[Serrated polyps of the duodenum. Three cases with immunohistological and molecular pathological findings]. / Serratierte Polypen des Duodenums. Drei Fälle mit immunhistologischen und molekularpathologischen Befunden.

We report on three cases of serrated polyps of the duodenum which were incidental endoscopic findings in three male patients with a median age of 70 years (range 63-84 years). Architecturally the histological findings in cases 1 and 2 were similar to hyperplastic polyps of the colon. In case 3 there was a low grade intraepithelial neoplasia which covered the whole polyp. This polyp relapsed after 2 years with similar histological findings. Immunohistochemically an increased proliferative activity was found in case 3 as well as associated overexpression of p16 (INK4a) and p53. No abnormal expression of MLH1 and ß-catenin was found in any of the polyps. Molecular pathological analysis showed a BRAF mutation (V600E) in case 3. A wild type sequence in the KRAS gene was found in all polyps. In conclusion, serrated polyps should be included in the diagnostic spectrum of benign duodenal polyps.

Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase.

The molecular mechanism of action of macrophage migration inhibitory factor (MIF), a cytokine with a critical role in the immune and inflammatory response, has not yet been identified. Here we report that MIF can function as an enzyme exhibiting thiol-protein oxidoreductase activity. Using a decapeptide fragment of MIF (MF1) spanning the conserved cysteine sequence motif Cys57-Ala-Leu-Cys60 (CALC), Cys-->Ser mutants (C57S MIF, C60S MIF, and C57S/C60S MIF) of human MIF (wtMIF), and alkylated wtMIF, we show that this activity is mediated by the CALC region and is important for the macrophage-activating properties of MIF. Both wtMIF and MF1 were demonstrated to form an intramolecular disulfide bridge. Using two common oxidoreductase assays, MIF was shown to enzymatically catalyze the reduction of insulin and 2-hydroxyethyldisulfide (HED). Examination of wtMIF and the mutants by far-UV circular dichroism spectroscopy (CD) together with denaturation studies showed that substituting or reducing the cysteine residues of CALC led to a reduced conformational stability of MIF but did not significantly change its overall conformation. A functional role for the CALC region was revealed by subjecting the mutants and alkylated wtMIF to the enzymatic assays. Mutant C60S did not have any enzymatic activity while mutant C57S had a reduced activity. Thiol-modified wtMIF that was alkylated under oxidizing conditions was found to have full enzymatic activity, whereas alkylation of wtMIF under reducing conditions completely eliminated MIF-mediated redox activity. Importantly, further physiological relevance of the disulfide motif was obtained by examining the mutants and alkylated MIF in an immunological assay that involved the macrophage-activating properties of MIF. In this test, mutant C60S was essentially inactive and mutant C57S was partly active, indicating together that at least some of the cytokine-like biological activities of MIF are dependent on the presence of cysteine 57 and 60. Again, use of the alkylated MIF species confirmed the role of the cysteine motif for this MIF activity. In conclusion, our results argue (a) that MIF exhibits enzymatic oxidoreductase activity, (b) that this activity is dependent on the presence of the catalytic center that is formed by cysteine residues 57 and 60, and (c) that certain MIF-mediated immune processes are due to the cysteine-mediated redox mechanism.

Structure activity studies of the cytokine macrophage migration inhibitory factor (MIF) reveal a critical role for its carboxy terminus.

Carboxy-truncated mutants of human MIF (MIF(1-104) and MIF(1-109)) were used in structure activity studies. CD spectroscopy revealed an overall structural similarity between the mutants and MIF. Denaturant-induced unfolding demonstrated that the C-terminus contributed significantly to the conformational stability of MIF. This appears to be due to the formation of two C-terminal beta-strands. The mutants were enzymatically active, exhibiting half of the enzymatic redox activity of MIF. However, immunological analysis showed that deletion of both 5 and 10 C-terminal residues resulted in loss of the macrophage activating properties of MIF, providing functional evidence that the C-terminus is important for immunological activity and trimer formation. A more detailed study of the C-terminus may assist in identifying the molecular basis for the immunological and enzymatic activities of MIF.

[Retrospective analysis of colorectal carcinomas reveals potential for improvement of diagnostic quality]. / [Indentifikation von Verbessurngspotenzial in der Diagnostik kolorecktaler karzinome mittels retrospektiver Analyse]

INTRODUCTION: The histopathological work-up of colorectal cancer is of critical importance for prognosis and therapy. Therefore we retrospectively analyzed all colorectal carcinomas diagnosed at our institution from January 2002 through March 2003. METHODS: 735 of a total of 777 carcinomas could be followed up. These tumors comprised 74 polypectomy specimens, 32 transanal microscopic surgery specimens, 322 surgical resection specimens analyzed at our own institution (subgroup A) and 263 surgical resection specimens that had been worked up at other pathological institutions (subgroup B) after the diagnosis of carcinoma had been established in our own institution in biopsies. 44 tumors were not treated locally until the end of the study. Quantitative quality parameters were analyzed statistically using Fisher's exact test. RESULTS: Between subgroups A and B significant differences were found within 1. the proportion of specimens with less than 12 lymph nodes examined (11 vs. 25 %), 2. for the detection of an R1 situation despite macroscopic R0 resection (9 vs. 1 %), 3. the proportion of early colonic cancers (9 vs. 4 %), 4. of mucinous adenocarcinomas (19 vs. 4 %), 5. of lymphatic (48 vs. 23 %) and venous invasion (25 vs. 8 %), 6. the frequency of a macroscopic description of mesorectum quality (85 vs. 14 %). In 7 of 32 surgically treated patients with a high-risk carcinoma originally found within a polypectomy specimen, residual tumor was detected in the surgical resection specimen (at the polypectomy site and/or in regional lymph nodes). CONCLUSION: Due to retrospective analysis of colorectal cancer specimens we were able to identify potential for improvement of diagnostic quality. Furthermore our data underline the importance of an oncological resection, if a high risk carcinoma has been detected within a polypectomy specimen.

Migration inhibitory factor induces killing of Leishmania major by macrophages: dependence on reactive nitrogen intermediates and endogenous TNF-alpha.

Macrophage migration inhibitory factor (MIF) is a product of activated T cells, anterior pituitary cells, and macrophages. MIF plays an important role in LPS-induced shock and delayed-type hypersensitivity. Furthermore, MIF exhibits a proinflammatory spectrum of action, promoting TNF-alpha production by macrophages, and counter-regulates glucocorticoid suppression of cytokine production. Here, we report that purified recombinant MIF activates murine macrophages to kill Leishmania major, with maximal effects at concentrations above 1 microg/ml. This MIF-mediated activation is specific, since it can be blocked completely by anti-MIF mAb. The MIF-mediated activation is dependent on TNF-alpha produced endogenously by macrophages, because the administration of anti-TNF-alpha antiserum markedly reduced the MIF effect. No MIF-mediated activation was observed in macrophages derived from TNF receptor p55 knockout mice, thus demonstrating the requirement of the smaller TNF receptor molecule for autocrine TNF-alpha signaling. A highly specific inhibitor of the inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl)lysine, dihydrochloride, also inhibited the action of MIF, suggesting an important role for iNOS in the antiparasitic properties of MIF. In line with this, no MIF-mediated activation was detected analyzing macrophages derived from iNOS-deficient mice. The effect of MIF was blocked completely by the macrophage-deactivating cytokines IL-10, IL-13, and TGF-beta. Finally, the expression of MIF mRNA and protein was up-regulated in lymph nodes of mice during the first week after infection with L. major. MIF therefore represents a cytokine involved not only in the recruitment of proinflammatory cells during infection but also in the complex regulation of the antimicrobial activity of these cells.

Specific antagonism of type I IL-4 receptor with a mutated form of murine IL-4.

IL-4 is a pleiotropic cytokine that is essential for the differentiation of Th2 cells and is critically involved in the pathogenesis of certain infectious and allergic diseases. We have produced and functionally characterized a mutant of murine IL-4 (IL-4.Y119D) as a potential antagonist of IL-4. The analysis of IL-4R binding revealed no differences between wild-type and mutated IL-4. Despite this finding, IL-4.Y119D was unable to induce proliferation of several IL-4-responsive T cell lines mediated via the type I IL-4R (IL-4Ralpha/common gamma chain (gamma c chain)) and specifically inhibited the proliferative effect of wild-type IL-4. In contrast, with IL-4.Y119D we found induction of MHC class II and CD23 molecules on resting splenic B cells as well as proliferation of B9 plasmocytoma cells. In addition, IL-4.Y119D induced mRNA for soluble IL-4R, leading to the release of soluble IL-4R protein by spleen cells. In macrophages, mutated IL-4 in combination with IFN-gamma induced TNF-alpha-dependent killing of Leishmania major parasites such as wild-type IL-4. The agonistic effects of IL-4.Y119D were observed on cells expressing the IL-13R alpha-chain, including an IL-13R alpha-chain transfected T cell line, but were absent in T cells that lack this molecule, indicating that IL-4.Y119D conveys its activity via the type II IL-4R (IL-4Ralpha/IL-13Ralpha). The described IL-4 mutant, therefore, represents a new tool to use in dissecting different IL-4 functions that are mediated by either type I or type II IL-4R complexes.