No abstract

In vitro binding assay and co-immunoprecipitation experimentWe prepared purified S-tagged recombinant LTA and T7-tagged galectin-2 derived from E. coli using the pET system (Novagen), and combined them. The co-immunoprecipitation experiments were performed using a monoclonal antibody against LTA (R&D Systems) coupled to HiTrapTM NHS-activated Sepharose HP (Amersham). We visualized the immune complex using T7 tag antibody (Stratagene) and horseradish peroxidase (HRP) conjugated with anti-mouse IgG antibody. For coimmunoprecipitation in mammalian cells, we transfected expression plasmids of Flag or S-tagged LTA, galectin-2 and LacZ (as a negative control) into COS7 cells (HSRRB; JCRB9127) or HeLa cells using Fugene. Immunoprecipitations were done in lysis buffer (20 mM Tris pH 7.5, with 150 mM NaCl, 0.1 % Nonident P-40). Twenty-four hours after transfection, cells were lysed, and immunoprecipitations were performed using anti-Flag tag M2 agarose (Sigma). We visualized the immune complex using HRP-conjugated S-protein (Novagen), anti-Flag M2 peroxidase conjugate (Sigma) or mouse monoclonal antibody against human a-tubulin (Molecular Probes) and HRP-conjugated anti-mouse IgG antibody. Confocal microscopyPolyclonal anti-human galectin-2 antisera were raised in rabbits using recombinant protein synthesized in E. coli. The antisera showed no cross-reactivity to structurally related molecules galectin-1 and galectin-3, analysed by western blot. Polyclonal antigalectin-2 antisera and either goat anti-human LTA IgG (R&D Systems) or mouse antihuman a-tubulin monoclonal IgM antibodies were used with Alexa secondary antibodies (Molecular Probes). U937 cells (HSRRB; JCRB9021) were stimulated for 30 min with phorbol myristate acetate (PMA) (20 ng ml 21 ) and fixed. They were subsequently incubated with the corresponding primary antibodies in phosphatebuffered saline containing 3% bovine serum albumin, and the corresponding Alexa secondary antibodies. siRNA and over-expression experimentsThe target sequences for galectin-2 (5 0 -AATCCACCATTGTCTGCAACT-3 0 ) were cloned into pSilencer 2.0-U6 siRNA vector (Ambion). For the over-expression experiment, the galectin-2 was cloned into pFlag-CMV5a vector. After transfection, Jurkat cells were stimulated with PMA (20 ng ml 21 ) for 24 h, and cells and supernatants were collected separately. LTA concentration was measured using an LTA-specific ELISA system (R&D Systems), and normalized by comparison with total protein concentration. The mRNA quantification procedure has been described previously 2 . Luciferase assayA DNA fragment, corresponding to nucleotides 3,188 to 3,404 of intron-1 of LGALS2, was cloned into pGL3-enhancer vector (Promega) in the downstream of SV40 enhancer in the 5 0 to 3 0 orientation. After 24 h transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). ImmunohistochemistryTissue samples were obtained from 16 patients with MI by elective directional coronary atherectomy. Immunohistochemical protocols were carried out a...

A variety of neutral serine proteases are important for the effector functions of immune cells. The neutrophil-derived serine proteases cathepsin G and neutrophil elastase are implicated in the host defense against invading bacterial and fungal pathogens. Likewise, the cytotoxic lymphocyte and NK cell granule-associated granzymes A and B are important for the elimination of virus-infected cells. The activation of many of these serine proteases depends on the N-terminal processing activity of the lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI). Although mice deficient in DPPI have defects in serine protease activation in multiple cellular compartments, the role of DPPI for human serine protease activation is largely undefined. Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive disease associated with loss-of-function mutations in the DPPI gene locus. In this study, we established that the loss of DPPI activity is associated with severe reduction in the activity and stability of neutrophil-derived serine proteases. Surprisingly, patients with PLS retain significant granzyme activities in a cytotoxic lymphocyte compartment (lymphokine-activated killer) and have normal lymphokine-activated killer-mediated cytotoxicity against K562 cells. Neutrophils from patients with PLS do not uniformly have a defect in their ability to kill Staphylococcus aureus and Escherichia coli, suggesting that serine proteases do not represent the major mechanism used by human neutrophils for killing common bacteria. Therefore, this study defines the consequences of DPPI deficiency for the activation of several immune cell serine proteases in humans, and provides a molecular explanation for the lack of a generalized T cell immunodeficiency phenotype in patients with PLS.

Questions of reproducibility and efficacy of histologic malignancy grading relative to alternative proliferation index measurements for outcome prediction remain unanswered. Microsections of specimens from the Cooperative Breast Cancer Tissue Resource (CBCTR) were evaluated by seven pathologists for reproducibility of grade and classification. Nuclear figure classification was assessed using photographs. Grade was assigned by the Bloom-Richardson method, Nottingham modification. Proliferation index was evaluated prospectively by deoxyribose nucleic acid precursor uptake with thymidine (autoradiographic) or bromodeoxyuridine (immunohistochemical) labeling index using fresh tissue from 631 node-negative breast cancer patients accessioned at St Luke's Hospital. A modified Nottingham-Bloom-Richardson grade was derived from histopathologic data. Median post-treatment observation was 6.4 years. Agreement on classification of nuclear figures (N ¼ 43) was less than good by kappa statistic (j ¼ 0.38). Grade was moderately reproducible in four trials (N ¼ 10,10,19, 10) with CBCTR specimens (j ¼ 0.50-0.59). Of components of Bloom-Richardson grade, agreement was least for nuclear pleomorphism (j ¼ 0.37-0.50), best for tubularity (j ¼ 0.57-0.83), and intermediate for mitotic count (j ¼ 0.45-0.64). Bloom-Richardson grade was a univariate predictor of prognosis in node-negative St Luke's patients, and was improved when mitotic count was replaced by labeling index (low, mid, or high). When labeling index was added to a multivariate model containing tumor size and vessel invasion, grade was no longer a significant predictor of tumor-specific relapse-free or overall survival. Mitotic index predicted best when intervals were lowered to 0-2, 3-10, and 410 mitotic figures per ten 0.18 mm 2 highpower fields. We conclude that Nottingham-Bloom-Richardson grades remain only modestly reproducible. Prognostically useful components of grade are mitotic index and tubularity. The Nottingham-BloomRichardson system can be improved by lowering cutoffs for mitotic index and by counting 20-30 rather than 10 high-power fields. Measurement of proliferation index by immunohistochemically detectable markers will probably give superior prognostic results in comparison to grade.

Loss-of-function defects in DNA mismatch repair (MMR), which manifest as high levels of microsatellite instability (MSI), occur in approximately 15% of all colorectal carcinomas (CRCs). This molecular subset of CRC characterizes patients with better stage-specific prognoses who experience no benefit from 5-fluorouracil chemotherapy. Most MMR-deficient (dMMR) CRCs are sporadic, but 15% to 20% are due to inherited predisposition (Lynch syndrome). High penetrance of CRCs in germline MMR gene mutation carriers emphasizes the importance of accurate diagnosis of Lynch syndrome carriers. Family-based (Amsterdam), patient/family-based (Bethesda), morphology-based, microsatellite-based, and IHC-based screening criteria do not individually detect all germline mutation carriers. These limitations support the use of multiple concurrent tests and the screening of all patients with newly diagnosed CRC. This approach is resource intensive but would increase detection of inherited and de novo germline mutations to guide family screening. Although CRC prognosis and prediction of 5-fluorouracil response are similar in both the Lynch and sporadic dMMR subgroups, these subgroups differ significantly with regard to the implications for family members. We recommend that new CRCs should be classified into sporadic MMR-proficient, sporadic dMMR, or Lynch dMMR subgroups. The concurrent use of MSI testing, MMR protein IHC, and BRAF c.1799T>A mutation analysis would detect almost all dMMR CRCs, would classify 94% of all new CRCs into these MMR subgroups, and would guide secondary molecular testing of the remainder.