of TMIF in neoplastic disease may provide information of prognostic value. Also, they provide the hope that if TMIF proves therapeutically effective in animal models, those results may be translated to human disease.

Cellular Hypersensitivity and Inflammation

Cellular hypersensitivity (cell-mediated immunity) is an immunologic reaction that is dependent upon the activity of sensitized lymphocytes rather than specific immunoglobulin. In most cases the lymphocytes do not act directly as effectors, but elaborate soluble products which are known as lymphokines. The classic example of a cell-mediated immune reaction is that of “delayed hypersensitivity,” which is the cutaneous manifestation of this form of response. It is also referred to, especially in the older literature, as “tuberculin” type hypersensitivity. The delayed reaction is a slowly evolving inflammatory reaction (24–48 h to reach maximal size) which develops at the site of injection of antigen into a previously sensitized individual. It appears grossly as an indurated lump and microscopically, at least in man and the guinea pig, as a collection of lymphocytes and macrophages which are usually collectively referred to as “mononuclear” cells. The delayed hypersensitivity reaction can be transferred to a normal, nonimmune individual by sensitized lymphocytes, but not by serum.

GENE EXPRESSION ANALYSIS OF A DEDIFFERENTIATED LIPOSARCOMA — DIFFERENCES BETWEEN HIGH AND LOW GRADE AREAS: ANALYSIS OF TWO CASES AND LITERATURE REVIEW

The phenomenon of dedifferentiation typically occurs in soft tissue sarcomas where a low grade or well-differentiated tumor shows an abrupt transformation to a high-grade sarcoma without lineage specificity. The biological behavior and metastatic potential of these tumors is dictated by the dedifferentiated phenotype. Tumor material was available from two dedifferentiated liposarcomas. We performed cDNA microarray analysis of a dedifferentiated liposarcoma in which the atypical lipomatous/well-differentiated and dedifferentiated portions were grossly distinct, to find differentially expressed genes in the dedifferentiated component compared to the well-differentiated component. There were 100 differentially expressed genes, both up- and down-regulated in the high grade sarcoma. In addition, we performed RT-PCR on selected genes in both cases to confirm the microarray findings. We discuss the expression patterns of these genes in comparison to other studies in the literature.

Inflammatory Lymphokines in Hypersensitivity Reactions

The immune system is responsible for both the detection of foreign substances and the response to them. Thus, immunologic function requires both recognition and reaction. Recognition involves the sequence of events by which the immune system is made aware of a foreign substance (antigen). It involves the interaction of the antigen with an appropriate cell surface receptor and leads to the activation of lymphocytes for both proliferation and differentiation. The products of differentiation include not only antibodies, but also memory cells, specialized effector cells, and a variety of nonantibody, hormonelike mediators. These latter agents, which have been shown to play a role in both afferent (inductive stages) and efferent (effector responses) manifestations of immunity, are known as lymphokines.

The tumor disappearance reaction: An in vivo effect of a noncytotoxic lymphokine active against tumor cells

A lymphokine, tumor migration inhibition factor (TMIF), that can inhibit the migration of tumor cells in vitro without cytotoxic effect has been described. TMIF can be produced in vitro or in vivo. In the present study, it is demonstrated that administration of TMIF can lead to a transient decrease in recoverable tumor cells from the peritoneal cavities of otherwise untreated mice. This result, which appears to be due to a direct effect of the mediator, rather than a consequence of inflammatory cell accumulation, is analogous to the effect of macrophage migration inhibition factor (MIF) on macrophages in the well known macrophage disappearance reaction. These findings demonstrate that TMIF can exert an effect in vivo predicted from its in vitro properties, and raise the possibility that this effect can be exploited in an appropriate therapeutic model.

Production of lymphokines affecting tumor cells by T-T hybridomas☆

T-Cell hybridomas were constructed by fusing BW5147, an AKR lymphoma, with concanavalin A-stimulated murine splenic lymphocytes. The hybrids which were formed were studied for their ability to produce a lymphokine which inhibits tumor cell migration (TMIF) as well as macrophage migration (MIF) using in vitro assays. Clones were identified which affected tumor cell motility without exerting similar effects on murine macrophages, although the opposite effect was not observed. Although noncoordinate production of these factors cannot be unequivocally established, these results demonstrate that clones can be constructed that preferentially secrete TMIF. In these experiments, we also tested the supernatants for another lymphokine effect on tumor cells; namely, the ability to inhibit tumor cell binding to endothelial monolayers. A number of clones were identified that lacked TMIF activity, but could inhibit the tumor cell-endothelial interaction, suggesting the possibility that these effects may be due to separate mediators.