Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas.

Sixteen monoclonal antibodies that were obtained after immunization of BALB/c mice with intact melanoma cells or extracts of melanoma cells were tested for reactivity with normal and malignant melanocytic cells in situ, using an immunoperoxidase technique on frozen tissue sections. Sections representing six histopathologically defined stages of tumor progression, ranging from normal melanocytes to highly malignant metastatic lesions, were used. Thirteen monoclonal antibodies (MAbs) did not stain normal melanocytes in situ, whereas three MAbs weakly stained between 1 and 12.5% of melanocytes in 6-22% of the skin sections examined. MAb B 73.1, which was produced by immunization of mice with human natural killer cells and which binds to the Fc receptor of natural killer cells and granulocytes, reacted exclusively with malignant cells that represent the last two stages of tumor progression, vertical growth phase (VGP) primary melanoma and metastatic melanoma. All other antibodies showed variable reactivity with benign proliferative lesions or radial growth phase (RGP), an early stage of primary melanoma. Staining by MAbs that were reactive with gangliosides, unknown antigens, receptors, and two proteins (120/94 kDa protein and 250 kDa glycoprotein) showed a gradual increase in subsequent stages of tumor progression. Two steps in tumor progression were characterized by significant quantitative changes in the expression of antigens detected by the MAbs used in this study. First, mature nevus cells showed significantly higher reactivity with a panel of six MAbs, when compared to normal melanocytes. Second, a separate panel of six MAbs discriminated between RGP and VGP primary melanoma cells. No significant differences in antigen expression were found between dysplastic nevus cells and RGP melanoma, except that some antigens (nerve growth factor receptor and GD2/GD3 gangliosides) appear to be expressed at lower levels in RGP lesions, nor did VGP primary and metastatic melanomas show significant differences in antigen expression. These results suggest that (a) tumor progression of melanocytic cells in vivo is accompanied by significant quantitative differences in the expression of antigens, (b) some of the antigens examined here are associated with biologically aggressive malignant lesions but not normal or premalignant melanocytic cells, and (c) RGP primary melanoma cells are antigenically more similar to nevus cells than to VGP primary melanoma cells.

Expression of melanoma-associated antigens in rapidly dividing human melanocytes in culture.

Conditions were established to induce rapid clonal growth of melanocytes from newborn foreskin. Surface antigen expression was analyzed using monoclonal antibodies derived by immunization of mice with melanoma cell, melanocyte, and placental membrane preparations. Unlike resting melanocytes in normal skin, cultured melanocytes expressed most major melanoma-associated antigens tested, e.g., nerve growth factor receptor, proteoglycan, transferrin-related Mr 97,000 protein antigen, Mr 120,000 protein, and gangliosides 9-O-acetyl GD3 and GD3. HLA-DR antigen and ganglioside GD2 were expressed at very low levels or not expressed. After several subpassages, most melanocyte cultures, including clones and melanocytes, initially sorted by rosetting with monoclonal antibody to nerve growth factor receptor, lost their characteristic bipolar morphology and expression of nerve growth factor receptor and Mr 97,000 antigen but continued to express high molecular weight proteins such as proteoglycan, Mr 130,000/105,000 and 120,000 antigen. The few melanocyte cultures that did maintain their characteristic bipolar to spindle morphology continued to express all melanoma-associated antigens and even began to express HLA-DR antigens. Melanocytes cultured in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also maintained their bipolar morphology, were often pigmented, and continued to express melanoma-associated antigens for several passages; they did not express HLA-DR antigen. Our studies indicate that rapidly proliferating melanocytes in culture undergo antigenic changes associated with malignancy.

Mouse ear spleen grafts: a model for intrasplenic immunization with minute amounts of antigen.

The production of monoclonal antibodies to protein antigens which can only be obtained in tiny amounts has been a major task, since classical in vivo immunization procedures are not always efficient. In order to circumvent this problem, two methods have been developed: (1) in vitro immunization, in which the immunogen is presented directly to spleen cell cultures; (2) intrasplenic immunization, a technique in which the immunogen is deposited in the spleen tissue. The latter approach requires less laboratory work and the risk of contamination, often a problem with in vitro cultures (Nilsson and Larsson, Immunol. Today (1990) 11, 10), is greatly reduced. Here, we describe a novel method of grafting neonatal spleens in the pinna of the mouse ear. Histological and functional studies show that these spleen grafts have white and red pulp and contain normal percentages of functional T and B cells. The results indicate that this procedure is extremely efficient in priming mice for a secondary humoral immune response, since very small amounts of soluble antigen (ovalbumin) were required. The data are discussed in terms of the advantages of this new technique over current procedures for intrasplenic immunization.

Anti-gamma delta T cell antibody blocks the induction and maintenance of oral tolerance to ovalbumin in mice.

The oral administration of antigens is one of the means of inducing tolerance in adult mammals. In this report, the role of gamma delta T cells in the induction and maintenance of orally-induced tolerance to ovalbumin was investigated. The injection of a monoclonal anti-gamma delta T cell monoclonal antibody blocked the induction of oral tolerance, because the secondary immune responses to ovalbumin in these animals were comparable to the corresponding responses in ovalbumin-immunized control mice. Furthermore, depletion of gamma delta T cells either in vivo or in vitro abolished already established oral-tolerance. The fact that the state of tolerance could be adoptively transferred to naive recipients by CD3+ alpha beta- gamma delta + spleen cells from tolerant mice. These results suggest that systemic oral tolerance is induced and actively maintained by mechanisms involving gamma delta T cells.

Administration of a nondepleting anti-CD25 monoclonal antibody reduces disease severity in mice infected with Trypanosoma cruzi.

The role of CD25+ regulatory T cells during the course of Trypanosoma cruzi infection has been previously analyzed, and the bulk of results have shown a limited role for this T cell subpopulation. In this study, we have used an IgM, nondepleting monoclonal antibody (mAb) aiming at blocking interleukin (IL)-2 activity on CD25+ T cells. The administration of this antibody 10 days before infection increased the resistance of outbred Swiss mice to the Colombian strain of T. cruzi. Anti-CD25-treated mice had lower parasitemia and augmented numbers of effector memory T cells. In addition, these animals showed higher numbers of splenic T cells secreting IFN-γ and TNF-α, both cytokines described to be involved in the resistance to T. cruzi infection. The same treatment also increased the numbers of splenic T cells that produced homeostatic and regulatory cytokines, such as IL-2 and IL-10, and CD4+CD25+ T cells. The administration of nondepleting anti-CD25 mAb at the beginning of the chronic phase, when parasites were cleared from the blood, halted the inflammatory process in the heart, without any signs of infection reactivation. These results indicate that nondepleting anti-CD25 monoclonal antibodies may be useful to treat chronic Chagas' disease.

A 3-D CAD tool for CT colonography.

Nowadays, biomedical images of the human body are an invaluable source of information that clinicians can use in order to make a correct and final diagnosis. Their use is widespread in almost every branch of medicine. In some cases, computer-aided diagnosis (CAD) tools have received approval for being used without any human intervention and validation, like, for example, in breast cancer detection relying on X-rays mammograms. In this paper we present a framework for building CAD tools based on the well-known software design patterns and an implementation of the first modules of a CAD tool for computed tomography (CT) colonography.

The role of the thymus in modulating gammadelta T cell suppressor activity during experimental Trypanosoma cruzi infection.

We have previously shown that splenic gammadelta T cells from young but not aged BALB/c mice possess suppressor activity in vivo and in vitro during the acute phase of Trypanosoma cruzi infection. The present work was undertaken to investigate the suppressor activity of gammadelta T cells from T. cruzi-infected euthymic or athymic mice and the role of the thymus in modulating non-adherent spleen cell suppressor activity during the acute phase of infection. Splenic gammadelta T cells from aged or athymic BALB/c mice reconstituted with total spleen cells or non-reconstituted did not exhibit suppressor activity when added to full allogeneic, mixed lymphocyte cultures. In contrast, splenic gammadelta T cells from young euthymic BALB/c mice showed suppressor activity in vitro. Thymectomy reduced the splenic gammadelta T cell suppressor activity of young BALB/c mice in a time-dependent manner, following a T. cruzi challenge. The continuous provision of thymocytes to aged mice, young thymectomized mice or total spleen cell-reconstituted athymic mice could re-establish the gammadelta T cell suppressor activity. Of particular significance was the observation that the depletion of gammadelta T cells during the acute phase of T. cruzi infection restored the capacity of these mice to mount a humoral immune response to a non-related antigen such as ovalbumin. These results indicate that gammadelta T cells of extrathymic origin cannot mediate suppression and that the thymus has a role in the regulation of suppression during the acute phase of T. cruzi infection.

Anti-IL-10 treatment does not block either the induction or the maintenance of orally induced tolerance to OVA.

Herein, the role of IL-10 in the induction and maintenance of oral tolerance was evaluated. The results show that: (1) mice treated with MoAb anti-IL-10 are permissive to the induction of oral tolerance to OVA; (2) anti-IL-10 treatment did not reverse the in vitro blocking of T cell proliferative response found in orally-tolerized mice; and (3) orally-induced tolerance could not be broken by anti-IL-10 treatment. Taken together, these results suggest that IL-10 is not a fundamental cytokine for the establishment and maintenance of oral tolerance.

Regulation of Trypanosoma cruzi infection in mice by gamma interferon and interleukin 10: role of NK cells.

Gamma interferon (IFN-gamma) plays an important role in experimental Trypanosoma cruzi infections, presumably by controlling the early replication of parasites in host macrophages. In this work, we show that NK cells represent an important cell type responsible for the production of most of the IFN-gamma in the early stage of T. cruzi infection and that the in vivo treatment of mice with anti-NK1.1 monoclonal antibody made resistant animals susceptible to the infection. Through in vitro experiments, we demonstrate that normal splenocytes from euthymic or athymic nude mice cultivated for 48 h with live T. cruzi trypomastigotes produced elevated levels of IFN-gamma. In addition, NK-depleted splenocytes show a drastic reduction of IFN-gamma production in response to live T. cruzi trypomastigotes. We also demonstrated that IFN-gamma production is dependent on a factor secreted by adherent cells. Supernatants of spleen cells from athymic nude mice are able to induce IFN-gamma production by normal splenocytes when cultured with trypomastigotes. The addition of anti-interleukin-10 to these cultures resulted in a marked increase in IFN-gamma production. On the other hand, the absence of NK cells led to an increased secretion of interleukin-10 upon in vitro stimulation with T. cruzi. Taken together, these results suggest that NK cells are the major source of IFN-gamma that could be involved in limiting the replication of T. cruzi in host macrophages during the early acute phase of the infection.

An age-related gamma delta T cell suppressor activity correlates with the outcome of autoimmunity in experimental Trypanosoma cruzi infection.

In this work the suppressive activity of splenic T cells from young and aged BALB/c mice infected with Trypanosoma cruzi were compared and correlated with the development of autoimmune myocarditis. The T cells from young adult BALB/c mice with acute T. cruzi infection exhibit suppressor activity when added to full allogeneic or Mls-disparate mixed lymphocyte cultures. This suppression could not be reverted by exogenous interleukin (IL)-2 and was not directly dependent on the presence of IL-4, IL-10 or transforming growth factor-beta. Further characterization of the T cell lineage responsible for the suppressor activity by in vitro and/or in vivo depletion with monoclonal antibody to alpha beta or gamma delta T cell receptor revealed that splenic gamma delta T cells function as suppressor lymphocytes in young T. cruzi-infected mice. In addition, these young adult BALB/c mice do not develop autoimmune myocarditis and showed a low incidence of syngeneic heart graft rejection in the early chronic phase of the infection. In contrast, T cells from acutely infected aged BALB/c mice lacked demonstrable T suppressor activity. Furthermore, these mice developed a severe autoimmune myocarditis as early as 2 months after the onset of the infection, when the majority of them reject syngeneic heart grafts. These findings suggest that a gamma delta T cell-mediated suppressor mechanism may operate in the avoidance of the breaking of tissue-specific tolerance during the acute infection. Moreover, such a mechanism is likely related to the immune system chronobiology.

NK1.1+ cells and T-cell activation in euthymic and thymectomized C57Bl/6 mice during acute Trypanosoma cruzi infection.

Natural killer (NK) cells may provide the basis for resistance to Trypanosoma cruzi infection, because the depletion of NK1.1 cells causes high levels of parasitemia in young C57Bl/6 mice infected with T. cruzi. Indeed, NK1.1 cells have been implicated in the early production of large amounts of interferon (IFN)-gamma, an important cytokine in host resistance. The NK1.1 marker is also expressed on special subpopulations of T cells. Most NK1.1+ T cells are of thymic origin, and their constant generation may be prevented by thymectomy. This procedure, by itself, decreased parasitemia and increased resistance in young mice. However, the depletion of NK1.1+ cells by the chronic administration of a monoclonal antibody (MoAb) (PK-136) did not increase the parasitemia or mortality in thymectomized C57Bl/6 mice infected with T. cruzi (Tulahuen strain). To study the cross-talk between NK1.1+ cells and conventional T cells in this model, we examined the expression of activation/memory markers (CD45RB) on splenic CD4+ and CD8+ T cells from young euthymic or thymectomized mice with or without depletion of NK1.1+ cells and also in aged mice during acute infection. Resistance to infection correlated with the amount of CD4+ T cells that are already activated at the moment of infection, as judged by the number of splenic CD4+ T cells expressing CD45RB(-). In addition, the specific antibody response to T. cruzi antigens was precocious and an accumulation of immunoglobulin (Ig)M with little isotype switch occurred in euthymic mice depleted of NK1.1+ cells. The data presented here suggest that NK1.1+ cells have important regulatory functions in euthymic, but not in thymectomized mice infected with T. cruzi. These regulatory functions include a helper activity in the generation of effector or activated/memory T cells.

Erythrocruorin of Glossoscolex paulistus (Oligochaeta, Glossoscolecidae): modulation of oxygen affinity by specific antibodies.

1) The Soret region absorption spectrum of erythrocruorin (ERC) obtained from Glossoscolex paulistus, shows that oxy-ERC has a maximum absorption peak at 416 nm while the deoxy-ERC from has a maximum at 427 nm. 2) In the presence of a specific antiserum (anti-ERC) and of anti-ERC immunoglobulin G raised in rabbits, there is a deviation to low wavelengths in the maximum absorption peak of deoxy-ERC while for the oxy form a red-shift is noticed. These shifts accompanied an increased affinity of the hemeprotein for oxygen, possibly because of changes in the overall macromolecular conformation. 3) A decrease in the oxygen affinity of erythrocruorin is observed when large amounts of non-specific serum are used. The same effect is observed in the presence of serum albumin, probably as a result of non-specific binding between the albumin and erythrocruorin. 4) The fluorimetric titration of erythrocruorin with anti-ERC Fab fragments results in a decrease in the intrinsic tryptophan fluorescence of the hemeprotein, a response indicative of a modification in the ERC's quaternary structure.