Inhibition of the immunoproteasome modulates innate immunity to ameliorate muscle pathology of dysferlin-deficient BlAJ mice.

Muscle repair in dysferlinopathies is defective. Although macrophage (Mø)-rich infiltrates are prominent in damaged skeletal muscles of patients with dysferlinopathy, the contribution of the immune system to the disease pathology remains to be fully explored. Numbers of both pro-inflammatory M1 Mø and effector T cells are increased in muscle of dysferlin-deficient BlAJ mice. In addition, symptomatic BlAJ mice have increased muscle production of immunoproteasome. In vitro analyses using bone marrow-derived Mø of BlAJ mice show that immunoproteasome inhibition results in C3aR1 and C5aR1 downregulation and upregulation of M2-associated signaling. Administration of immunoproteasome inhibitor ONX-0914 to BlAJ mice rescues muscle function by reducing muscle infiltrates and fibro-adipogenesis. These findings reveal an important role of immunoproteasome in the progression of muscular dystrophy in BlAJ mouse and suggest that inhibition of immunoproteasome may produce therapeutic benefit in dysferlinopathy.

Detection and genotyping of HPV-DNA through different types of diagnostic platforms in liquid-based cervical-cytology samples.

BACKGROUND: At present cervical cancer represents the second most common cancer in women worldwide and it reaches a global mortality rate of 52%. Only the early detection and the adequate treatment of pre-neoplastic lesions and early-stage cervical cancer decrease the mortality rate for this type of cancer. Cervical carcinoma screening, as a method of second prevention, is currently feasible through molecular research of high-risk HPV genotypes and in lots of organized screening programs the Pap-test is performed only in women with positive HPV-test. Currently, there are various diagnostic platforms detecting and molecular genotyping HPV, which are based on different procedures, determining uneven viral genotypes panels and using diverse type of vials to collect and store the samples. Previous studies have pointed out that DNA-HPV test can be negative in pre-neoplastic lesions, even of high grade, or in presence of cervical cancer. Therefore, it's important to assess the risk of false negative diagnoses using DNA-HPV molecular test, because in this circumstance women do not undergo immediately Pap-test, but they are submitted to second round screening with DNA-HPV test after 5 years: this protocol could increase the incidence of "interval cancers". The present study aims at comparing the results of HPV detection and genotyping on liquid based cervical cytology, using some of the most relevant diagnostic platforms in commerce. METHODS: The study is based on a group of patients which went to their private gynecologist in a contest of opportunistic screening. The vial used in the examined population has been EASYPREP® preservative solution (YD Diagnostics CORP-Republic of Korea); liquid-based cervical cytology sampling has been done using a single device (plastic brush), allowing to collect simultaneously cytological material from exocervix and endocervix (Rovers® Cervex-Brush®). The diagnostic platforms employed have been the following: A) Digene HC2 HPV DNA Test, on RCS System (QIAGEN); B) BD Onclarity™ HPV test, on automate platform BD Viper™ LT (Becton Dickinson); C) Xpert® HPV, on GeneXpert® Infinity Systems platform (Cepheid). Every platform researched high-risk HPV genotypes panels (hr-HPV). Part of the clinical records has also been analyzed through PCR and genes L1 and E6/E7 complete sequencing, in order to further typing the viral population. RESULTS: We have examined 1284 samples of women aged 16 to 73 years: 1125 have been tested using HC2 procedure, 272 samples with Onclarity method, 159 with Xpert® method and 55 samples have been analyzed using PCR and sequencing of gene L1 and gene E6/E7. HPV-DNA was detected with Onclarity method in 15,07%, with Xpert® method in 13,83% and using HC2 procedure in 12,27% of samples. The comparison between the three molecular methods revealed diagnostic discrepancies in 3,14% of our records between Onclarity test and Xpert® method and in 2,20% (6/272) between HC2 test and Onclarity test. Globally, in 431 tests, compared using different diagnostic platforms, discrepant diagnoses, referring to hr-HPV presence or to detected genotype, have been observed 11 times (2,55%). Genotype 16 appeared the most expressed in the positive samples (20,99%), whereas genotype 18 resulted the less expressed in the examined population (4,94%). DISCUSSION: The present study highlights the following: 1) Positive results' percentage for high-risk HPV-DNA genotypes, deriving from the three diagnostic platforms used and with the same vial to collect and store samples, does not significantly vary on the basis of the type of equipment and it is congruent with the Italian percentage already detected during organized screening programs. 2) Even the molecular diagnostic approach could give false negative results, preventing the detection in the screened population of cervical HPV-related lesions and theoretically endangering women to develop "interval cancer". 3) In the population examined, genotype 16 has been the most expressed, whereas genotype 18 was among the less frequently detected. Other genotypes often noticed have been: 56-59-66 (Onclarity P3 group), 31, 51 and 35-39-68 (Onclarity P2 group). This remark emphasizes the importance of HPV infection and genotypes distribution's continuous monitoring, considering that HPV-vaccines planned in Italy in the "National vaccination prevention program 2017-2019" are not specific for the majority of these genotypes. 4) The necessity to improve the screening program to identify cervical carcinomas and pre-neoplastic cervical lesions is remarked by the detection during HPV-test of possible coinfection (present at least in 8,76% of our records). In fact, the risk of development of cervical cancer might be associated with type-specific interactions between genotypes in multiple infections and, in addition, other genotypes, not targeted by quadrivalent HPV-vaccine, can increase the risk of cervical carcinoma. 5) As there's a different combination of HPV-genotypes in diagnostic categories used by the HPV screening platforms, it's important that anyone who is in charge of this diagnostic analysis promotes among clinicians the adequate rendition of the laboratory's data in the patient records, reporting both the diagnostic result and the method through which it has been obtained.

Fetal and placental chromosomal mosaicism revealed by QF-PCR in severe IUGR pregnancies.

A number of genetic and environmental factors are taken into account as responsible for intrauterine growth restriction (IUGR); nevertheless, the relevance of genetic alteration in IUGR aetiology remains to be determined. The aim of this study was to investigate using a combined cytogenetic-molecular approach, improved by a new application of QF-PCR method, the presence of mosaic chromosomal changes in fetal/placental samples from 12 pregnancies with unexplained severe IUGR. This multiple approach allowed us to reveal and quantify subtle chromosomal mosaicisms with less than 5% of trisomic cells even in cases in which cytogenetic and FISH analyses failed to reveal them. These are three pregnancies with a mosaic trisomy for chromosomes 7, 2 and 14; the former case presented matUPD7 and was previously described in this journal (Placenta 22 (2001) 813) in association with pre- and postnatal growth restriction. It is intriguing that chromosomes 7, 2 and 14 are known or suspected to harbour imprinted genes, so that an unbalanced gene dosage in a subset of cells during embryonic development could lead to an early impairment of placental function. Our findings indicate that extensive molecular and cytogenetic studies of IUGR fetal and placental tissues are necessary to reveal at least part of the heterogeneous genetic lesions implicated in IUGR phenotypes.

Loss of heterozygosity of the NOS3 dinucleotide repeat marker in atherosclerotic plaques of human carotid arteries.

We have investigated 28 atherosclerotic plaques of human carotid arteries with a panel of 39 microsatellite markers for the presence of LOH. The objective of this research was to verify if LOH, described in association with tumorigenic process, could be involved also in benign fibroproliferative disease. Seventy percent of samples demonstrated allelic imbalance: 50% of cases showed LOH at a minimum of one locus, 3.5% at a minimum of two loci and 14.3% at three or more loci. The percentages of LOH ranged between 3.8 and 14.3% and the highest involved polymorphic marker is the NOS3 internal dinucleotide repeat. Our results indicate that, like tumorigenesis, the atherogenic process could also involve LOH mechanism. Furthermore, the finding regarding the NOS3 internal polymorphism suggests a possible role of the gene as cofactor in formation of the atheromas.

Post-zygotic origin of complete maternal chromosome 7 isodisomy and consequent loss of placental PEG1/MEST expression.

Maternal UPD of chromosome 7 is associated with pre- and postnatal growth retardation (IUGR, PNGR) and Silver-Russell syndrome (SRS [MIM 180860]). We report a case of IUGR in a newborn with SRS stigmata. Using combined haplotyping and cytogenetic-FISH studies we characterized the lymphocytes, umbilical cord and four placental cotyledons. The results are consistent with complete maternal isodisomy 7 and trisomy 7 mosaicism of post-zygotic origin. The trisomic cell line was prevalent in trophoblast cells from two placental cotyledons. Trisomy 7 of post-zygotic origin is a frequent finding, but maternal isodisomy 7, due to trisomic rescue has never been reported. PEG1/MEST expression was evaluated on placenta cDNA and a specific transcript was revealed only in the cotyledons with a high percentage of trisomic cells and the presence of the paternal chromosome 7 contribution, but not in the placental biopsies with maternal isodisomy 7. The histological features of the four placental fragments revealed that isodisomy 7 correlates with a pattern of cotyledonary hyper-ramification due to an increase of the branching angiogenesis, which could be the result of a defect of angiogenesis caused by the absence of PEG1 product. The severe hypo-ramification of the two cotyledons, showing trisomy 7 mosaicism, may be due to the triplicate dosage of genes on chromosome 7. The delayed fetal growth could be the phenotypic effect of the imbalance between imprinted and non-imprinted genes on chromosome 7 in the fetus or the result of abnormal placental function during pregnancy.

Chromosome 7 monosomy and deletions in myeloproliferative diseases.

We studied deletion and monosomy of chromosome 7 in 150 patients with myeloproliferative diseases. We found 8/150 patients with monosomy 7 by cytogenetics and 4/150 with deletions of the long arm of chromosome 7 by restriction fragment length polymorphism (RFLP) analysis performed with Southern and polymerase chain reaction. To overcome limitation of RFLP analysis, we restricted loss of heterozygosity study with microsatellites to 45 patients, observing deletion 7q31.1 in 7/45 patients. In all patients with molecular alterations the deletion was observed only in myeloid cells, while the monosomy was detected in both myeloid precursor and lymphocytes. This finding suggests a CD34-totipotent stem cell origin for the monosomy and a colony forming unit - granulocyte, erythrocyte, monocyte, megakaryocytes (CFU-GEMM) stem cell origin for the deletions.

Integrin laminin receptor profile of pulmonary squamous cell and adenocarcinomas.

The differential expression of laminin receptors has been shown to modulate the invasive capability of malignant cells. We have investigated the reactivity of human pulmonary squamous carcinomas (SSC, n = 20) and adenocarcinomas (ADC, n = 20) with monoclonal antibodies to the cytoplasmic and extracellular domains of the integrin subunits alpha3 and alpha6. Integrins containing these subunits are laminin receptors. Monoclonal antibodies to beta1 and beta4 subunits, the beta1C splice variant of beta1, as well as to Ki-67, were also used. Reverse transcription polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was done to detect possible mutations in the cytodomains. All carcinomas expressed alpha3 extensively; alpha3 expression predominated (40 of 40) over alpha6 (25 of 40). In all alpha6-positive carcinomas, alpha6A was expressed, whereas alpha6B was weakly expressed only in some of them. No mutations of the intracytoplasmic domain A of alpha3 and of the A or B intracytoplasmic domains of alpha6 were shown. Notably, in normal bronchial epithelium, alpha6 colocalized with beta4, whereas in the tumors, alpha6A frequently overlapped with beta1 in a circumferential pattern; alpha6beta1 coexpression was also shown by coprecipitation experiments. Strong and extensive beta4 reactions were invariably polarized at the cell/stroma interface in SCC and ADC. An inverse correlation was found between the expression of beta1C and Ki-67. The prevalence of alpha6A in pulmonary SCC and ADC is in contrast with previous results in colonic ADC in which alpha6B prevails, and alpha6 predominates over alpha3. The absence of mutations of the cytodomains suggests that the integrin subunits of these carcinomas are potentially active. Predominance of alpha3 over alpha6 and of alpha6A over alpha6B may contribute to explain the aggressive and metastatic behavior of lung carcinomas.

Human cholangiocarcinoma development is associated with dysregulation of opioidergic modulation of cholangiocyte growth.

BACKGROUND/AIMS: Incidence of cholangiocarcinoma is increasing worldwide, yet remaining highly aggressive and with poor prognosis. The mechanisms that drive cholangiocyte transition towards malignant phenotype are obscure. Cholangiocyte benign proliferation is subjected to a self-limiting mechanism based on the autocrine release of endogenous opioid peptides. Despite the presence of both, ligands interact with delta opioid receptor (OR), but not with microOR, with the consequent inhibition of cell growth. We aimed to verify whether cholangiocarcinoma growth is associated with failure of opioidergic regulation of growth control. METHODS: We evaluated the effects of OR selective agonists on cholangiocarcinoma cell proliferation, migration and apoptosis. Intracellular signals were also characterised. RESULTS: Activation of microOR, but not deltaOR, increases cholangiocarcinoma cell growth. Such an effect is mediated by ERK1/2, PI3K and Ca(2+)-CamKIIalpha cascades, but not by cAMP/PKA and PKCalpha. microOR activation also enhances cholangiocarcinoma cell migration and reduces death by apoptosis. The anti-apoptotic effect of microOR was PI3K dependent. CONCLUSIONS: Our data indicate that cholangiocarcinoma growth is associated with altered opioidergic regulation of cholangiocyte biology, thus opening new scenarios for future surveillance or early diagnostic strategies for cholangiocarcinoma.

A common T/C polymorphism in the promoter region of the beta T-cell receptor gene.

Polymorphisms in the T-cell receptor genes can provide important information for the study of the immune response system, particularly for autoimmune diseases. This report characterizes a common T to C polymorphism in the promoter of the beta 2 constant chain of the T-cell receptor, which abolishes a recognition site for BglII restriction endonuclease.

An Ava I polymorphism in the TP53 gene.

TP53 gene plays a major role in the process of malignant transformation and tumour progression so that abnormalities such as point mutation or allelic loss of this gene are a common finding in different tumour types. Most of the mutations identified cover a conserved region of the gene, spanning from exon 4 to exon 9. The present report describes a novel polymorphism, 12 nucleotides downstream the splicing junction of exon/intron 9 identified in a cohort of 103 Italian healthy blood donors. The polymorphism results in the creation of a new restriction site for Ava I.

Molecular changes in hepatocellular dysplastic nodules on microdissected liver biopsies.

The genetic profile of dysplastic hepatocellular nodules arising in cirrhosis is poorly understood. We assessed loss of heterozygosity (LOH) and microsatellite instability (MI) in 10 dysplastic nodules (4 low-grade and 6 high-grade) with surrounding cirrhosis and in 10 hepatocellular carcinomas (HCC). Six microsatellite loci were selected and investigated on microdissected needle biopsies. Twenty-four (24.4%) informative loci showed allelic loss, while MI was seen in 3 loci only (3%). The most involved sites were located on chromosomes 4q (54.5%) and 8p (50%). LOH was documented in 16.6%, cirrhotic, 50% low-grade dysplastic nodules (LGDN), 83% high-grade dysplastic nodules (HGDN), and 70% malignant nodules. LOH at multiple loci was increasingly seen from cirrhotic to HGDN, but not from the latter to HCC. The fractional allelic loss (FAL) was significantly increased in dysplastic and neoplastic nodules as compared with cirrhosis (P <.01). The progressive accumulation of genetic changes in cirrhotic, dysplastic, and malignant hepatocellular nodules is in keeping with a multistep process of carcinogenesis; within this spectrum, HGDN can be considered advanced precursors of HCC.

Molecular alterations of Barrett's esophagus on microdissected endoscopic biopsies.

Alterations in proto-oncogenes and tumor suppressor genes play a role in the sequence from Barrett's metaplasia to esophageal adenocarcinoma. The present study aims to ascertain whether molecular abnormalities take place in Barrett's metaplasia and low-grade dysplasia and to correlate them with the histological features of the esophageal mucosa. Forty-one formalin-fixed, paraffin-embedded endoscopic esophageal biopsies were classified according to the type of metaplastic changes (noncolumnar fundic and cardial metaplasia; columnar metaplasia, with and without intestinal features). After microdissection samples were examined for loss of heterozygosity (LOH) using polymorphic markers on 5q (D5S82), corresponding to APC (adenomatous polyposis coli) gene, 13q (CA repeat in intron 2 position 14815 to 14998 of the retinoblastoma gene), 17p (D17S513) corresponding to p53 locus, and for p53 mutations. Molecular alterations including LOH, allelic imbalance, and microsatellite instability could be detected in all types of metaplastic changes and sporadically in the squamous epithelium adjacent to the metaplastic tissue. Molecular alterations involving microsatellites D5S82 and the CA repeat inside the retinoblastoma gene were more frequent in nonintestinal metaplasia whereas those involving the p53 locus took place in columnar intestinal metaplasia and in low-grade dysplasia. Clonal changes were demonstrated in different metaplastic areas in three patients. Genetic alterations comprising LOH and microsatellite instability characterize Barrett's mucosa and appear related to the type of metaplastic change. Some of them precede the development of intestinal metaplasia, suggesting that genetic alterations take place earlier than previously thought.

Molecular disorders in transitional vs. peripheral zone prostate adenocarcinoma.

Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been shown to be mechanisms for tumor-suppressor gene inactivation in human oncogenesis. In our study, we examined LOH and MSI using 16 polymorphic markers of DNA for chromosomes 1, 3, 7, 8, 10 and 11. Microdissected tumor samples were isolated from 32 patients, representing 11 foci of incidentally discovered prostate cancer of the transitional zone (TZ), 12 prostate cancer of the peripheral zone (PZ) and 10 of high-grade PIN. We found loss of heterozygosity in the TZ group in 91% of informative cases (10/11) with al least 1 marker compared to 58% of cases (7/12) in PZ group and 70% of cases (7/10) in the HGPIN group. Chromosome 7 showed the highest rate of allelic loss in all 3 categories, with loss of 43% of loci in PIN, 37% in TZ tumors and 31% in PZ tumors. At chromosome 11, LOH was detected in 26% of loci in the TZ group, in 7% of loci in the PZ group and in 13% of loci in the PIN group. On chromosome 8, the PZ and HGPIN group showed allelic loss in 22% and 21% of loci, respectively, compared to 10% detected in the TZ group. The TZ group showed a significant higher rate of allelic instability compared to that observed in tumor samples from the peripheral zone: 73% of cases (8/11) showed genetic alterations (RER+ phenotype) in at least 4 loci analyzed compared to 8% and 10% in the PZ and HGPIN groups, respectively (p = 0.0006). These data suggest that transitional zone carcinoma and peripheral zone carcinoma display distinct and specific genetic alterations in different chromosomes. This diversity may help explain biologic and clinical differences between carcinomas arising in these distinct zones of the prostate. Also our results strongly suggest that the RER+ mutator phenotype could be linked to early development of transitional zone prostate carcinoma.