Molecular genetic analysis of BAX and cyclin D1 genes in patients with malignant glioma

INTRODUCTION AND OBJECTIVES: Brain tumorigenesis is a complex process involving multiple genetic alterations. Cyclin D1 and BAX genes are two of the most important regulators in controlling the normal proliferation and apoptosis of cells, respectively. In this study, we analysed the possibilities of involvement of cyclin D1 and BAX genes in the gliomagenesis. METHODS AND RESULTS: In determining gene alterations of exon 4 of cyclin D1 gene and exon 6 of BAX gene, all samples were amplified by polymerase chain reaction (PCR) and subsequently by direct sequencing. Our results showed a frameshift mutation (G base deletion) at nucleotide 82 of codon 28 in exon 4 of the cyclin D1 gene and another frameshift mutation with a deletion of C base at nucleotide 153 of exon 6 of the BAX gene in two separate cases of a glioblastoma multiform (WHO Grade IV) sample. CONCLUSION: These findings suggest that both cyclin D1 and BAX genes alteration are rarely found in brain tumors. However, the alteration might cause a significant effect of the normal protein production and this might contribute to the development of brain tumorigenesis in Malaysian patients.     

Correlation of CXCL12 Expression and FoxP3+ Cell Infiltration with Human Papillomavirus Infection and Clinicopathological Progression of Cervical Cancer

Human cervical cancer is an immunogenic tumor with a defined pattern of histopathological and clinical progression. Tumor-infiltrating T cells contribute to immune control of this tumor; however, cervical cancer dysregulates this immune response both through its association with human papillomavirus (HPV) infection and by producing cytokines and chemokines. Animal tumor models have revealed associations between overproduction of the chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) and dysregulation of tumor-specific immunity. We therefore proposed that CXCL12 expression by cervical precancerous and cancerous lesions correlates with histopathological progression, loss of immune control of the tumor, and HPV infection. We found a significant association between cancer stage and CXCL12 expression for squamous and glandular lesions as well as with the HPV16+ (high-risk) status of the neoplastic lesions. Cancer progression was correlated with increasing levels of FoxP3 T-cell infiltration in the tumor. FoxP3 and CXCL12 expression significantly correlated for squamous and glandular neoplastic lesions. These observations were supported by enzyme-linked immunosorbent assay and Western blotting. In addition, we demonstrated CXCL12 expression by dyskaryotic cells in ThinPrep cervical smears. This study robustly links increased CXCL12 expression and FoxP3⁺-cell infiltration to HPV infection and progression of cervical cancer. It supports the detection of CXCL12 in cervical smears and biopsies as an additional biomarker for this disease.Human cervical cancer is an immunogenic tumor, and several tumor-specific antigens have been identified in recent years.^1,2,3 Persistent infection with high-risk human papillomavirus (HPV) (types 16 and 18) has been shown to be an important risk factor for malignant transformation of the cervical epithelium.^4,5 Tumor immunity in this context results from the positive recognition of tumor antigens by immature dendritic cells, which subsequently migrate to draining lymph nodes and interact with and activate both helper CD4⁺ T cells and effector antigen-specific CD8⁺ T cells.¹ Activated and targeted antigen-specific cytotoxic CD8⁺ T cells subsequently circulate through the peripheral blood to the tumor and then selectively transmigrate into the cancer itself and kill tumor cells. Immune control of cervical cancer is therefore critically dependent on the activation and migration of tumor-specific cytotoxic T cells into the tumor and the subsequent killing of neoplastic cells. The success of the immune response in destroying cervical cancer is thought to depend on the relationship between the kinetics of tumor growth and the efficacy of tumor-specific cytotoxic T cells (in balance with regulatory T cells) in infiltrating the tumor and killing neoplastic cells over time. It is well established that the magnitude of T-cell infiltration within certain forms of cancer correlates with a favorable prognosis.⁶Tumors, including cervical cancer, evade immune recognition using a number of different mechanisms, including the down-regulation of major histocompatibility class I antibodies, the immunological ignorance to tumor antigens, the lack of costimulatory molecules and antigen loss, or the expression of inhibitory molecules.^7,8 Several studies have also shown that tumors, including cervical cancer, overexpress specific chemokines, such as stromal cell-derived factor 1 (SDF-1 or CXCL12), that are thought to dysregulate the immune response to the tumor.^9,10,11 Chemokines are a superfamily of 8- to 11-kDa proteins found in humans that have been shown to be critical in causing directional movement of immune cells in vitro and in determining the localization of leukocytes in models of inflammatory, immune-mediated diseases, and cancer.^11,12,13 Chemokines have been shown to act as chemoattractants for leukocyte subpopulations including T cells and dendritic cells. Chemokines signal via G_αi protein-coupled receptors on the cell surface and subsequently induce directed cell movement in response to a gradient of the chemokines.¹⁴The chemokine CXCL12 is a known T-cell chemoattractant that selectively binds the receptor CXCR4.^9,10,11 Until recently CXCL12 was thought to have chemoattractant activity only for T cells. We recently demonstrated that the chemokine CXCL12 could serve as a bidirectional cue, attracting T cells at low concentrations and repelling at high concentrations in vitro and in vivo.¹⁵ Certain murine models of melanoma and ovarian cancer indicate that the effect of CXCL12 on the tumor immune response is dose dependent.¹⁶ Low levels of CXCL12 expression result in infiltration of the tumor by T cells, a delay in tumor growth, and the development of a long-lived tumor-specific T cell-mediated immune response. In contrast, high levels of CXCL12 expression result in reduced infiltration of the tumor by tumor antigen-specific T cells¹⁷ and the suppression of antitumor immune responses via various mechanisms including the intratumoral accumulation of FoxP3⁺ suppressor T cells.^18,19We hypothesized that secretion of CXCL12 by cervical tumors is correlated with progression of the tumor and loss of immune control. To explore this hypothesis we first examined whether clinicopathological progression of cervical cancer is associated with an increase in CXCL12 expression and a concomitant reduction in immune cell infiltration into the tumor as the tumor progresses from a preinvasive to an invasive state. CXCL12 expression and T-cell infiltration were studied in tissue sections of normal cervix, preinvasive, microinvasive, and invasive squamous cervical cancer, and preinvasive and invasive adenocarcinoma using immunohistochemical (IHC) staining methods in concert with quantitative digital image analysis, Western blotting, and enzyme-linked immunosorbent assay (ELISA). Because HPV is associated with cervical cancer, we also examined the relationship between HPV status and CXCL12 expression in normal, preinvasive, and invasive cervical cancer. In this way we demonstrated that CXCL12 is a robust biomarker for clinical progression that is correlated both with HPV infection and the accumulation of FoxP3⁺ T cells in the tumor.     

Genetic aspects of valvulopathies

Valvular dystrophies due to myxoid degeneration are common and potentially serious cardiac pathologies. They constitute a heterogeneous group of which the most usual is idiopathic mitral valvular prolapse (Barlow's disease). The majority of mitral valvular prolapses are sporadic, but there are several familial forms. Transmission is usually autosomal dominant with incomplete penetrance and variable expression. The first chromosomal location to be identified was on the 16p11-13 chromosome. Since then, two other loci have been identified on the 11p15.4 and 13q31-32 chromosomes. Our team has recently identified the first gene responsible for myxoid valvulopathy linked to the X chromosome, from a large family of 318 members. This is the gene that codes for filamin A, which is a cytoskeleton protein. The frequency of mutations in this gene is still unknown, but out of 7 families in which transmission was compatible with X-linked transmission, mutations were discovered in 4 of the families. Thanks to a genetic epidemiological approach, we have also demonstrated that there are familial forms of aortic stenosis, which are probably common. Identification of the genes implicated in these common forms of valvular pathology is important, as it will allow a better understanding of the pathophysiology of these valvular disorders and could lead to better therapeutic management in the future.     

Marginal Accuracy of Monolithic and Veneered Zirconia Crowns Fabricated by Conventional and Digital Workflows

Purpose

To compare the marginal accuracy of zirconia crowns fabricated by different workflows (conventional and digital) and designs (monolithic and veneered).

Materials and Methods

A prepared maxillary first molar was used for the study. Four workflow combinations were evaluated: (1) intraoral scanning and monolithic zirconia (IOS-M), (2) intraoral scanning and veneered zirconia (IOS-V), (3) conventional impression and monolithic zirconia (IMP-M), and (4) conventional impression and veneered zirconia (IMP-V). All of the specimens had similar designs. The veneered groups had a buccal cutback for esthetic veneer application. A total of 10 crowns were produced in each workflow. The vertical and horizontal marginal accuracies were measured with a traveling microscope. Depending on the normality of the data, one-way analysis of variance test or Kruskal-Wallis test were applied to evaluate the differences among the groups (α = 0.05).

Results

The most superior vertical marginal accuracy was observed for IOS-V (mean = 22.5 μm; SD = 6.7 μm), followed by IMP-V (mean = 23.9 μm; SD = 7.8 μm), IOS-M (mean = 28.7 μm; SD = 10.3 μm), and IMP-M (mean = 39.8 μm; SD = 22.0 μm), respectively (p < 0.001). The IOS-M had the greatest mean horizontal discrepancies (mean = 23.9 μm; SD = 4.3 μm) followed by IMP-M (mean = 21.3 μm; SD = 5.7 μm), IMP-V (mean = 19.2 μm; SD = 5.3 μm) and IOS-V (mean = 17.6 μm; SD = 5.7 μm) (p < 0.001).

Conclusions

Monolithic zirconia crowns fabricated digitally had superior marginal accuracy than monolithic zirconia crowns fabricated conventionally. Esthetic buccal veneering of predominantly monolithic zirconia copings improved the vertical and horizontal marginal accuracies.     

The use of enzyme-linked immunosorbent assay for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom and venom antibodies.

The specificity and sensitivity of an indirect and two (an ‘ordinary’ and a ‘rapid’) double sandwich enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom were examined. The three assays were equally sensitive and the accuracy of the assays was not substantially affected by individual variation in the venom composition. The specificity of the assays was examined against 26 venoms from snakes of the families Viperidae and Elapidae. While the double sandwich ELISA procedures were sufficiently specific to be used in the clinical immunodiagnosis of C. rhodostoma bite in Malaysia, the indirect ELISA procedure exhibited extensive cross-reactivity with other Malaysian pit viper venoms. Attempts were made to improve the specificity of the indirect ELISA procedure for the quantitation of C. rhodostoma venom. A ‘low ELISA cross-reactivity’ venom fraction (termed VF52) was isolated from C. rhodostoma venom by repeated Sephadex G-100 gel filtration chromatography. The indirect ELISA procedure using antibodies to VF52 as immunoreagent showed an improvement in specificity. The use of the indirect ELISA procedure for the detection of C. rhodostoma antibodies was also examined and the results show that the assay was sufficiently specific to be used for retrospective diagnosis of C. rhodostoma bite in Malaysia, in particular when VF52 was used as the coating antigen.     

Nitrous oxide pollution in the operating room. A comparison of two modes of ventilation

The use of laryngeal mask airway (LMA) as an alternative to the endotracheal tube (ETT) is becoming more popular in the practice of anesthesia. It is undeniable that this device has numerous advantages over endotracheal tube, however it does not provide an airtight seal between the airway and atmospheric gases. This may lead to pollution of the operating room environment with nitrous oxide. One hundreds adult patients undergoing general anaesthesia were divided into two groups. The airway in Group I was maintained with LMA with spontaneous ventilation and ETT with intermittent positive pressure ventilation (IPPV) was used for Group II. The result demonstrated that the ETT group recorded concentrations of nitrous oxide that were well above the NIOSH recommended eight hour time weighted average of 25ppm throughout the duration of surgery when compared to patients using LMA.     

Serum selenium level and other risk factors for breast cancer among patients in a Malaysian hospital

Objective The aim of this study is to investigate the association between breast cancer and serum selenium level as well as other risk factors for breast cancer. Methods A matched case-control study was conducted in a hospital in Malaysia from July 2000 to January 2001 and from May 2001 to June 2001. Sixty-two newly diagnosed breast cancer patients were selected as the cases. Each control, selected from the same hospital population was matched to each case according to age, ethnic group, and menopausal status. Results The mean selenium concentration among the cases was significantly lower than that among the control. There was a significant association (p<0.05) between breast cancer and low selenium serum level, nulliparity (OR=5.5,95% CI=1.22 to 24.81), exposure to cigarette smoke (OR=2.2, 95% CI=1.04 to 4.65) and use of oral contraceptives (OR=3.0, 95% CI=1.09 to 8.25) as determined by the McNemar test. Multivariate analysis showed that nulliparity (OR=10.08, 95% CI=1.48 to 68.52) and use of oral contraceptives (OR=3.66, 95% CI=1.36 to 9.87) were associated with increased breast cancer risk. An increased selenium concentration contributes to a reduced risk of breast cancer (OR=0.89, 95% CI=0.84 to 0.94). Conclusion The results suggest that use of oral contraceptive pills, being nulliparous, and a low serum selenium level are associated with breast cancer.