Investigating the genetic characteristics of CAD: Is there a role for myocardial perfusion imaging techniques?

Several environmental and genetic factors have been found to influence the development and progression of coronary artery disease (CAD). Although the effects of the environmental hazards on CAD pathophysiology are well documented, the genetic architecture of the disease remains quite unclear. A number of single-nucleotide polymorphisms have been identified based on the results of the genome-wide association studies. However, there is a lack of strong evidence regarding molecular causality. The minority of the reported predisposing variants can be related to the conventional risk factors of CAD, while most of the polymorphisms occur in non-protein-coding regions of the DNA. However, independently of the specific underlying mechanisms, genetic information could lead to the identification of a population at higher genetic risk for the long-term development of CAD. Myocardial single-photon emission computed tomography (SPECT) and positron emission tomography (PET) are functional imaging techniques that can evaluate directly myocardial perfusion, and detect vascular and/or endothelial dysfunction. Therefore, these techniques could have a role in the investigation of the underlying mechanisms associated with the identified predisposing variants, advancing our understanding regarding molecular causality. In the population at higher genetic risk, myocardial SPECT or PET could provide important evidence through the early depiction of sub-clinical dysfunctions, well before any atherosclerosis marker could be identified. Notably, SPECT and PET techniques have been already used for the investigation of the functional consequences of several CAD-related polymorphisms, as well as the response to certain treatments (statins). Furthermore, therefore, in the clinical setting, the combination of genetic evidence with the findings of myocardial SPECT, or PET, functional imaging techniques could lead to more efficient screening methods and may improve decision making with regard to the diagnostic investigation and patients' management.

Expression profile of the GA733 gene family in colorectal cancer: correlation with clinicopathological parameters.

GA733-1/-2/-3 genes have been detected in various types of cancer, although their role has not been fully clarified. GA733-2 and GA733-1 have been correlated with lymph node metastases in laryngeal cancer and liver metastases, respectively. Only a few studies have elucidated the mechanisms regulating GA733-1/-2 expression and their effect on colorectal cancer. Therefore, the expression pattern and the role of the aforementioned molecules in colorectal carcinogenesis were evaluated in this study. Tissue samples were obtained from 40 patients with colorectal cancer with no liver metastases. GA733-1/-2 mRNA levels were evaluated by quantitative real-time polymerase chain reaction. GA733-1/-2 gene expression in noncancerous/cancerous tissues was also correlated with clinicopathological parameters. The GA733-1 mRNA levels were very low; however, the GA733-1 mRNA transcripts were higher in cancerous tissues than in normal tissues (median ratio, 0.004391/0.00093; range, 0.000001- 0.025139/0.000001-0.007761), respectively (P = 0.012). GA733-2 gene expression was higher in noncancerous tissues than in cancerous tissues (median ratio 273.31/115.64; range, 65.24-1,486.41/11.58-1,189.14; P = 0.0000195). Lower GA733-2 expression in cancer tissues appeared to correlate with lymph node metastases (P < 0.05). GA733-1 gene expression was significantly higher in cancerous samples; conversely, the GA733-2 mRNA levels were higher in noncancerous tissues, and were significantly correlated with lymph node perforation in colorectal cancer (P < 0.05). Therefore, GA733-1/-2 mRNA expression levels appear to be a potential predictive marker of tumorigenesis.

Modeling of signaling pathways in chondrocytes based on phosphoproteomic and cytokine release data.

OBJECTIVE: Chondrocyte signaling is widely identified as a key component in cartilage homeostasis. Dysregulations of the signaling processes in chondrocytes often result in degenerative diseases of the tissue. Traditionally, the literature has focused on the study of major players in chondrocyte signaling, but without considering the cross-talks between them. In this paper, we systematically interrogate the signal transduction pathways in chondrocytes, on both the phosphoproteomic and cytokine release levels. METHODS: The signaling pathways downstream 78 receptors of interest are interrogated. On the phosphoproteomic level, 17 key phosphoproteins are measured upon stimulation with single treatments of 78 ligands. On the cytokine release level, 55 cytokines are measured in the supernatant upon stimulation with the same treatments. Using an Integer Linear Programming (ILP) formulation, the proteomic data is combined with a priori knowledge of proteins' connectivity to construct a mechanistic model, predictive of signal transduction in chondrocytes. RESULTS: We were able to validate previous findings regarding major players of cartilage homeostasis and inflammation (e.g., IL1B, TNF, EGF, TGFA, INS, IGF1 and IL6). Moreover, we studied pro-inflammatory mediators (IL1B and TNF) together with pro-growth signals for investigating their role in chondrocytes hypertrophy and highlighted the role of underreported players such as Inhibin beta A (INHBA), Defensin beta 1 (DEFB1), CXCL1 and Flagellin, and uncovered the way they cross-react in the phosphoproteomic level. CONCLUSIONS: The analysis presented herein, leveraged high throughput proteomic data via an ILP formulation to gain new insight into chondrocytes signaling and the pathophysiology of degenerative diseases in articular cartilage.

Smoking affects mRNA expression of bone morphogenetic proteins in human periosteum.

Periosteum is important for bone homoeostasis through the release of bone morphogenetic proteins (BMPs) and their effect on osteoprogenitor cells. Smoking has an adverse effect on fracture healing and bone regeneration. The aim of this study was to evaluate the effect of smoking on the expression of the BMPs of human periosteum. Real-time polymerase chain reaction was performed for BMP-2,-4,-6,-7 gene expression in periosteal samples obtained from 45 fractured bones (19 smokers, 26 non-smokers) and 60 non-fractured bones (21 smokers, 39 non-smokers). A hierarchical model of BMP gene expression (BMP-2 > BMP-6 > BMP-4 > BMP-7) was demonstrated in all samples. When smokers and non-smokers were compared, a remarkable reduction in the gene expression of BMP-2, -4 and -6 was noticed in smokers. The comparison of fracture and non-fracture groups demonstrated a higher gene expression of BMP-2, -4 and -7 in the non-fracture samples. Within the subgroups (fracture and non-fracture), BMP gene expression in smokers was either lower but without statistical significance in the majority of BMPs, or similar to that in non-smokers with regard to BMP-4 in fracture and BMP-7 in non-fracture samples. In smokers, BMP gene expression of human periosteum was reduced, demonstrating the effect of smoking at the molecular level by reduction of mRNA transcription of periosteal BMPs. Among the BMPs studied, BMP-2 gene expression was significantly higher, highlighting its role in bone homoeostasis.

Predictive role of cytokine gene polymorphisms for the development of femoral head osteonecrosis.

INTRODUCTION: Osteonecrosis (ON) is a multifactorial disease that leads to hip destruction. Lately, much focus has been at femoral head preservation with nonsurgical methods. In this study we examined the polymorphisms of IL-1α, IL-1R, IL-1RA, IL-4Rα, IL-1ß, IL-12, γIFN, TGF-ß, TNF-a, IL-2, IL-4, IL-6 and IL-10 genes for evaluation of their contribution in ON. MATERIAL AND METHODS: DNA was extracted from 112 ON patients and 438 healthy donors. Analysis of the polymorphisms was completed using the PCR-SSP method. Statistical analysis was performed using the χ ^{2} test to compare the genotype and allelic frequency distribution. RESULTS: The CT and GA genotypes of the IL-1α (-889) and TNF-a (-238) genes were found higher in the patients (51.8% and 10.8%, respectively) compared to the healthy donors (39.7% and 2.1%, respectively). In TGF-ß codon 25, the G to C polymorphism in the homozygous state was found in 1.8% of the patients and the C allele frequency was 8.9%, whereas the G allele frequency was 91.1%. Also, at the IL-10 (-1082) gene the GG genotype was 16.2% in the controls whereas in the patients was 7.2%. CONCLUSIONS: Based on the above, we showed that certain genotypes of the IL-1α, TGF-ß, IL-10 and TNF-a genes could be related in the pathogenesis of a complicated disease, such as osteonecrosis. The presence of one of the above mentioned polymorphisms or the simultaneous carriage of more than one may further increase the risk for osteonecrosis, especially in those at high risk, such as patients receiving corticosteroids.

FSH modulatory effect on human granulosa cells: a gene-protein candidate for gonadotrophin surge-attenuating factor.

Previous evidence indicates a homology of gonadotrophin surge-attenuating factor (GnSAF) to the carboxyl terminal of human serum albumin (HSA) and the ability of human granulosa cells to produce mRNA transcripts corresponding to this fragment, but the underlying mechanism is still unknown. This study investigated the role of FSH in vitro in the expression of the carboxyl terminal of HSA by human luteinized granulosa cells. Cells were cultured on poly-l-lysine-coated microscope slides in the absence or presence of 10 ng/ml FSH, followed by in-situ hybridization and immunocytochemistry. In the presence of FSH, mRNA transcripts corresponding to the carboxyl terminal of the HSA gene and corresponding protein could be detected in comparable intensity to that seen by hepatic HepG2 cells (positive control). Significantly lower expression was detected in granulosa cells cultured without FSH addition (P<0.01), but no expression was detected in HeLa cells. These results demonstrate for the first time that FSH stimulates the expression of the carboxyl terminal fragment of the HSA gene and corresponding protein in human luteinized granulosa cells. Therefore, the carboxyl terminal of HSA has a functional role in the ovary and this further supports the notion that this HSA fragment is a GnSAF-bioactive entity.

Different patterns of p16 immunoreactivity in cervical biopsies: correlation to lesion grade and HPV detection, with a review of the literature.

p16 is one extensively studied marker in gynecological pathology. However, its routine application in the diagnosis of squamous intraepithelial lesions of the uterine cervix may present difficulties for the general pathologist. The aim of the present study was to examine a series of 100 cervical biopsies/LEEP specimens, with detailed HPV-typing, for patterns of p16 immunoreactivity and possible correlations with morphology and HPV types. Four patterns of immunopositivity were recognized, according to the distribution of positively stained cells, and these correlated to lesion grade. A review of the pertinent literature concerning p16 immunoreactivity in squamous intraepithelial lesions and nonneoplastic epithelia of the uterine cervix is included in an effort to summarize the existing data and the remaining questions at both the practical and theoretical level.

Leptin receptor expression in neoplastic and normal ovarian and endometrial tissue.

PURPOSE: The objective of this study was to investigate the expression of leptin receptors in benign and malignant tumors of the ovaries and endometrium and its association with body mass index (BMI). METHODS: Histological uterine and ovarian samples of normal and neoplastic tissue from 35 patients aged 37-72 years were examined for the expression of leptin receptors with the method of RT-PCR. T. RESULTS: A BMI > 30 was correlated with increased expression of leptin receptors. Both Ra and Rb receptors were expressed in normal and neoplastic tissues. A statistically significant difference in leptin receptor expression was detected between normal and neoplastic tissue, with expression being around 5-fold higher in neoplatic tissue. CONCLUSION: Endometrial neoplasms and long leptin isoform receptor expression were associated with an increased BMI. A role of long isoform in endometrial carcinogenesis is proposed.

Modulating effect of leptin on basal and follicle stimulating hormone stimulated steroidogenesis in cultured human lutein granulosa cells.

BACKGROUND: In vitro data have shown conflicting results in terms of the effect of leptin on granulosa cells steroidogenesis. AIM: The aim of the present study was to investigate the effect of low and high doses of leptin on basal and FSH-induced steroids secretion by human luteinized granulosa cells in culture. MATERIALS AND METHODS: Granulosa cells were obtained from normal women undergoing in vitro fertilization (IVF) treatment and were cultured in serum-free conditions for 72 h. A one-way analysis of variance design was set to study the effect of leptin on basal and FSH-induced steroidogenesis. RESULTS: Leptin affected basal estradiol and progesterone secretion in a dose-related manner. In particular, leptin at low concentrations stimulated the secretion of estradiol (1 and 10 ng/ml) and progesterone (10 ng/ml), while at a high concentration (100 ng/ml) it suppressed the secretion of both steroids. A dose-related effect of leptin on FSH-induced steroidogenesis was not evident, since only the suppressive effect of the high concentration of leptin (100 ng/ml) reached statistical significance for both steroids. CONCLUSIONS: These results demonstrate that leptin affects the secretion of steroids in luteinized granulosa cells in a dose-dependent manner. Although a physiological role for leptin is possible, it is suggested that this protein is a mediator of negative rather than positive influential interactions on ovarian function that may compromise fertility.

L1 (CAM) (CD171) in ovarian serous neoplasms.

PURPOSE OF THE INVESTIGATION: The evaluation of L1 (CAM) as a tumor progression marker and as a prognostic factor in serous ovarian tumors. METHODS: L1 (CAM) protein expression was assessed by immunohistochemistry and Western blot in serous ovarian tumors [cystadenomas (n = 20), borderline tumors (n = 14) and carcinomas (n = 47)], and was correlated with stage,grade, progression-free survival time (PFS) and overall survival. RESULTS: L1 (CAM) immunoreactivity correlated significantly with stage and grade. It increased from benign tumors to early carcinomas and to advanced stage carcinomas progressively and significantly. In Stage III G3 carcinoma patients, low L1 (CAM) expressing tumors exhibited better response to chemotherapy and were associated with statistically significantly longer PFS (p = 0.002). CONCLUSION: L1 (CAM) expression represents a novel diagnostic marker in serous ovarian neoplasms that shows characteristics of tumor progression. L1 expression was associated with chemotherapy response.

Retinoid receptor alpha and Beta expression in serous ovarian tumors.

The expression of retinoid acid receptors alpha (RARalpha) and beta (RARbeta) and estrogen receptor alpha (ERalpha) was assessed by immunohistochemistry and Western blotting in normal ovaries, serous cystadenoma (n = 20), serous borderline (n = 14), and serous ovarian cancer (n = 47) and was correlated in cancer cases with stage, grade, progress-free survival (PFS), and survival. RARalpha was increasingly expressed in benign cystadenomas, borderline, and low-stage and advanced-stage neoplasms (p < 0.001). In stage III, G3 serous carcinoma, increased RARalpha expression was an independent prognostic factor associated with lower chemoresponse to first-line chemotherapy (taxol and carboplatin) and shorter PFS (p < 0.002).RARbeta and ERalpha expression did not correlate with RARalpha tumor characteristics or PFS and survival.

Expression of human serum albumin (HSA) mRNA in human granulosa cells: potential correlation of the 95 amino acid long carboxyl terminal of HSA to gonadotrophin surge-attenuating factor.

INTRODUCTION: According to previous studies, gonadotrophin surge-attenuating factor (GnSAF), which is assumed to be produced in human granulosa cells, has a homology with the carboxyl terminal of the human serum albumin (HSA) protein. In an attempt to validate these findings, whole or partial expression of the HSA gene was studied by RT-PCR analysis in human granulosa cells from women undergoing IVF treatment. METHODS: RT-PCR analysis of HSA RNA transcripts in luteinized granulosa cells was done in order to investigate the possible expression of the HSA gene. To ensure the specificity of PCR products, restriction enzyme and sequence analysis were performed. Western blot analysis was carried out to detect the possible expression of the albumin gene in granulosa cells. RESULTS: RT-PCR analysis and sequencing analysis of cDNA from granulosa cells revealed bands identical with those from the positive control for the amino as well as the carboxyl terminal corresponding to HSA gene at the cytoplasmic level. CONCLUSION: We have demonstrated that human granulosa cells express the carboxyl and amino terminal part of the HSA gene in levels comparable to those found in human hepatocytes. It is suggested that the coding gene for GnSAF may be a result of an alternative expression of HSA gene.

Telomerase reverse transcriptase mRNA expression in peripheral lymphocytes of patients with chronic HBV and HCV infections.

SUMMARY: Telomerase activity is present at low levels in peripheral lymphocytes (PL) and is upregulated upon activation, possibly protecting PL from telomere shortening. As decreased telomere length is considered a sign of cellular senescence, telomerase may, therefore, play an important role on immune function, organ regeneration and carcinogenesis. So far, quantification of human telomerase reverse transcriptase levels (hTERT) in PL, has not been reported. We determined hTERT mRNA levels in PL of hepatitis B virus (HBV) and hepatitis C virus (HCV) patients, in an attempt to address whether hTERT transcripts in PL are altered in these viral diseases, which are characterized by immune dysfunction and increased incidence of hepatocarcinogenesis. hTERT mRNA levels in PL of HBV (n = 17), HCV (n = 24) patients and healthy controls (n = 22) were quantified by real-time polymerase chain reaction. We observed significantly lower hTERT mRNA levels in HBV and HCV patients compared with healthy individuals (P < 0.05). hTERT mRNA levels were not associated with the patients' clinical status (inactive, hepatitis and cirrhosis). Also no correlation was observed between hTERT mRNA expression, and HBV and HCV replicative activity. In the inactive group (n = 18) we observed a negative correlation between hTERT mRNA expression and disease duration (rs = -0.52, P < 0.03). We performed for the first time an accurate quantification of hTERT mRNA expression in PL of HBV and HCV patients. The observed low levels of hTERT mRNA expression in the above patients may suggest its involvement in the immunopathogenesis of chronic viral hepatitis.

The role of human telomerase catalytic subunit mRNA expression in cervical dysplasias.

Telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression were investigated in cervical specimens and were correlated with cytologic findings and the presence of human papilloma virus (HPV) infection. Telomerase activity was evaluated by the telomeric repeat protocol assay and hTERT mRNA expression was evaluated by reverse transcriptase polymerase chain reaction (PCR). HPV DNA was detected by PCR, as well as restriction endonuclease digestion. HPV DNA was detected in all 82 specimens with abnormal cytologic findings and in 4 of 34 normal samples. Low-grade squamous intraepithelial lesions (LGSILs) were present in 74 of 82 specimens (90.2%) and high-grade squamous intraepithelial lesions (HGSILs) were present in 8 of 82 (9.75%) specimens. Seven of the eight HGSIL (87.5%) and 26 of 74 LGSIL (35.1%) specimens were hTERT positive, whereas all normal specimens were hTERT mRNA negative. Telomerase activity was detected in 21 of 74 (28.4%) LGSIL/atypical squamous epithelial cells of undetermined significance (ASCUS) and in five of eight (62.5%) HGSIL samples. A correlation was observed among telomerase activity, hTERT mRNA expression, and high-risk HPV infection in HGSIL samples (P < 0.001). High-risk HPV infection assessment showed 75% sensitivity and 72.2% specificity for HGSILs. Telomerase activity assessment in cervical smears showed sensitivity and negative predictive value (NPV) for HGSILs 62.5% and 96.7%, whereas specificity and positive predictive value (PPV) were 80.5% and 19.2%, respectively. hTERT mRNA expression assessment showed 87.5% sensitivity and 98.7% NPV for HGSILs, whereas specificity and PPV were 76% and 21.2%, respectively. Based on the above-described telomerase assessment values, it is suggested that the telomerase system might not be an appropriate diagnostic marker for cytology, given that the final evaluation must rely on a combination of all available test assessment data, clinical diagnosis, as well as the follow-up of all LGSIL samples that were positive for telomerase activation.

Absence of leptin expression and secretion by human luteinized granulosa cells.

Whether leptin is secreted by the human ovary is not known. The available data on leptin gene (ob gene) expression by human granulosa cells are conflicting. The aim of the present study was first to re-examine the expression of leptin messenger RNA (mRNA) by human granulosa cells and second to investigate if these cells have the ability to secrete leptin in cultures. Human luteinized granulosa cells were obtained from normal women undergoing in vitro fertilisation treatment after ovarian stimulation and follicle aspiration. The expression of ob gene was studied by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) both in primary granulosa cells treated immediately after oocyte recovery and in cells cultured up to 24 h under baseline and hormonally stimulated conditions (FSH: 100 ng/ml, LH: 100 ng/ml). ob mRNA transcripts were not detected in luteinized granulosa cells, while they were present in adipose tIssue cDNA. Actin gene expression was detected in all studied samples. Using a sensitive radioimmunoassay (lower limit of detection 0.05 ng/ml), leptin was undetectable in the culture media at all points during the 72 h cultures, while at the same time significant amounts of oestradiol and progesterone were produced particularly after the addition of androstendione (1 microM) to the incubation media. These results demonstrate for the first time that leptin is not secreted by human luteinized granulosa cells in cultures. From a physiological point of view, this may contribute to the development of the optimal follicular environment for oocyte maturation during the preovulatory period.

Molecular analysis of transferrin receptor mRNA expression in acute myeloid leukaemia.

Transferrin receptor (TfR, CD71) is an integral membrane glycoprotein that mediates cellular uptake of iron. In most tissues, TfR expression is correlated positively with proliferation and regulated at the post-transcriptional level. The available data regarding the pattern of TfR gene expression in haematological malignancies are very limited. In the present study, we evaluated TfR gene expression at the molecular level in bone marrow (BM) samples of 44 patients with de novo acute myeloid leukaemia (AML) at diagnosis with BM blasts > 85%. TfR mRNA levels were determined by densitometric analysis of quantitative reverse transcription polymerase chain reaction products corresponding to TfR exons 15-17. Each sample was tested in at least two independent experiments. In 13/44 patients, TfR messages were not detected (this is probably an underestimate as some positive results may be attributed to residual normal erythroid cells present in the samples). In 17/44, TfR mRNA levels were low-intermediate, and were high in the remaining patients (14/44). TfR mRNA positivity was significantly associated with older age. No statistically significant correlations were found either with specific French-American-British (FAB) subtypes or attainment of complete remission, incidence of relapse and survival (after adjusting accordingly for age and FAB subtype). The absence of TfR mRNA transcripts in a significant minority of cases suggests that alternative mechanisms of iron uptake may function in AML blast cells.

Valproic acid, trichostatin and their combination with hemin preferentially enhance gamma-globin gene expression in human erythroid liquid cultures.

BACKGROUND AND OBJECTIVES: In addition to conventional therapy, current treatment of thalassemia and sickle cell anemia includes inducers of hemoglobin F synthesis (hydroxyurea, erythropoietin, azacytidine and butyrate). However, because of concerns about the dose-limiting myelotoxicity, potential carcinogenicity and high cost of the above agents, an intensive search for less toxic or more effective drugs is ongoing. In this study we tested the effect of valproic acid and trichostatin, alone or in combination with hemin, on gamma chain synthesis in human erythroid liquid cultures. DESIGN AND METHODS: The agents were tested on erythroid human liquid cultures derived from normal peripheral blood, peripheral blood from beta(s)/beta(thal) patients, normal cord blood and normal bone marrow samples. The effect of the agents was expressed as increase of gamma/gamma+beta m-RNA, measured with competitive reverse transcriptase-polymerase chain recation (RT-PCR), or as increase of HbF, measured by high performance liquid chromatography (HPLC). RESULTS: Addition of valproic acid or trichostatin to human erythroid cell cultures preferentially enhanced gamma mRNA synthesis in all blood samples (2.9 to 3.5-fold). The addition of hemin enhanced the effect up to 10-fold. INTERPRETATION AND CONCLUSIONS: Valproic acid, trichostatin and their combination with hemin (all three FDA-approved drugs) preferentially increase gamma-globin chain synthesis and may be helpful for the treatment of hemoglobinopathies.