CTLA4Fcε, a novel soluble fusion protein that binds B7 molecules and the IgE receptors, and reduces human in vitro soluble CD23 production and lymphocyte proliferation.

Immunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fcε, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fcε appeared as a 70,000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fcε binds to IgE receptors FcεRI and FcεRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fcε to FcεRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fcε to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fcε - but not IgE - induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fcε caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.

Fluorescent in situ hybridization heating pretreatment: the key is temperature control.

Fluorescent in situ hybridization (FISH) is a very useful tool for diagnostic and prognostic purposes in pathology. However, many laboratories still experience troubles when applying FISH to paraffin material. To overcome these difficulties, different pretreatments which include enzymatic digestion have been described. Usually, previous to digestion, a heating step is performed. The aim of this study was to compare the efficiency of the heating step with different buffers and different heating methods. We conclude that the main factor in the heating pretreatment is the temperature control, irrespective of the buffer used. Best results are obtained with any buffer by heating the slides to 99°C for 15 min followed by 10 min at room temperature.

Global site-specific neddylation profiling reveals that NEDDylated cofilin regulates actin dynamics.

Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.

Publisher Correction: Global site-specific neddylation profiling reveals that NEDDylated cofilin regulates actin dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Thyroid Papillary Microtumor: Validation of the (Updated) Porto Proposal Assessing Sex Hormone Receptor Expression and Mutational BRAF Gene Status.

Given the high incidence and excellent prognosis of many papillary thyroid microcarcinomas, the Porto proposal uses the designation papillary microtumor (PMT) for papillary microcarcinomas (PMCs) without risk factors to minimize overtreatment and patients' stress. To validate Porto proposal criteria, we examined a series of 190 PMC series, also studying sex hormone receptors and BRAF mutation. Our updated Porto proposal (uPp) reclassifies as PMT incidental PMCs found at thyroidectomy lacking the following criteria: (a) detected under the age of 19 years; (b) with multiple tumors measuring >1 cm adding up all diameters; and (c) with aggressive morphologic features (extrathyroidal extension, angioinvasion, tall, and/or hobnail cells). PMCs not fulfilling uPp criteria were considered "true" PMCs. A total of 102 PMCs were subclassified as PMT, 88 as PMC, with no age or sex differences between subgroups. Total thyroidectomy and iodine-131 therapy were significantly more common in PMC. After a median follow-up of 9.6 years, lymph node metastases, distant metastases, and mortality were only found in the PMC subgroup. No subgroup differences were found in calcifications or desmoplasia. Expression of estrogen receptor-α and estrogen receptor-ß, progesterone receptor, and androgen receptor was higher in PMC than in nontumorous thyroid tissue. BRAF mutations were detected in 44.7% of PMC, with no differences between subgroups. In surgical specimens, the uPp is a safe pathology tool to identify those PMC with extremely low malignant potential. This terminology could reduce psychological stress associated with cancer diagnosis, avoid overtreatment, and be incorporated into daily pathologic practice.

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.

Fluorescence in situ hybridization analysis of the ALK gene in 2,045 non-small cell lung cancer patients from North-Western Spain (Galicia).

Identification of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements is a standard diagnostic test in patients with advanced non-small cell lung cancer (NSCLC). The current study describes the experience of ALK rearrangement detection of a referral center in the public health care system of Galicia in North-Western Spain. The fluorescence in situ hybridization (FISH) patterns of the ALK gene and the clinical and pathological features of these patients are reported. This study is also of interest for comparative purposes due to the relative geographical isolation of the area, which could have contributed to particular genetic features. A total of 2,045 tissue samples from NSCLC patients were collected between October 2010 and July 2015 and tested for ALK rearrangements by FISH. Examination of 1,686 paraffin-embedded tissue specimens and 395 cytological samples (306 cell block preparations and 53 cytological smears) was conducted, and any associations between the FISH results and clinicopathological features were assessed. The rate of successful evaluation was marginally higher in tissue samples than in cytological samples (92.9% vs. 84.1%); this difference was not significant. ALK rearrangements were identified in 82 patients(4%): 65 (79.3%) in tissue specimens, 15 (18.3%) in cell block samples and 2 (2.4%) in cytological smears. This genetic translocation appeared to be associated with a non-smoking history, younger age, female gender, stage IV and adenocarcinoma histological type. The findings demonstrate that ALK evaluation by FISH is feasible in tissue and cytological samples. The clinical and pathological features of the ALK-positive series of patients are similar to those previously reported in the literature.

BRAF mutation in solid cell nest hyperplasia associated with papillary thyroid carcinoma. A precursor lesion?

We describe a case of solid cell nest hyperplasia associated with papillary thyroid carcinoma in a 48-year-old man with goiter. The entire gland was examined; in 1 section, the cells of 1 solid cell nest were in close contact with a follicular variant of papillary microcarcinoma. A second follicular variant of papillary microcarcinoma, 1 follicular adenoma, hyperplastic nodules, and some lymphoid aggregates were also found. Scattered p63-positive cells were found in the second papillary microcarcinoma. After microdissection, the same BRAF(V600E) mutation was found both in a pool of 5 solid cell nests and in the adjacent papillary microcarcinoma. BRAF(V600E) mutation and the previously unreported BRAF(G593D) mutation along with p.G606G silent change were found in the second papillary microcarcinoma, but no mutations were detected in the follicular adenoma or in the 2 other pools of solid cell nests screened for BRAF gene mutations. These findings support a histogenetic link between the main cells of solid cell nests and papillary thyroid carcinoma, and suggest solid cell nest hyperplasia as a precursor lesion of papillary thyroid carcinoma.