Phenotyping, functional characterization, and developmental changes in canine intestinal intraepithelial lymphocytes

Little is currently known about the lymphocyte populations in the normal and diseased canine gut. The aim of this study was thus the phenotypical and functional characterization of canine intestinal intraepithelial lymphocytes (IEL). IEL were isolated from full-thickness biopsies of 15 adult Swiss Beagle dogs (mean age 8.2 +/-2.8 years) and compared to mesenteric lymph node cells. The phenotypical characterization by multi-parameter flow cytometry revealed that canine IEL differ substantially from lymph node T cells, and consist of various unconventional lymphocyte subsets, unique to mucosal surfaces. These include gammasigma T cells, and CD4(-)CD8(-) and CD8alphaalpha(+) T cells. IEL populations in adult dogs were also compared to those isolated from neonatal Beagle dogs. Analysis revealed a high frequency of undifferentiated CD4(-)CD8(-) T cells in newborn dogs whereas mature CD4(+) and CD8(+) T cells predominate in adult dogs, indicating maturation of the intestinal immune system during development. As IEL in other species are thought to exhibit regulatory functions, we investigated the role of IEL on the activation-induced proliferation of lymph node T cells. While IEL alone did not show activation-induced proliferation, they significantly inhibited the proliferation of activated lymph node T cells in a cell number-dependent manner. These findings are the first to demonstrate that canine intestinal IEL have an immunoregulatory phenotype, which may contribute to the maintenance of intestinal immune homeostasis and may, therefore, be lost in canine chronic enteropathies.

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8alphaalpha TCRalphabeta IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required. Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (>85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Prediction of outcome in patients with low-grade squamous intraepithelial lesions by fluorescence in situ hybridization analysis of human papillomavirus, TERC, and MYC

BACKGROUND Cytology is an excellent method with which to diagnose preinvasive lesions of the uterine cervix, but it suffers from limited specificity for clinically significant lesions. Supplementary methods might predict the natural course of the detected lesions. The objective of the current study was to test whether a multicolor fluorescence in situ hybridization (FISH) assay might help to stratify abnormal results of Papanicolaou tests. METHODS A total of 219 liquid-based cytology specimens of low-grade squamous intraepithelial lesions (LSIL), 49 atypical squamous cells of undetermined significance (ASCUS) specimens, 52 high-grade squamous intraepithelial lesion (HSIL) specimens, and 50 normal samples were assessed by FISH with probes for the human papillomavirus (HPV), MYC, and telomerase RNA component (TERC). Subtyping of HPV by polymerase chain reaction (PCR) was performed in a subset of cases (n=206). RESULTS There was a significant correlation found between HPV detection by FISH and PCR (P<.0001). In patients with LSILs, the presence of HPV detected by FISH was significantly associated with disease progression (P<.0001). An increased MYC and/or TERC gene copy number (>2 signals in>10% of cells) prevailed in 43% of ASCUS specimens and was more frequent in HSIL (85%) than in LSIL (33%) (HSIL vs LSIL: P<.0001). Increased TERC gene copy number was significantly correlated with progression of LSIL (P<.01; odds ratio, 7.44; area under the receiver operating characteristic curve, 0.73; positive predictive value, 0.30; negative predictive value, 0.94) CONCLUSIONS: The detection of HPV by FISH analysis is feasible in liquid-based cytology and is significantly correlated with HPV analysis by PCR. The analysis of TERC gene copy number may be useful for risk stratification in patients with LSIL.

Morphoproteomic evidence of constitutively activated and overexpressed mTOR pathway in cervical squamous carcinoma and high grade squamous intraepithelial lesions.

Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.

Hyperplasia to neoplasia sequence of duodenal and pancreatic neuroendocrine diseases and pseudohyperplasia of the PP-cells in the pancreas.

Hyperplastic changes of the neuroendocrine cell system may have the potential to evolve into neoplastic diseases. This is particularly the case in the setting of genetically determined and hereditary neuroendocrine tumor syndromes such as MEN1. The review discusses the MEN1-associated hyperplasia-neoplasia sequence in the development of gastrinomas in the duodenum and glucagon-producing tumors in the pancreas. It also presents other newly described diseases (e.g., glucagon cell adenomatosis and insulinomatosis) in which the tumors are (or most likely) also preceded by islet cell hyperplasia. Finally, the pseudohyperplasia of PP-rich islets in the pancreatic head is defined as a physiologic condition clearly differing from other hyperplastic-neoplastic neuroendocrine diseases.

Immunodeficiency and the risk of cervical intra-epithelial neoplasia 2/3 and cervical cancer: A nested case-control study in the Swiss HIV Cohort Study.

HIV-infected women are at increased risk of cervical intra-epithelial neoplasia (CIN) and invasive cervical cancer (ICC), but it has been difficult to disentangle the influences of heavy exposure to HPV infection, inadequate screening, and immunodeficiency. A case-control study including 364 CIN2/3 and 20 ICC cases matched to 1,147 controls was nested in the Swiss HIV Cohort Study (1985-2013). CIN2/3 risk was significantly associated with low CD4+ cell counts, whether measured as nadir (odds ratio (OR) per 100-cell/μL decrease=1.15, 95% CI: 1.08, 1.22), or at CIN2/3 diagnosis (1.10, 95% CI: 1.04, 1.16). An association was evident even for nadir CD4+ 200-349 versus ≥350 cells/μL (OR=1.57, 95% CI: 1.09, 2.25). After adjustment for nadir CD4+, a protective effect of >2-year cART use was seen against CIN2/3 (OR versus never cART use=0.64, 95% CI: 0.42, 0.98). Despite low study power, similar associations were seen for ICC, notably with nadir CD4+ (OR for 50 versus >350 cells/μL= 11.10, 95% CI: 1.24, 100). HPV16-L1 antibodies were significantly associated with CIN2/3, but HPV16-E6 antibodies were nearly exclusively detected in ICC. In conclusion, worsening immunodeficiency, even at only moderately decreased CD4+ cell counts (200-349 CD4+ cells/μL), is a significant risk factor for CIN2/3 and cervical cancer. This article is protected by copyright. All rights reserved.

Molecular markers of human papillomavirus (HPV) infection and epidemiologic risk factors for squamous intraepithelial lesions (SIL) of the cervix

Infection with certain types of HPV is a necessary event in the development of cervical carcinoma; however, not all women who become infected will progress. While much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation, little is known about the additional factors needed to drive the process. Currently, conventional cervical screening is insufficient at identifying women who are likely to progress from premalignant lesions to carcinoma. Aneuploidy and chromatin texture from image cytometry have been suggested as quantitative measures of nuclear damage in premalignant lesions and cancer, and traditional epidemiologic studies have identified potential factors to aid in the discrimination of those lesions likely to progress. ^ In the current study, real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, LSIL, and HSIL. Quantitative cytometry was used to gather information about the DNA index and chromatin features of cells from the same women. Logistic regression modeling was used to establish predictor variables for histologic grade based on the traditional epidemiologic risk factors and molecular markers. ^ Prevalence of mRNA transcripts was lower among women with normal histology (27%) than for women with LSIL (40%) and HSIL (37%) with mean levels ranging from 2.0 to 4.2. The transcriptional activity of HPV 18 was higher than that of HPV 16 and increased with increasing level of dysplasia, reinforcing the more aggressive nature of HPV 18. DNA index and mRNA level increased with increasing histological grade. Chromatin score was not correlated with histology but was higher for HPV 18 samples and those with both HPV 18 and HPV 16. However, chromatin score and DNA index were not correlated with mRNA levels. The most predictive variables in the regression modeling were mRNA level, DNA index, parity, and age, and the ROC curves for LSIL and HSIL indicated excellent discrimination. ^ Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from cervical swabs. Epidemiological modeling of malignant progression in the cervix should include molecular markers, as well as the traditional epidemiological risk factors. ^

RB-mediated suppression of spontaneous multiple neuroendocrine neoplasia and lung metastases in Rb+/− mice

Alterations in pathways mediated by retinoblastoma susceptibility gene (RB) product are among the most common in human cancer. Mice with a single copy of the Rb gene are shown to develop a syndrome of multiple neuroendocrine neoplasia. The earliest Rb-deficient atypical cells were identified in the intermediate and anterior lobes of the pituitary, the thyroid and parathyroid glands, and the adrenal medulla within the first 3 months of postnatal development. These cells form gross tumors with various degrees of malignancy by postnatal day 350. By age of 380 days, 84% of Rb+/− mice exhibited lung metastases from C-cell thyroid carcinomas. Expression of a human RB transgene in the Rb+/− mice suppressed carcinogenesis in all tissues studied. Of particular clinical relevance, the frequency of lung metastases also was reduced to 12% in Rb+/− mice by repeated i.v. administration of lipid-entrapped, polycation-condensed RB complementary DNA. Thus, in spite of long latency periods during which secondary alterations can accumulate, the initial loss of Rb function remains essential for tumor progression in multiple types of neuroendocrine cells. Restoration of RB function in humans may prove an effective general approach to the treatment of RB-deficient disseminated tumors.

Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apcmin (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apcmin, and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apcmin/+ epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apcmin/+ epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apcmin/+-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.

A resistant genetic background leading to incomplete penetrance of intestinal neoplasia and reduced loss of heterozygosity in ApcMin/+ mice

Previous studies of Min/+ (multiple intestinal neoplasia) mice on a sensitive genetic background, C57BL/6 (B6), showed that adenomas have lost heterozygosity for the germ-line ApcMin mutation in the Apc (adenomatous polyposis coli) gene. We now report that on a strongly resistant genetic background, AKR/J (AKR), Min-induced adenoma multiplicity is reduced by about two orders of magnitude compared with that observed on the B6 background. Somatic treatment with a strong mutagen increases tumor number in AKR Min/+ mice in an age-dependent manner, similar to results previously reported for B6 Min/+ mice. Immunohistochemical analyses indicate that Apc expression is suppressed in all intestinal tumors from both untreated and treated AKR Min/+ mice. However, the mechanism of Apc inactivation in AKR Min/+ mice often differs from that observed for B6 Min/+ mice. Although loss of heterozygosity is observed in some tumors, a significant percentage of tumors showed neither loss of heterozygosity nor Apc truncation mutations. These results extend our understanding of the effects of genetic background on Min-induced tumorigenesis in several ways. First, the AKR strain carries modifiers of Min in addition to Mom1. This combination of AKR modifiers can almost completely suppress spontaneous intestinal tumorigenesis associated with the Min mutation. Second, even on such a highly resistant genetic background, tumor formation continues to involve an absence of Apc function. The means by which Apc function is inactivated is affected by genetic background. Possible scenarios are discussed.

Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia

DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2′-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase–PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.