Exogenous retroelement integration in sperm and embryos affects preimplantation development.

Retroelement transcripts are present in male and female gametes, where they are typically regulated by methylation, noncoding RNAs and transcription factors. Such transcripts are required for occurrence of retrotransposition events, while failure of retrotransposition control may exert negative effects on cellular function and proliferation. In order to investigate the occurrence of retrotransposition events in mouse epididymal spermatozoa and to address the impact of uncontrolled retroelement RNA expression in early preimplantation embryos, we performed in vitro fertilization experiments using spermatozoa preincubated with plasmid vectors containing the human retroelements LINE-1, HERVK-10 or the mouse retroelement VL30, tagged with an enhanced green fluorescence (EGFP) gene-based cassette. Retrotransposition events in mouse spermatozoa and embryos were detected using PCR, FACS analysis and confocal microscopy. Our findings show that: (i) sperm cell incorporates exogenous retroelements and favors retrotransposition events, (ii) the inhibition of spermatozoa reverse transcriptase can decrease the retrotransposition frequency in sperm cells, (iii) spermatozoa can transfer exogenous human or mouse retroelements to the oocyte during fertilization and (iv) retroelement RNA overexpression affects embryo morphology and impairs preimplantation development. These findings suggest that the integration of exogenous retroelements in the sperm genome, as well as their transfer into the mouse oocyte, could give rise to new retrotransposition events and genetic alterations in mouse spermatozoa and embryos.

Imprinted genes in myeloid lineage commitment in normal and malignant hematopoiesis.

Genomic imprinting is characterized by the parent-of-origin monoallelic expression of several diploid genes because of epigenetic regulation. Imprinted genes (IGs) are key factors in development, supporting the ability of a genotype to produce phenotypes in response to environmental stimuli. IGs are highly expressed during prenatal stages but are downregulated after birth. They also affect aspects of life other than growth such as cognition, behavior, adaption to novel environments, social dominance and memory consolidation. Deregulated genomic imprinting leads to developmental disorders and is associated with solid and blood cancer as well. Several data have been published highlighting the involvement of IGs in as early as the very small embryonic-like stem cells stage and further during myeloid lineage commitment in normal and malignant hematopoiesis. Therefore, we have assembled the current knowledge on the topic, based mainly on recent findings, trying not to focus on a particular cluster but rather to have a global view of several different IGs in hematopoiesis.

Apolipoprotein E polymorphisms and ischaemic stroke: a two-center Greek study.

BACKGROUND AND PURPOSE: Apolipropotein E(apoE) is a plasma protein exhibiting three common isoforms (E2, E3, E4). Its involvement in lipoprotein metabolism may have an impact on stroke occurrence. As results in the literature are inconclusive further studies are needed to elucidate its role. Our objective was to study the role of apoE isoforms and the interplay with environmental risk factors in patients with first ischaemic stroke occurrence in the Greek population. METHODS: Three hundred and twenty-nine patients with first-ever ischaemic stroke were included in our study. Strokes of cardioembolic origin and patients with autoimmune or prothrombotic syndromes were excluded. A control group of 361 subjects with no stroke history were also included in our study. Risk factors (hyperlipidemia, hypertension, diabetes mellitus and smoking) were assessed. ApoE alleles were determined in all subjects participating in the study. RESULTS: Genotype ε3/ε3 was found to have a protective role against stroke occurrence compared with other genotypes (odds ratio 0.674, 95% confidence interval 0.480-0.946) especially in the female patient subgroup. In multivariate analysis after adjustment for age, body mass index (BMI), hypertension, dyslipidemia, diabetes mellitus and smoking, the role of genotype was limited and outweighed by risk factors in both genders. No association between apoE alleles and BMI, cholesterol, triglycerides or high-density lipoprotein plasma levels was noted. CONCLUSIONS: Our study was indicative of a protective role of the ε3/ε3 genotype, especially in female patients. However, risk factors such as age, BMI, hypertension, dyslipidemia, diabetes mellitus and smoking have a strong impact on stroke occurrence and outweigh the protective role of the ε3/ε3 genotype.

Anterior ischemic optic neuropathy in a patient with Crohn's disease and aberrant MTHFR and GPIIIa gene variants.

Large spectrums of ophthalmic manifestations from the anterior to the posterior segment have been so far reported in patients with inflammatory bowel disease. Anterior ischemic optic neuropathy is caused by acute ischemic infarction of the optic nerve head and is distinguished in two different types, non-arteritic anterior ischemic optic neuroparhy (NAION) which is the most frequent type and arteritic anterior ischemic optic neuropathy. Non-arteritic anterior ischemic optic neuroparhy may result in severe visual field loss. We present the case of a 69 year-old man with known history of Crohn's disease that was referred to the Department of Ophthalmology after noticing sudden blurred vision of his left eye. Ophthalmologic examination revealed a corrected visual acuity of 8/10 OS and 10/10 OD. Pupil examination showed a relative afferent pupillary defect of the left pupil and fluoroangiography revealed hyperfluorescence of the left optic disc, indicating edema and NAION attack on his left eye. Genetic analysis showed that the patient was homozygous for MTHFR C677T genetic polymorphism and A1/A2 heterozygous for GPIIIa polymorphism.

Frequent abnormalities of the immune system in gliomas and correlation with the WHO grading system of malignancy.

AIM: To investigate the cellular and humoral immunity status of gliomas, and their association with the WHO grading system. MATERIAL AND METHODS: We have conducted a case-control study of 49 patients with gliomas and 30 healthy controls. We used ELISA assays, radial immunodiffusion, indirect immunofluorescence, latex test and flow cytometry assays to estimate preoperative in serum the immunological profile. RESULTS: Patients with glioma had significantly reduced amounts of IL2 (p=0.000), TNF-a (p=0.033), IgG (p=0.011), IgA (p=0.027),C4 (p=0.026) ,CD3+ (p=0.001), CD4+ (p=0.000), CD8+ (p=0.002), ratio CD4/CD8 (p=0.000), CD19+ (p=0.04) and elevated IL10 (p=0.05) compared with healthy controls. No statistically significant differences were observed concerning viral agents, total NK cells, IgM, IgE, IL16, granzyme-b, RF, ANA, ENA, anti-dsDNA and anti-cardiolipin antibodies. A higher WHO grade, after controlling for age and gender, was associated with decreased number of CD3+ (p=0.011), CD4+ (p=0.015), CD8+ (p=0.048) and ratio CD4/CD8 (p=0.027), as well as with decreased IL2 (p=0.018), C4 (p=0.02), and IgG (p=0.05). IL2 and CD4+ counts were significant predictors of grade. CONCLUSIONS: A shift from Th1 to Th2, a CD3+ and CD19+ lymphocytopenia, a diminished fraction CD4/CD8 and a reduced amount of immunoglobulins and complement were observed in the patients with gliomas. A higher WHO grade of the tumor was associated with greater impairments of immunity. Since defects of both humoral and cellular immunity were equally observed and significant predictors of grade were assessed, a preoperative evaluation of the immune system of patients with gliomas is being proposed.

A twisted kiss: in vitro and in vivo evidence of genetic variation and suppressed transcription of the metastasis-suppressor gene KiSS1 in early breast cancer.

UNLABELLED: KiSS-1 is ametastasis suppressor gene, its inactivation linked to advanced tumor stage and dismal prognosis. We studied its mutational status ,transcription and protein expression in human cancer cell lines and patients with early breast cancer.
Tumor tissue DNA and messenger RNA (mRNA) of KiSS1 exons III and IV from the human cancer cell lines Hela, Jurkat, A549, W138t, MCF-7 and from formalin-fixed resected breast adenocarcinomas from 50 women were analysed by means of PCR-SSCP, RT-PCR and sequencing. Tumor tissue was stained for KiSS1 protein expression by means of the streptavidin-biotin complex immunoperoxidase assay. Presence of KiSS1 mutation, mRNA levels and protein staining were examined for correlations with patient/tumor characteristics.
A transversion in exon IVa replacing cytosine with guanine was identified 242 base pairs from the translation start site (242C>G) in the cell lines MCF-7, A549 and in 5/50 tumors (10%), resulting in substitution of proline by arginine (P81R) and alteration of the protein tertiary structure. As the substitution was present in germ-line DNA in 3/5 breast cancer patients harbouring the polymorphism in their tumor, the incidence of tumour-specific somatic mutation was 4% among the 50 patiens with early breast cancer. Although the P81R substitution was associated with reduced KiSS1 protein immunoreactivity (56% in wild-type tumors versus 20% in KiSS1-variant tumours) and with axillary nodal involvement (55% in wild-type versus 80% in KiSS1-variant tumors), the correlations did not reach statistical significance. KiSS1 mRNA was detected in only 15/48 tumours (31%) and showed no correlation with mutation or protein expression. Twenty-six tumors stained for KiSS1 protein, in contrast to the universal strong staining seen in normal breast parenchyma and placental tissues. At amedian follow-up of 38 months, relapses occurred in 20% of women with non wild-type tumors versus 13% of women with wild-type KiSS1 tumors (p=0.7). Presence of KiSS1 mutation, mRNA levels and protein expression did not have prognostic significance for relapse-free survival.
In conclusion, altered nucleotide sequence and repression of transcription are two potential mechanisms of suppression of the anti-metastatic effects of KiSS1 in early breast cancer: Confirmation in larger cohorts and study of functional effects of the 242C>G exon IVa mutation are warranted. KEYWORDS: KiSS1, metastasis-suppressor gene, breast cancer, mutation, transcription.

Targeting c-KIT, PDGFR in cancer of unknown primary: a screening study for molecular markers of benefit.

AIMS: In view of available targeted therapies, we investigated the presence of c-kit, PDGFR gene mutations and protein expression in cancer of unknown primary (CUP) in order to study their contribution in pathogenesis, their prognostic value and potential as therapeutic targets. METHODS: Mutations in hot spots c-kit exon 11 and PDGFR exons 12 and 18 were studied in paraffin-embedded tumour samples from 50 patients with CUP by means of PCR-based single-strand conformational polymorphism and protein expression by means of streptavidin-biotin immunoperoxidase assays. Molecular markers were screened for possible correlations with patient outcome. RESULTS: No shifted band was detected in any of the polyacrylamide gel electrophoreses, indicating absence of c-kit exon 11 and PDGFR exon 12, 18 mutations. Immunohistochemical analysis in 37 tumours revealed positive membranous CD117 expression in 30 samples (81%) of which five exhibited strong (+3), four moderate (+2) and 21 weak (+1) staining. PDGFRa protein staining was seen in 15 out of 30 (50%) cases, mostly weak (13) and rarely moderate (1) or strong (1). The expression of KIT or PDGFRa protein did not correlate with the clinical outcome of the patients in our cohort. CONCLUSIONS: In a moderate-sized CUP patient cohort, KIT or PDGFRa protein overexpression is rare, does not have gross prognostic significance for survival and is not associated with presence of activating mutations.

Effect of the hemodialysis session on bcl-2 expression in peripheral blood mononuclear cells in vivo.

bcl-2 is a proto-oncogene with a regulatory role in many conditions due to its marked inhibitory action on apoptosis. Reports regarding the effect of hemodialysis (HD) on apoptosis of mononuclear cells and in association with bcl-2 expression in particular, are controversial. The aim of the present study was to examine in vivo the influence of an HD session on bcl-2 expression of lymphocytes and monocytes. We measured quantitative bcl-2 expression with flow cytometry, in terms of antibodies bound per cell, in blood samples taken from 44 HD patients before and after an HD session. 27 patients (group I) were dialyzed with synthetic-type membranes and 17 (group II) with cellulose-type membranes. bcl-2 expression increased statistically significantly in lymphocytes (1,616 +/- 718 to 1,894 +/- 715 molecules/cell, p < 0.01) at the end of HD. Monocyte expression of bcl-2 was lower than in lymphocytes and almost did not change after the HD session (654 +/- 446 to 698 +/- 375 molecules/cell, p = NS). Comparison between the two groups did not reveal a significant difference in either the baseline bcl-2 expression or in the value of the increase after HD. We conclude that HD seems to decrease lymphocyte apoptosis independent of the biocompatibility of the dialyzer membrane.

p53 enhances the Delta-24 conditionally replicative adenovirus anti-glioma effect.

Previous studies have demonstrated that the conditionally replicative adenovirus Ad5Delta24 is a powerful cytolytic agent against glioma selectively affecting cells with a defective p16/Rb/E2F pathway. The p53 protein is also known to be an apoptotic factor for glioma cells. In this study, we examined the simultaneous delivery of the combination of exogenous p53 and Ad5Delta24 adenovirus in glioma cells. Infecting cells with low doses of adenovirus p53 and Ad5Delta24 resulted in an additive effect on cell death. The cell death induced by both agents was independent of the p53 status of cells. Flow cytometry revealed that the potent anti-tumor effect induced by the mixture of Ad5CMV-p53 and Ad5Delta24 adenoviruses was due to a combination of apoptosis and cell lysis. Our results indicate that Ad5CMV-p53 enhances the oncolytic effect of the Ad5Delta24 adenovirus, and the combination of adenovirus Ad5Delta24 and Ad5CMV-p53 may thus be a potential therapeutic tool for gliomas.

A single-center, retrospective study of management and outcome of 45 elderly AML patients, diagnosed in 2001.

Acute myeloid leukemia (AML) predominantly affects older adults, a population with a poor prognosis, due to age, comorbidities and forms of disease. We present a retrospective study of 45 patients older than 60 years of age, with AML, who were diagnosed and/or treated in our clinic in the year 2001. Our study refers to 32 men, 63-80 years of age and 13 women, 62-85 years of age. Fourteen of them were diagnosed as de novo leukemia while 31 developed secondary leukemia, due to myelodysplasia, chronic myeloid leukemia and essential thrombocytemia. A therapeutic protocol that included 2 courses of induction chemotherapy with idarubicin 8mg/m2 for 3 days, aracytin 100 mg/m2 for 5 days and etoposide 75 mg/m2 for 5 days, followed by 2 courses of consolidation chemotherapy with aracytin 800 mg/m2/d for 4 days, was administered. In patients with acute promyelocytic leukemia we additionally administered all trans retinoic acid. Those with erythroleukemia also received erythropoietin, 10,000 IU 3 times a week. All patients received supportive therapy with blood products and G-CSF during blood marrow aplasia. Four patients refused therapy and three patients received only blood product support because of poor performance status. Nine out of the 38 patients who received chemotherapy (23.7%) achieved a complete remission after treatment, while, 13 out of 38 (34.2%) only a partial one (overall remission rate: 57.9 %). Ten patients relapsed in <6 months and 12 patients relapsed in >6 months. Patients who received only supportive treatment died 2-5 months after initial diagnosis. During therapy, 16 patients (42.1%) died due to: infection, cerebrovascular or gastrointestinal bleeding and acute myocardial infarction. In conclusion, it appears that a high percentage of the elderly patients with AML, despite the unfavourable prognosis, responded to chemotherapy (57.9%) and achieved longer survival durations compared to patients who refused therapy or received supportive treatment alone. Unfortunately, a large number of them exhibited serious complications during treatment, with a mortal outcome. Close follow-up and supportive care highly contributed to an improvement of treatment outcome in elderly patients with acute myeloid leukemia.

T-cell receptor gammadelta-large granular lymphocytic leukemia associated with an aberrant phenotype and TCR-Vbeta20 clonality.

Large granular lymphocytic (LGL) leukemia is a rare heterogenous disorder of mature lymphocytes with a characteristic morphology, multiple autoimmune disorders and indolent clinical course. Most cases exhibit a T-cell phenotype of CD3, CD8 and CD57 positivity, while the minority exhibit a CD2, CD56, and CD16 positive NK-cell phenotype. We report a case of a 71-year-old female suffering from a TCRgammadelta positive T-cell leukemia with a morphology compatible to LGL leukemia. She referred to the hospital for investigation of mild anemia, lymphocytosis, neutropenia and hyperglobulinemia. Peripheral blood and bone marrow were occupied by mature large granular lymphocytes with abundant azurophilic granules. The immunophenotype was CD3+, CD2+, CD5+, CD7+, CD4-, CD8-, CD16-, CD56-, CD57- and the Vbeta repertoire analysis showed clonal reactivity with Vbeta20 mAb. The patient was diagnosed as having T-LGL and was treated with G-CSF. So far, she experiences an indolent clinical course. To our knowledge, this is a rare case of TCRgammadelta positive T-LGL leukemia with the aberrant immunophenotype of CD3+, CD4-, CD8-, CD16-, CD56-, CD57-.

Recurrent episodes of deep vein thrombosis in a young man.

We present the case of a 32-year-old man suffering from recurrent episodes of deep vein thrombosis (DVT). He was heterozygous for the G1691A mutation in the Factor V gene. His father was heterozygous for the same mutation and had a unique episode of DVT after a fracture of the tibia. Genetic predisposition significantly influences the prevalence of thrombotic events, however, additional unknown factors may be involved in the initiation and recurrence of venous thromboembolism.

Prevalence of the G20210A prothrombin gene mutation in Northwestern Greece and association with venous thromboembolism.

AIM: The G20210A mutation of the prothrombin gene is a genetic risk factor for venous thromboembolism (VTE). Variability exists in the mutation prevalence in both normal individuals and VTE patients. The aim of this study was to determine the mutation prevalence in Northwestern Greece and evaluate its association with VTE. METHODS: Presence of the G20210A mutation was investigated using DNA analysis in 176 consecutive patients with a history of venous thrombosis or pulmonary embolism and in 300 healthy controls, all Caucasian residents of Northwestern Greece. RESULTS: The mutation was present 12 patients (6.8%) and 8 controls (2.7%). The odds ratio for presence of the mutation versus the normal genotype in VTE was 2.7 (95% CI: 1.1 to 6.7), which was statistically significant. The prevalence of the G20210A prothrombin gene mutation in Northwestern Greece is 2.7% (95% CI: 0.8% to 4.4%) with an allele frequency of 1.3% (95% CI: 0.4% to 2.3%). CONCLUSION: The G20210A mutation of the prothrombin gene is associated with VTE in the Caucasian residents of this geographic region.

A case of HLA-DR negative B-precursor acute lymphoblastic leukemia.

We present the case of a 9-year-old girl from northwestern Greece admitted to our Hospital because of malaise, low-grade fever, intermittent hip joint pain, anemia, leukopenia and thrombocytopenia. The examination of a bone marrow aspirate revealed the predominance of blast cells (97%) with FAB L1 morphology, immunopheno-typically positive for CD19 (95%), CD10 (95%), CD22 (95%), CD13 (82%), CD34 (95%) and CD38 (72%), with dim expression of CD45 and of the intracellular antigen terminal deoxynucleotidyl transferase (Tdt). Only 10% of the blasts expressed HLA-DR. Staining for CD2, CD3, CD5, CD7, CD20, CD23, CD33, CD14, CD15, AC133 and KOR-SA3544 was negative. Blast cells were lacking surface immunoglobulin expression and bcr/abl rearrangements were not detected. Cell cycle analysis revealed a diploid cell population. Karyotypic abnormalities were not identified. The lack of expression of HLA-DR and the presence of myeloid antigen CD13 indicated that it was a rare case of B-precursor ALL with aberrant immunophenotypic characteristics.

Lack of association between the C677T mutation in the 5,10-methylenetetrahydrofolate reductase gene and venous thromboembolism in Northwestern Greece.

BACKGROUND: Hyperhomocysteinemia has been associated with venous thrombosis. Under known and unknown conditions the C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene is accompanied by elevated levels of homocysteine. However, the relationship of this mutation with venous thromboembolism (VTE) remains controversial. The purpose of this study was to evaluate the association of the MTHFR mutation with VTE. METHODS: The presence of the C677T mutation in the MTHFR gene was investigated in a population of 176 consecutive patients with a history of venous thromboembolism and in a control group of 300 healthy subjects, using DNA analysis. RESULTS: The prevalence of homozygosity in the patient group was 13.6% and in healthy subjects 10%. The odds ratio for venous thromboembolism in the presence of the homozygous genotype (677TT) was 1.4 (95% confidence interval (C.I.), 0.8 to 2.5), which was not statistically significant. CONCLUSIONS: Homozygosity for the T677 allele of the MTHFR gene, although slightly more prevalent in patients compared to controls, has not been found in association with venous thromboembolism.

Rheumatoid arthritis and B-cell chronic lymphocytic leukemia.

The association between lymphoproliferate malignancies, especially lymphoma, and rheumatoid arthritis (RA) has been confirmed by several studies. However; there are few reports of RA patients who developed B-cell chronic lymphocytic leukemia (B-CLL) and vice versa. We report a patient with B-CLL who developed RA and another with RA who presented with B-CLL during follow-up. We discuss the incidence of B-CLL among the RA population and the possible interaction of the pathogenetic mechanisms of these two entities.

Polycystic ovarian syndrome and thrombophilia.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is associated with insulin-induced plasminogen activator inhibitor-1 (PAI-1) elevations. Since thrombophilic states correlate with high miscariage rates, as does PCOS, this study aimed at looking for thrombophilic predisposition in PCOS women compared with non-PCOS controls. METHODS: The prevalence of antithrombin III, protein S and protein C deficiencies, as well as factor V Leiden, prothrombin G20210A factor and methylene tetrahydrofolate reductase (MTHFR) mutations, was compared between two different groups of women, one with PCOS (n = 30) and one without PCOS (n = 45). RESULTS: Median proportions of activated protein C, S and antithrombin III as well as the activated protein C ratios were within normal ranges in both samples. There was no evidence that the genetic analysis for factor V Leiden or prothrombin factor differed between the two samples. The odds ratio (OR) of bearing a mutation on the MTHFR gene was 1.2-fold higher [95% confidence interval (CI) 0.470-3.065] in women with PCOS than in women without (P = 0.83). Although this difference is not statistically significant, it might indicate a slightly higher prevalence of heterozygous genotypes in women with PCOS (OR = 1.197, 95% CI 0.473-3.034). CONCLUSIONS: Molecular risk factors of hereditary thrombophilia do not show increased prevalence in women with PCOS in comparison with women in the general population. The existence of a possible trend towards higher prevalence of MTHFR mutation in women with PCOS needs further study, particularly regarding homocysteine levels.

Skin and gingival lesions in a young woman with B-prolymphocytic leukemia (B-PLL).

B-PLL has not been often associated with diffuse skin involvement or oral lesions. We present a 32 year-old woman in whom skin and gingival manifestations were the prominent clinical signs of disease relapse.