CD4+ T cells play an important role in acute experimental pancreatitis in mice

BACKGROUND & AIMS: Few data are available on the potential role of T lymphocytes in experimental acute pancreatitis. The aim of this study was to characterize their role in the inflammatory cascade of acute pancreatitis. METHODS: To type this issue, acute pancreatitis was induced by repeated injections of cerulein in nude mice and in vivo CD4(+) or CD8(+) T cell-depleted mice. The role of T lymphocyte-costimulatory pathways was evaluated using anti-CD40 ligand or anti-B7-1 and -B7-2 monoclonal blocking antibodies. The role of Fas-Fas ligand was explored using Fas ligand-targeted mutant (generalized lymphoproliferative disease) mice. Severity of acute pancreatitis was assessed by serum hydrolase levels and histology. Intrapancreatic interleukin 12, interferon gamma, Fas ligand, and CD40 ligand messenger RNA were detected by reverse-transcription polymerase chain reaction. Intrapancreatic T lymphocytes were identified by immunohistochemistry. RESULTS: In control mice, T cells, most of them CD4(+) T cells, are present in the pancreas and are recruited during acute pancreatitis. In nude mice, histological lesions and serum hydrolase levels are significantly decreased. T-lymphocyte transfer into nude mice partially restores the severity of acute pancreatitis and intrapancreatic interferon gamma, interleukin 12, and Fas ligand gene transcription. The severity of pancreatitis is also reduced by in vivo CD4(+) (but not CD8(+)) T-cell depletion and in Fas ligand-targeted mutant mice. Blocking CD40-CD40 ligand or B7-CD28 costimulatory pathways has no effect on the severity of pancreatitis. CONCLUSIONS: T lymphocytes, particularly CD4(+) T cells, play a pivotal role in the development of tissue injury during acute experimental pancreatitis in mice.

Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology.

Fifty-one in vivo characterized autonomous single adenomas have been studied for functional parameters in vitro, for gene and protein expression and for pathology, and have been systematically compared to the corresponding extratumoral quiescent tissue. The adenomas were characterized by a high level of iodide trapping that corresponds to a high level of Na+ /iodide symporter gene expression, a high thyroperoxidase mRNA and protein content, and a low H2O2 generation. This explains the iodide metabolism characteristics demonstrated before, ie, the main cause of the "hot" character of the adenomas is their increased iodide transport. The adenomas spontaneously secreted higher amounts of thyroid hormone than the quiescent tissue and in agreement with previous in vivo data, this secretion could be further enhanced by thyrotropin (TSH). Inositol uptake was also increased but there was no spontaneous increase of the generation of inositol phosphates and this metabolism could be further activated by TSH. These positive responses to TSH are in agreement with the properties of TSH-stimulated thyroid cells in vitro and in vivo. They are compatible with the characteristics of mutated TSH receptors whose constitutive activation accounts for the majority of autonomous thyroid adenomas in Europe. The number of cycling cells, as evaluated by MIB-1 immunolabeling was low but increased in comparison with the corresponding quiescent tissue or normal tissue. The cycling cells are observed mainly at the periphery; there was very little apoptosis. Both findings account for the slow growth of these established adenomas. On the other hand, by thyroperoxidase immunohistochemistry, the whole lesion appeared hyperfunctional, which demonstrates a dissociation of mitogenic and functional stimulations. Thyroglobulin, TSH receptor, and E-cadherin mRNA accumulations were not modified in a consistent way, which confirms the near-constitutive expression of the corresponding genes in normal differentiated tissue. On the contrary, early immediate genes expressions (c-myc, NGF1B, egr 1, genes of the fos and jun families) were decreased. This may be explained by the proliferative heterogeneity of the lesion and the previously described short, biphasic expression of these genes when induced by mitogenic agents. All the characteristics of the autonomous adenomas can therefore be explained by the effect of the known activating mutations of genes coding for proteins of the TSH cyclic adenosine monophosphate (cAMP) cascade, all cells being functionally activated while only those at the periphery multiply. The reason of this heterogeneity is unknown.

Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

Expression of galectin-3 in the tumor immune response in colon cancer.

The role of tumor-associated macrophages (TAMs) is controversial. Although most studies on different cancer types associate them with a poorer prognosis, interestingly in colon cancer, most articles indicate that TAMs prevent tumor development; patients with high TAMs have better prognosis and survival rate. M1-polarized macrophages produce high level of tumor necrosis factor-alpha, interleukin-1 beta or reactive oxygen species, which can effectively kill susceptible tumor cells. In contrast, M2-polarized macrophages can secrete different factors that promote tumor cell growth and survival or favor angiogenesis and tissue invasion. Considering the beneficial role of TAMs in colon cancer, we speculated that they may not display the M2 polarization commonly observed in tumor microenvironment, but rather develop M1 properties. Therefore, we used an in vitro model to analyze the effects of supernatants from M1-polarized macrophages on DLD-1 colon cancer cells. Our data indicate that the conditioned medium from LPS-activated macrophages (CM-LAM) contains a high level of granulocyte-macrophage colony-stimulating factor, interleukins-1 beta, -6, -8 and tumor necrosis factor-alpha, and that it exerts a marked growth inhibitory activity on DLD-1 cells. Prolonged exposure to CM-LAM results in cell death by apoptosis. Such exposure to CM-LAM leads to the modulation of gal-3 expression: we observed a marked downregulation of gal-3 mRNA and protein expression following CM-LAM treatment. We also describe that the knockdown of gal-3 sensitizes DLD-1 cells to CM-LAM. These data suggest an involvement of gal-3 in the response of colon cancer cells to proinflammatory stimuli, such as the conditioned medium from activated macrophages.

Properties of the protein matrix revealed by the free energy of cavity formation.

The classical picture of the hydrophobic stabilization of proteins invokes a resemblance between the protein interior and nonpolar solvents, but the extent to which this is the case has often been questioned. The protein interior is believed to be at least as tightly packed as organic crystals, and was shown to have very low compressibility. There is also evidence that these properties are not uniform throughout the protein, and conflicting views exist on the nature of sidechain packing and on its influence on the properties of the protein.

Regulation of the alpha-fetoprotein promoter: Ku binding and DNA spatial conformation

This work shows that the proximal promoter of the mouse Afp gene contains a Ku binding site and that Ku binding is associated with down-regulation of the transcriptional activity of the Afp promoter. The Ku binding site is located in a segment able to adopt a peculiar structured form, probably a hairpin structure. Interestingly, the structured form eliminates the binding sites of the positive transcription factor HNF1. Furthermore, a DNAse hypersensitive site is detected in footprinting experiments done with extracts of AFP non-expressing hepatoma cells. These observations suggest that the structured form is stabilised by Ku and is associated with extinction of the gene in AFP non-expressing hepatic cells.

Immediate early gene expression in dog thyrocytes in response to growth, proliferation, and differentiation stimuli.

In dog thyroid cells, insulin or IGF-1 induces cell growth and is required for the mitogenic action of TSH through cyclic AMP, of EGF, and of phorbol esters. HGF per se stimulates cell proliferation and is thus the only full mitogenic agent. TSH and cAMP enhance, whereas EGF phorbol esters and HGF repress differentiation expression. In this study, we have investigated for each factor and regulatory cascade of the intermediate step of immediate early gene induction, that is, c-myc, c-jun, jun D, jun B, c-fos, fos B, fra-1, fra-2, and egr1; fra-1 and fra-2 expressions were very low. TSH or forskolin increased the levels of c-myc, jun B, jun D, c-fos, and fos B while decreasing those of c-jun and egr1. Phorbol myristate ester stimulated the expression of all the genes. EGF and HGF stimulated the expression of all the genes except jun D and for EGF fos B. All these effects were obtained in the presence and in the absence of insulin, which shows that insulin is not necessary for the effects of the mitogens on immediate early gene expression. The definition of the repertoire of early immediate genes inductible by the various growth cascades provides a framework for the analysis of gene expression in tumors. (1) Insulin was able to induce all the protooncogenes investigated except fos B. This suggests that fos B could be the factor missing for insulin to induce mitogenesis. (2) No characteristic pattern of immediate early gene expression has been observed for insulin, which induces cell hypertrophy and is permissive for the action of the other growth factors. These effects are therefore not accounted for by a specific immediate early gene expression. On the other hand, insulin clearly enhances the effects of TSH, phorbol ester, and EGF on c-myc, junB, and c-fos expression. This suggests that the effect of insulin on mitogenesis might result from quantitative differences in the transcription complexes formed. (3) c-myc, c-fos, and jun B mRNA induction by all stimulating agents, whether inducing cell hypertrophy, or growth and dedifferentiation, or growth and differentiation, suggests that, although these expressions are not sufficient, they may be necessary for the various growth responses of thyroid cells. (4) The inhibition of c-jun and egr1 mRNA expression, and the marked induction of jun D mRNA appear to be specific features of the TSH cAMP pathway. They might be related to its differentiating action. (5) fos B, which is induced by TSH, forskolin, phorbol ester, and HGF but not by insulin, could be involved in the mitogenic action of the former factors.

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.

From clonal selection to immune networks: induction of silent idiotypes.

Journal Article

Idiotypic games within the immune network.

In this paper, we have considered the problem of selection of available repertoires. With Ab2 as immunogens, we have used the idiotypic cascade to explore potential repertoires. Our results suggest that potential idiotypic repertoires are more or less the same within a species or between different species. A given idiotype "à la Oudin" can become a recurrent one within the same outbred species or within different species. Similarly, an intrastrain crossreactive idiotype can be induced in other strains, even though there is a genetic disparity between these strains. The structural basis of this phenomenon has been explored. We next examined results showing the loss and gain of recurrent idiotypes without any intentional idiotypic manipulation. A recurrent idiotype can be lost in a syngeneic transfer and a private one can become recurrent by changing the genetic background. The change of available idiotypic repertoires at the B cell level has profound influences on the idiotypic repertoires of suppressor T cells. All these results imply that idiotypic games are played by the immune system itself, a strong suggestion that the immune system is a functional idiotypic network.

Modulation of the complement system in monocytes contributes to tuberculosis-associated immune reconstitution inflammatory syndrome.

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is a common complication in HIV-TB co-infected patients receiving combined antiretroviral therapy (cART). This study investigated a putative contribution of monocytes to the development of TB-IRIS.