CHEK2 gene alterations independently increase the risk of death from breast cancer in Bulgarian patients

Checkpoint kinase 2 (CHEK2) is a?DNA damage-activated protein kinase implicated in cell cycle checkpoint control. The significance of CHEK2 alterations for breast cancer incidence and clinical behavior is not clear. In this study we determined the mutational spectrum and the level of promoter hypermethylation of CHEK2 gene in a?group of 145 Bulgarian patients with breast cancer. A?special emphasis was put on the clinical impact of CHEK2 alterations for breast cancerogenesis.PCR-SSCP-sequencing analysis of the entire coding sequence of CHEK2 gene was performed to estimate the mutational profile of tumor samples. Methylation-sensitive SSCP was applied to determine the methylation status in CpG clusters implicated in CHEK2 silencing. Clinical significance of CHEK2 alterations was evaluated using standard statistical methods.Mutations in CHEK2 were identified in 9.65 % of the patients. Two novel missense substitutions Thr476Met (C >T) and Ala507Gly (C>G), and a?novel silent variant Glu79Glu (A>G) were registered. However, hypermethylation was not found in any of the studied cases. Comparison with clinical characteristics showed that CHEK2 positive women have predominantly lobular type of breast carcinoma (р=0.04) and PR+ status (p=0.092). CHEK2 mutations correlated significantly with ATM+ status (p=0.046). All patients with the Glu79Glu variant were progesterone receptor positive (p=0.004). A?decrease in overall survival (p = 0.6301) and a?threefold increased independent risk of death (HR = 3.295, 95%CI 0.850-12.778, p?= 0.085) in CHEK2+patients was found.Our data indicate the significance of CHEK2 gene alterations in contrast to promoter hypermethylation in breast cancerogenesis. Specificity of CHEK2 mutational profile for the Bulgarian population was found. Though CHEK2 mutational status correlated with more favorable clinical characteristics, including positive progesterone receptor and lobular histological type, it independently increased the risk of death in these patients. Keywords: breast cancer, CHEK2, mutation, hypermethylation, clinico-pathological characteristics, overall survival.     

Contact sensitivity to hair dyes can be detected by the consumer open test

To avoid allergic reactions to hair dyes, an open test ("skin sensitivity test" or "dab test") is recommended 48 hours before the hair colouring procedure. We have investigated in a multicenter study, under standardized conditions and medical supervision, the validity of this test as a practical method to detect allergy to paraphenylenediamine (PPD)-containing hair dyes in 30 PPD patch test-positive and 30 PPD patch test-negative subjects. A defined volume of a marketed hair colouring product containing 1.8% PPD was applied in an open patch test to the retroauricular area. The grading method comprised the dermatologist's clinical evaluation and objective numerical scores. Allergic reactions were elicited in all PPD-positive subjects with a maximal intensity on Day 2; no allergic reactions were elicited in PPD-negative subjects. Increased severity of reactions by the dermatologist's clinical evaluation was correlated strongly with increasing numerical scores. The "skin sensitivity test" can be considered as an effective method to detect type IV hair dye allergy and as such, as an important factor in its secondary prevention.     

Clinical application of a fiber-reinforced post system

The tooth structure of an endodontically treated tooth was restored with a core on a post, both prefabricated with fiber-reinforced polymer-ceramic material (Sculpture/FibreKor) and subsequently with a metal-free crown. The tooth restoration is described in detail. It includes root canal preparation, polyvinyl siloxane impression taking, laboratory fabrication of the core on the post, adhesive cementation of the post-and-core system in the root canal, fabrication and cementation of the restorations, adjustment, and finishing. The success of this metal-free system is based on its increased flexural and tensile strength. Its application is safe, effective, and reliable. The high-quality aesthetics of the restorations is due to the translucency of the post-and-core material.     

Possibilities for predictive measurement of the transthoracic impedance in defibrillation.

Transthoracic electrical defibrillation is administered by high voltages and currents applied through large size electrodes. Therefore, the defibrillator load impedance becomes an essential factorfor the efficacy of the procedure. Attempts at prediction of transthoracic impedance by pre-shock measurement with low-amplitude high-frequency current have yielded apparently promising results. A reassessment was undertaken of the comparison between transthoracic impedance measured over a wide frequency range (bioimpedance spectroscopy) and measured during the shock. An estimation of the possibilities for pre-shock 'prediction ' of the impedance was performed, to allow adequate selection of the defibrillation energy or current with the intention of increasing the possibility for positive results with the first shock. Data were obtained from experimental fibrillation/defibrillation cycles on dogs andfrom cardioversion of atrial fibrillation or flutter in patients. The final results suggest that high-frequency low-amplitude impedance measurements cannot predict the corresponding value during the shock with very high accuracy, as differences up to 15-17% were found using biphasic pulses in patients. However, the method can be used for approximate assessments.     

Altered vitronectin receptor (alphav integrin) function in fibroblasts adhering on hydrophobic glass.

Function of integrins is crucial for adhesion, movement, proliferation, and survival of cells. In a recent study we found impaired fibronectin receptor function on hydrophobic substrata (G. Altankov et al. J Biomater Sci Polym Edn 1997;8:712-740). Here, we have studied the distribution and function of the vitronectin receptor (alphav integrin) in fibroblasts adhering on hydrophilic glass and hydrophobic octadecyl glass (ODS). The morphology of fibroblasts and the organization of actin cytoskeleton were studied and found to be altered on ODS, where the cells did not spread and possessed condensed actin. Pretreatment of the surfaces with serum or pure vitronectin improved cell morphology on both substrata, resulting in the development of longitudinal actin stress fibers. It was found with biotinylated vitronectin that comparable quantities of vitronectin were adsorbed from single vitronectin solutions or serum on glass and on hydrophobic ODS. The organization of the vitronectin receptors on the ventral cell surface was investigated in permeabilized cells showing normal focal adhesions in fibroblasts plated on glass but none of these structures on ODS. The distribution of alphav integrin on the dorsal cell surface was studied on nonpermeabilized living cells after antibody tagging. While fibroblasts adhering on plain or serum-treated glass developed a linear organization of alphav integrin, cells on plain and serum-treated ODS were not able to reorganize the vitronectin receptor. Studies on signal transduction with antiphosphotyrosine antibodies revealed co-localization of alphav integrin and phosphotyrosine in focal adhesions on glass and serum-treated glass. However, signaling was almost absent on plain ODS and weak on serum-treated ODS. It was concluded that alterations in vitronectin receptor function on the ventral cell surface caused by the hydrophobic material surface inhibit signal transfer and subsequent intracellular events that are important for the organization and function of integrins.     

Reduced expression of arrestin beta 2 by graft monocytes during acute rejection of rat kidneys

During acute rejection, numerous pro-inflammatory and cytotoxic monocytes accumulate in the vasculature of experimental renal allografts. Arrestins (ARRBs) are cellular regulators of inflammation, but nothing is known about their expression during rejection. Intravascular mononuclear graft leukocytes were isolated 4 days after kidney transplantation. ARRB1 and ARRB2 mRNA expression was reduced in blood leukocytes from allografts undergoing acute rejection, whereas on the protein level only ARRB2 was changed. Flow cytometry and confocal microscopy revealed ARRB1 and ARRB2 expression by monocytes and T cells, with a selective decrease in ARRB2 expression in monocytes during acute rejection. I-κB directly interacted with ARRB2 and the levels of both proteins strongly correlated. Concomitantly, the mRNA expression of NF-κB targeted genes increased. Our results suggest that activation of blood monocytes in renal isografts is dampened by high ARRB2 levels. During acute rejection, ARRB2 levels are reduced and classical monocyte activation is enabled via NF-κB activation.     

A double‐blind,randomized assessment of the irritant potential of sunscreen chemical dilutions used in photopatch testing*

Background: The maximum concentration of organic sunscreen filters in current usage that does not lead to irritant reactions when performing photopatch testing is not known. Such irritant reactions can be misinterpreted as positive photoallergic contact dermatitis reactions. Objective: To determine the frequency of irritant reactions to 19 organic sunscreen filters in current use. Patients/Methods: Ninety‐four healthy volunteers were photopatch tested using the European consensus methodology to three different concentrations (2%, 5%, and 10%) of 19 organic sunscreen filters at the Photobiology Unit in Dundee, UK. Results: Of the 94 subjects recruited, 80 were analysed after withdrawals and exclusions. Of the 19 organic sunscreen filters studied, only 2 compounds led to irritant reactions in ≥5% subjects. Five per cent and 10% benzophenone‐4 led to irritant reactions in four and six subjects, respectively. Five per cent methylene bis‐benzotriazolyl tetramethylbutylphenol led to irritant reactions in six subjects, but unlike benzophenone‐4, this was not in a dose‐dependent fashion. Conclusions: When performing photopatch testing according to the European consensus methodology with these 19 organic sunscreen filters, a 10% concentration is suitable for all filters, except benzophenone‐4, which should be tested at a concentration of 2%.     

Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity

Contact hypersensitivity (CHS) is a T cell-mediated skin inflammation induced by epicutaneous exposure to haptens in sensitized individuals. We have previously reported that CHS to dinitrofluorobenzene in mice is mediated by major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. In this study, we show that CD8(+) T cells mediate the skin inflammation through their cytotoxic activity. The contribution of specific cytotoxic T lymphocytes (CTLs) to the CHS reaction was examined both in vivo and in vitro, using mice deficient in perforin and/or Fas/Fas ligand (FasL) pathways involved in cytotoxicity. Mice double deficient in perforin and FasL were able to develop hapten-specific CD8(+) T cells in the lymphoid organs but did not show CHS reaction. However, they did not generate hapten-specific CTLs, demonstrating that the CHS reaction is dependent on cytotoxic activity. In contrast, Fas-deficient lpr mice, FasL-deficient gld mice, and perforin-deficient mice developed a normal CHS reaction and were able to generate hapten-specific CTLs, suggesting that CHS requires either the Fas/FasL or the perforin pathway. This was confirmed by in vitro studies showing that the hapten-specific CTL activity was exclusively mediated by MHC class I-restricted CD8(+) T cells which could use either the perforin or the Fas/FasL pathway for their lytic activity. Thus, cytotoxic CD8(+) T cells, commonly implicated in the host defence against tumors and viral infections, could also mediate harmful delayed-type hypersensitivity reactions.