Intracellular antibodies for proteomics

The intracellular antibody technology has many applications for proteomics studies.

The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology.

We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins.

The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.     

Search for poliovirus long-term excretors among patients affected by agammaglobulinemia

Patients with agammaglobulinemia may excrete enteroviruses, including vaccine-derived poliovirus, for prolonged periods of time. This poses a risk to the patients but it also may pose a risk to the population after eradication of poliovirus and the cessation of routine vaccination. To assess this risk, a pilot study was performed to identify potential poliovirus long-term excretors in a cohort of 38 patients with a definite/presumptive diagnosis of X-linked agammaglobulinemia (XLA). Stool samples were analyzed to detect any polio or other enteroviruses replicating in the gut and neutralizing antibodies against polioviruses were measured in the sera. No viruses were isolated from the stool samples and most sera had neutralizing antibody levels against all three poliovirus serotypes considered by the WHO to be protective in immunocompetent individuals. This suggests that long-term excretion of enteroviruses in patients with agammaglobulinemia is relatively uncommon.     

Analysis of aberrant somatic hypermutation (SHM) in non-Hodgkin's lymphomas of patients with chronic HCV infection

Hepatitis C virus (HCV) and aberrant somatic hypermutation (SHM) have each been suggested to contribute to the development of B-cell non-Hodgkin's lymphoma (NHL). The incidence of PIM-1, PAX-5, RhoH/TTF, and c-MYC mutations in tumour biopsy specimens from 32 HCV-infected B-cell NHL patients was analysed to determine whether the extent of aberrant SHM among these patients differed from that previously reported for HCV-negative B-cell NHL patients. Mutation of PIM-1, PAX-5, RhoH/TTF, and c-MYC was detected in 4 (13%), 5 (16%), 4 (13%), and 4 (13%) of 32 samples, respectively. In HCV-positive B-cell NHL patients, the frequency of aberrant SHM was lower than that already found in HCV-negative B-cell NHL patients. This indicates that, unlike B-cell lymphomas from HCV-negative patients, aberrant SHM may not contribute significantly to malignant transformation in HCV-associated B-cell lymphomas.     

KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes

Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP-dependent potassium (K(ATP)) channel of the pancreatic beta-cell, were found in patients with PNDM. Closure of the K(ATP) channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non-autoimmune, insulin-requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK-PDMI, KCNJ11-PDMI) to avoid confusion between patients with early-onset, autoimmune type 1 diabetes.     

Diversity of genomic breakpoints in TFG-ALK translocations in anaplastic large cell lymphomas: identification of a new TFG-ALK(XL) chimeric gene with transforming activity

Anaplastic large cell lymphomas are associated with chromosomal aberrations involving the anaplastic lymphoma kinase (ALK) gene at 2p23 that result in the expression of novel chimeric ALK proteins with transforming properties. In most of these tumors, the t(2;5)(p23;q35) generates the NPM-ALK fusion gene. However, several studies have now demonstrated that genes other than NPM may be fused to the ALK gene. We have recently described two different ALK rearrangements involving the TRK-fused gene (TFG) in which the same portion of ALK was fused to different length fragments of the 5' TFG region. These two rearrangements encoded chimeric proteins of 85 kd (TFG-ALK(S)) and 97 kd (TFG-ALK(L)), respectively. In this study, we have identified a new ALK rearrangement in which the catalytic domain of ALK was fused to a larger fragment of the TFG gene (TFG-ALK(XL)), encoding for a fusion protein of 113 kd. Genomic analysis of these three TFG-ALK rearrangements revealed that the TFG breakpoints occur at introns 3, 4, and 5, respectively, whereas the ALK breakpoints always occur in the same intron. No homologous regions or known recombination sequences were found in these regions. Transfection experiments using NIH-3T3 fibroblasts showed a similar transforming efficiency of TFG-ALK variants compared with NPM-ALK. In addition, in common with NPM-ALK, the TFG-ALK proteins formed stable complexes with the signaling proteins Grb2, Shc, and PLC-gamma. In conclusion, these findings indicate that the TFG may use a variety of intronic breakpoints in ALK rearrangements generating fusion proteins of different molecular weights, but with similar transforming potential than NPM-ALK.     

Co-expression of Epstein-Barr virus latent membrane protein and vimentin in “aggressive” histological subtypes of Hodgkin's disease

Summary The presence of Epstein-Barr virus (EBV) genome in Hodgkin's and Reed-Sternberg (HRS) cells, as detected using in situ hybridization (ISH) with biotinylatedBamHI V probes, along with the expression of EBV-encoded latent membrane protein (LMP) and vimentin was examined in paraffin-embedded sections of 39 immunomorphologically characterized cases of Hodgkin's disease (HD). ISH demonstrated EBV in HRS cells in 15 of 39 cases, whereas LMP expression was detected in 11 of 39 cases, only in the presence of EBV genome detection. With the exception of 1 case, in which HRS cells expressed B-cell-associated antigens, the LMP-positive cases included specimens in which HRS cells were of non-B, non-T phenotype. LMP expression showed a stronger association with lymphocyte depletion (LD) (3/3) and mixed cellularity (MC) (6/11) than with lymphocyte predominance (0/5) or nodular sclerosis (2/20) subtypes. Vimentin expression on HRS cells was found in all the LMP-expressing cases and only in a fraction (13/28) of LMP-negative cases. This study supports the view that HD represents a heterogeneous group of diseases also in terms of EBV association, LMP expression being strongly related to the aggressive LD and MC histological subtypes. In light of the supposed interactions between vimentin and LMP, their coexpression on HRS cells, as detected in this study, provides further evidence for a significant role of EBV in the development of a proportion of HD cases.     

Cystic Medullary Thyroid Carcinoma: Report of a Case with Morphological and Clinical Correlations

Cystic lesions of the thyroid are common findings. Although many thyroid cysts are of benign, some cases of hemorrhagic degenerative changes occur in neoplastic nodules, mostly follicular neoplasms and papillary carcinomas. The occurrence of hemorrhagic changes in medullary carcinomas has never been documented with aspirative cytological and histological pictures to the best of our knowledge. A case of medullary thyroid carcinoma with a large central hemorrhagic cyst is described, and the literature regarding the pathogenesis of this regression and the occurrence of cystic neoplasms in the thyroid is reviewed.     

ALK Expression Defines a Distinct Group of T/Null Lymphomas (“ALK Lymphomas”) with a Wide Morphological Spectrum

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffin sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma “common” type (75%), “lymphohistiocytic” (10%), “small cell” (8.3%), “giant cell” (3.3%), and “Hodgkin’s like” (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin’s-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and “Hodgkin’s-like” features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype (“ALK lymphomas”), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.