Increased level of DNA damage in some organs of obese Zucker rats by γ‐H2AX analysis

In a recent study, we showed that lymphocytes of obese Italian children/adolescents displayed levels of double strand breaks (DSB), assayed as serine 139‐phosphorylated histone H2AX (γ‐H2AX), about eightfold higher than normal weight controls, and that 30% of this damage‐generated micronuclei. These findings suggested that obese children could be at increased risk of obesity‐mediated cancer later in life. We therefore aimed to assess the level of γ‐H2AX in a genetic animal model of obesity (Zucker rat) to identify a genotoxic/carcinogenic risk in some organs. The DSB marker was studied in 3‐ to 4‐week‐old rats and in 9‐ to 13‐week‐old rats. Paraffin‐embedded sections of heart, thyroid, liver, pancreas, lung, kidney, esophagus, and gut from the fa?/fa? (obese) and the fa+/fa? (lean) control animals were processed for immunohistochemistry detection of γ‐H2AX. Pancreas (0.0624 ± 0.0195), lung (0.1197 ± 0.0217), esophagus (0.1230 ± 0.0351), kidney (0.1546 ± 0.0149), and gut (0.1724 ± 0.0352) of 9‐ to 13‐week‐old obese rats showed a higher proportion of γ‐H2AX‐positive nuclei, than their lean counterparts (0.0092 ± 0.0033, 0.0416 ± 0.0185, 0.0368 ± 0.0088, 0.0686 ± 0.0318, and 0.0703 ± 0.0239, respectively). No difference was seen in the 3‐ to 4‐week‐old age group with regard to obesity, indicating that the DNA damage increased with older age of the rats. We hypothesize that the organs of the obese animals showing high levels of DSB could represent target tissues for the development of obesity‐related cancers. Environ. Mol. Mutagen. 58:477–484, 2017. © 2017 Wiley Periodicals, Inc.     

Comparison of PAX-2, RCC antigen, and antiphosphorylated H2AX antibody (gamma-H2AX) in diagnosing metastatic renal cell carcinoma by fine-needle aspiration

Diagnosing metastatic renal cell carcinoma (RCC) by fine-needle aspiration (FNA) can be challenging. Existing antibodies supporting a diagnosis of RCC, including CD10 and RCC-Ma, have problems with specificity and interpretation. In this report, we evaluate the use of two newer immunostains, PAX-2 and gamma-H2AX, which to our knowledge have not been studied in FNA material, in the diagnosis of metastatic RCC and in comparison with RCC-Ma. 29 cases of metastatic RCC were identified as well as a TMA of an additional 30 RCC cases. In the case cohort, RCC-Ma in a membranous pattern of staining identified 15/27 (56%) metastatic RCC, although interpretation was made difficult in many cases due to focality of staining and non-specific cytoplasmic staining. PAX-2 stained 23/29 (79%) of tumors in a nuclear stain, most strongly. Gamma-H2AX stained 19/26 (73%) of metastatic RCC strongly in a nuclear stain. In the TMA, strong, diffuse nuclear staining with gamma-H2AX was present in 22/30 RCC (73%). If weak staining was also included as positive, 26/30 (87%) were positive. PAX-2 stained RCC TMA with a lower percentage at 56%, including weaker staining intensity. Both PAX-2 and gamma-H2AX demonstrated patchy staining of normal renal tubules, PAX-2 to a greater extent. Both PAX-2 and gamma-H2AX are sensitive markers for the diagnosis of metastatic RCC, with improved ease of interpretation when compared with RCC-Ma. A combination of all 3 markers identified 87% of cases, and failure to stain for both PAX-2 and gamma-H2AX suggests against, but does not disprove, a diagnosis of RCC.     

γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9

Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry‐based assay (MultiFlow? DNA Damage Kit—p53, γH2AX, Phospho‐Histone H3) can distinguish direct‐acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. [ 2016 ]: Environ Mol Mutagen 57:171‐189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk‐away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen. 57:546–558, 2016. © 2016 Wiley Periodicals, Inc.     

α‐Methylacyl‐CoA racemase (AMACR) in fine‐needle aspiration specimens of prostate lesions

The elevated expression of P504S gene and its product α‐methylacyl‐CoA racemase (AMACR) can serve as a molecular marker for prostate cancer. The goal of this study is to investigate P504S/AMACR expression in fine‐needle aspiration smears and correlate it with cytological diagnosis. Immunocytochemistry was performed in 35 patients with morphological diagnosis of prostate carcinoma (n = 16), atypia (n = 15), and benign hyperplasia (n = 4). Among 16 malignant cases there were two low‐grade, eight intermediate, and six high‐grade prostate carcinomas. Cytoplasmic positivity is analyzed qualitatively as predominantly diffuse or focal and quantitatively as <5%, 5–50%, and >50% of cells. Benign cases showed no P504S/AMACR expression. Positive staining was recorded in 75% of malignant cases, but in the majority of them it was weak and focal or diffuse and in a small amount of cells. The most intensive staining was seen in low‐grade carcinomas and some atypical cases. This observation indicates a correlation between P504S/AMACR expression and differentiation of cells. P504S/AMACR staining might be of great value in cytodiagnosis of prostate lesions as well as an example of the characterization of cells at the molecular level using fresh tissue obtained by fine‐needle aspiration. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

β‐catenin mutation in ovarian solid pseudopapillary neoplasm

Primaly solid pseudopapillary neoplasm (SPN) of the ovary is a rare tumor; recently 6 cases have been reported. Its pathogenesis, however, remains largely unclear. We report an additional case of primary ovarian SPN of an 18‐year‐old girl. The aim of this study is to define the difference between pancreatic and ovarian SPN by histological and molecular examination. Microscopically the tumor predominantly showed a solid pattern and focally a pseudopapillary pattern. The tumor cells showed two patterns of abundant eosinophilic cytoplasm and intracytoplasmic vacuoles. Immunohistochemistry of the tumor was positive for β‐catenin (nuclear and cytoplasmic reactivity), α1‐antitrypsin, vimentin, CD56, synaptophysin (focal weak), CD10. Mutation analyses revealed a point mutation, c.110C >T, in exon 3 of the the β‐catenin gene (CTNNB1), which causes the replacement of serine with phenylalanine at codon 37. A Ser37 point mutation is known to be one of the oncogenic somatic mutations in pancreatic SPN and the major oncogenic β‐catenin mutation. Ovarian SPN of our case was similar to pancreatic SPN histologicaly and had the same genomic characteristics. We expected that both ovarian and pancreatic SPNs shared the same oncogenesis related to Wnt/β‐catenin pathway for tumorgenesis.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Hepatoblastoma—An attempt of histological subtyping on fine‐needle aspiration material

Hepatoblastoma (HB) is classified into epithelial, mixed (epithelial/mesenchymal), and small‐cell (anaplastic) type. Wnt/β‐catenin pathway plays a key role in hepatic development, regeneration, and tumorigenesis, and HB is known to present β‐catenin mutations (50–90%). The present study was undertaken to delineate the cytomorphologic features of HB and to evaluate the feasibility of subtyping of HB on fine‐needle aspiration cytology (FNAC). The expression of β‐catenin in these tumors was also evaluated both of histopathologic sections and on the aspirated material. Thirty‐three cases with fine‐needle aspirates of HB were retrieved over a period of 12 years. Cytologic diagnosis was reviewed in the light of clinicoradiological data, response to therapy, and subsequent histopathology. Immunochemistry for β‐catenin was performed in 19 of 33 cases on histopathologic sections (n = 10)/cell blocks (n = 6)/cytosmears (n = 3). Based on the cytologic features, the cases were divided into fetal HB (n = 17), embryonal HB (n = 4), combined epithelial HB (n = 8), and mixed HB (n = 4). Four cases of histopathologically proven mixed HB were reported as pure epithelial HB on FNAC, as mesenchymal elements were not represented in the cytology smears. Cytoplasmic as well as nuclear staining for β‐catenin was noted in a total of 10 of 19 cases. FNAC can accurately categorize epithelial HB; however, in mixed type, the accuracy depends on number of areas sampled. Cell block can be of help to perform ancillary investigations especially β‐catenin for both diagnostic and therapeutic purposes. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

KRAS mutation analysis on cytological specimens of metastatic colo‐rectal cancer

Recent evidences showed that metastatic colorectal cancer (CRC) patients with tumors harboring a KRAS gene mutation do not derive benefit from the administration of epidermal growth factor receptor‐directed monoclonal antibodies. Typically, the specimens available for KRAS mutational analysis are formalin‐fixed paraffin‐embedded (FFPE) primary tumor tissue blocks. However, in patients with rectal tumours undergoing neoadjuvant therapy, the source of FFPE material is limited. In this setting, CRC cytological samples taken from the metastatic site may be exploited. However, these specimens show at least some degree of necrosis; thus, their suitability for the KRAS assay needs to be tested. Here, we show that 18/19 (94.7%) metastatic CRC smears were perfectly adequate for codon 12 and 13 KRAS mutational analysis by direct gene sequencing. Only one case (5.3%) showing abundant necrotic debris and poor cellular preservation was not informative for KRAS status. Codon 12 gene mutations were found in 4/18 (22.2%) of the adequate cases (c35G>T n = 2; c34G>T n = 1; c35G>A n = 1). Concordance between cytological and FFPE samples, both available in 13 patients, occurred in 92.3% (12/13) of the cases. Thus, whenever histological specimens of CRC are notavailable, KRAS testing may be reliably performed on cytological specimens. Diagn. Cytopathol. 2010;38:869–873. © 2010 Wiley‐Liss, Inc.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

E-cadherin and α-, β-, and γ-catenin protein expression in relation to metastasis in human breast carcinoma

In the metastatic process, various cell–cell adhesion molecules seem to play an important role. E-cadherin, a transmembrane protein with an extracellular and an intracellular domain, is one of the key players involved in cell–cell adhesion. The function of E-cadherin in preventing metastasis in tumour development is believed to be dependent on intracellular catenins. In a previous study, the expression of E-cadherin was examined in a series of human breast carcinomas. In that study, down-regulation of E-cadherin failed to correlate with lymph node and/or distant metastasis. In the present study, the expression of α-, β-, and γ-catenins has been examined in a subset of the same tumours in order to evaluate their possible role in breast cancer metastasis. Tumour tissues from 90 primary breast carcinomas were immunostained for α-, β-, and γ-catenins. Reduced or absent immunoreactivity in the tumour tissue was seen in 63 (70·0 per cent) for α-catenin, in 50 (55·6 per cent) for β-catenin, and in 50 (55·6 per cent) for γ-catenin. Reduced expression of each of the catenins alone failed to correlate to metastasis. However, when all of the four proteins (E-cadherin, α-catenin, β-catenin, and γ-catenin) were analysed as one group, a significant association was seen between reduction in immunoreactivity of at least one of these four proteins and the presence of metastases. These results indicate that if one of these proteins is down-regulated, the function of the others in suppressing metastasis is altered. A significant association was seen between lobular invasive tumours and β-catenin expression. © 1998 John Wiley & Sons, Ltd.     

Synthesis and Characterization of Polyrotaxanes Comprising γ‐CDs and Distal Azide‐Terminated PHEMA Using Propargylamine Monosubstituted β‐CDs as End Stoppers

To highlight whether γ‐cyclodextrins (CDs) facilitate propargylamine monosubstituted β‐CDs (PA‐β‐CDs) as end stoppers to get threaded onto a distal azide terminated poly(2‐hydroxyethylmethacrylate) (PHEMA) homopolymer (PH‐46‐2N₃) to create linear and hyperbranched polyrotaxanes (PRs) via the in situ copper‐catalyzed azide/alkyne cycloaddition (CuAAC), PH‐46‐2N₃ is self‐assembled with a varying amount of γ‐CDs in water and then subjected the CuAAC with PA‐β‐CDs to end‐cap the resulting γ‐CD‐PHEMA polypseudorotaxanes (PPRs) into the γ‐CD‐PHEMA PRs. It demonstrates that γ‐CDs cannot promote PA‐β‐CDs to be entrapped on the PHEMA chain most likely due to their different cavity size and molecular framework and linear PRs are always formed with up to 29% γ‐CD coverage ratio along the PHEMA axis thereof.     

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell‐contact‐dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4⁺CD25⁺ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.