TNF-α gene polymorphisms: association with age-related macular degeneration in Russian population

AIM: To study polymorphisms in promotor regions of tumor necrosis factor (TNF)-α TNF-863A/C (rs1800630), TNF-308A/G (rs1800629), and TNF-238A/G (rs361525) in patients with age-related macular degeneration (AMD) and associations of complex TNF-α genotypes with AMD. METHODS: One hundred and two patients (82 women, 20 men; mean age 64.2±1.2y) with AMD and 100 healthy age- and sex-matched controls (82 women, 18 men; 60±1.4y) were included in the study. All subjects were Caucasian, all subjects and their parents were inhabitants of Russia. Genomic DNA was obtained from EDTA-preserved blood using the standard phenol-chloroform method. Polymorphisms were detected by polymerase chain reaction followed by the restriction fragment length polymorphism method. The following TNF-α genotypes were studied: TNF-α-238 AA, GA, GG, TNF-α-308 AA, GA, GG, TNF-α-863 AA, CA, CC. RESULTS: Differences in TNF-α-863 and TNF-α-238 genotypes frequencies in patients with AMD and healthy controls were not found. The distribution of TNF-α-308 AA and TNF-α-308 GA genotypes was significantly different between the studied group and the controls [odds ratios (OR) =0.22, P=0.0287 and OR=2.91, P=0.0063, respectively]. TNF-863CC/TNF-308GA and TNF-308GA/TNF-238GG genotypes were associated with the increased risk of AMD (OR=2.48, P=0.0332 and OR=2.51, P=0.0187, respectively). Five genotypes combinations appeared to be protective. CONCLUSION: In the present study, single nucleotide polymorphisms and complex polymorphisms of one of the key inflammatory cytokines TNF-α, and a number of significant associations of these polymorphisms with AMD in Russian population have been shown. Complex analysis of genotypes could be important in AMD risk factors detection and studying pathogenesis.     

Effectiveness of inhaled hypertonic saline application for sputum induction to improve Mycobacterium tuberculosis identification in patients with pulmonary tuberculosis

Wiener Medizinische Wochenschrift - This study assessed the effectiveness and diagnostic significance of hypertonic saline sputum induction for improving Mycobacterium tuberculosis (MTB) detection....     

Simulation with cells in vitro of tamoxifen treatment in premenopausal breast cancer patients with different CYP2D6 genotypes

Background and Purpose

Tamoxifen is a prodrug that is metabolically activated by 4-hydroxylation to the potent primary metabolite 4-hydroxytamoxifen (4OHT) or via another primary metabolite N-desmethyltamoxifen (NDMTAM) to a biologically active secondary metabolite endoxifen through a cytochrome P450 2D6 variant system (CYP2D6). To elucidate the mechanism of action of tamoxifen and the importance of endoxifen for its effect, we determined the anti-oestrogenic efficacy of tamoxifen and its metabolites, including endoxifen, at concentrations corresponding to serum levels measured in breast cancer patients with various CYP2D6 genotypes (simulating tamoxifen treatment).

Experimental Approach

The biological effects of tamoxifen and its metabolites on cell growth and oestrogen-responsive gene modulation were evaluated in a panel of oestrogen receptor-positive breast cancer cell lines. Actual clinical levels of tamoxifen metabolites in breast cancer patients were used in vitro along with actual levels of oestrogens observed in premenopausal patients taking tamoxifen.

Key Results

Tamoxifen and its primary metabolites (4OHT and NDMTAM) only partially inhibited the stimulant effects of oestrogen on cells. The addition of endoxifen at concentrations corresponding to different CYP2D6 genotypes was found to enhance the anti-oestrogenic effect of tamoxifen and its metabolites with an efficacy that correlated with the concentration of endoxifen; at concentrations corresponding to the extensive metabolizer genotype it further inhibited the actions of oestrogen. In contrast, lower concentrations of endoxifen (intermediate and poor metabolizers) had little or no anti-oestrogenic effects.

Conclusions and Implications

Endoxifen may be a clinically relevant metabolite in premenopausal patients as it provides additional anti-oestrogenic actions during tamoxifen treatment.     

Physical and Numerical Simulations of Closed Die Hot Forging and Heat Treatment of Forged Parts

The paper describes physical and numerical simulations of a manufacturing process composed of hot forging and controlled cooling, which replace the conventional heat treatment technology. The objective was to investigate possibilities and limitations of the heat treatment with the use of the heat of forging. Three steels used to manufacture automotive parts were investigated. Experiments were composed of two sets of tests. The first were isothermal (TTT) and constant cooling rate (CCT) dilatometric tests, which supplied data for the identification of the numerical phase transformation model. The second was a physical simulation of the sequence forging-cooling on Gleeble 3800, which supplied data for the validation of the models. In the numerical part, a finite element (FE) thermal-mechanical code was combined with metallurgical models describing recrystallization and grain growth during forging and phase transformations during cooling. The FE model predicted distributions of the temperature and the austenite grain size in the forging, which were input data for further simulations of phase transformations during cooling. A modified JMAK equation was used to calculate the kinetics of transformation and volume fraction of microstructural constituents after cooling. Since the dilatometric tests were performed for various austenitization temperatures before cooling, it was possible to include austenite grain size as a variable in the model. An inverse algorithm developed by the authors was applied in the identification procedure. The model with optimal material parameters was used for simulations of hot forging and controlled cooling in one of the forging shops in Poland. Distributions of microstructural constituents in the forging after cooling were calculated. As a consequence, various cooling sequences during heat treatment could be analyzed and compared.     

Overexpression of inducible nitric oxide synthase in the kidney of the spontaneously hypertensive rat

In kidney, nitric oxide (NO) produced by nitric oxide synthase (NOS) regulates sodium and water excretion and renal medullary blood flow. However, excessive NO production causes nitrative damage and oxidative stress. Since oxidative stress may be linked to hypertension, we examined the expression and activity of inducible NOS (iNOS) in the kidney of the spontaneously hypertensive rat (SHR) and compared our findings to control normtotensive Wistar Kyoto (WKY) rat. Compared with WKY rat, there was significant (p < .05) overexpression (by 96%) and increased (2-fold) activity of iNOS in the cortex but not in the outer medulla, of SHR kidney; in the inner medulla, there was a 6.9-fold increase in iNOS activity in SHR. Increased expression (by 104%) and activity (3.3-fold) of iNOS was specifically observed in proximal tubules (PTs) of the cortex, accompanied by higher (2-fold) tissue nitrite levels. Although certain antioxidant enzymes such as catalase and Mn-superoxide dismutase were overexpressed, glutathione peroxidase was underexpressed in SHR PTs. Overexpression of the inducer of the iNOS promoter, nuclear factor-kappaB (NF-kappaB), with elevated nitrotyrosinylated proteins, further confirmed an elevated state of iNOS-induced oxidative stress in SHR kidneys, possibly signifying its role in the maintenance of essential hypertension seen in these animals.     

Early and persistent impaired percent alpha variability on continuous electroencephalography monitoring as predictive of poor outcome after traumatic brain injury

OBJECT: Early prediction of outcomes in patients after they suffer traumatic brain injury (TBI) is often nonspecific and based on initial imaging and clinical findings alone, without direct physiological testing. Improved outcome prediction is desirable for ethical, social, and financial reasons. The goal of this study was to determine the usefulness of continuous electroencephalography (EEG) monitoring in determining prognosis early after TBI, while the patient is in the intensive care unit. METHODS: The authors hypothesized that the reduced percentage of alpha variability (PAV) in continuous EEG tracings indicates a poor prognosis. Prospective continuous EEG monitoring was performed in 89 consecutive patients with moderate to severe TBI (Glasgow Coma Scale [GCS] Scores 3-12) from 0 to 10 days after injury. The PAV was calculated daily, and the time course and trends of the PAV were analyzed in comparison with the patient's Glasgow Outcome Scale (GOS) score at the time of discharge. In patients with GCS scores of 8 or lower, a PAV value of 0.1 or lower is highly predictive of a poor outcome or death (positive predictive value 86%). The determinant PAV value was obtained by Day 3 after injury. Persistent PAV values of 0.1 or lower over several days or worsening of the PAV to a value of 0.1 or lower indicated a high likelihood of poor outcome (GOS Scores 1 and 2). In comparison with the combination of traditional initial clinical indicators of outcome (GCS score, pupillary response to light, patient age, results of computerized tomography scanning, and early hypotension or hypoxemia), the early PAV value during the initial 3 days after injury independently improved prognostic ability (p < 0.01). CONCLUSIONS: Continuous EEG monitoring performed with particular attention paid to the PAV is a sensitive and specific method of prognosis that can indicate outcomes in patients with moderate to severe TBI within 3 days postinjury.     

The changing nature of murder in Russia

The death rate from homicide in Russia increased rapidly during the 1990 s. It is now about 20 times higher than in western Europe and is among the highest recorded anywhere in the world. However, this issue has received little attention so far from public health researchers or policymakers.This paper describes the changing nature of homicide during the 1990 s in Russia as a whole and, in more detail, in the Udmurt Republic. The study uses data from three sources: routine mortality data for Russia from 1970 to 1999; statistics on criminal investigations and convictions in Russia between 1990 and 1997; and an in-depth study of homicide trial records in the Udmurt Republic in 1989-1991 and 1998.Deaths from homicide increased between 1970 and 1985, falling slightly during the 1985 anti-alcohol campaign and then resuming their increase until 1994. Another fall in the late 1990 s was arrested in 1998, with an increase in 1999. By 1999 the age standardised homicide death rate in Russia was 81% higher than in 1990, an increase almost twice that of all causes of death combined. Throughout the 1990 s about 10% of those convicted of homicide were female. Of those homicides leading to convictions in the Udmurt Republic, 71% of those killed by males were male, as were 76% of those killed by females. Killings of women by men often involved sexual assaults. In Russia as a whole, about 80% of those convicted of homicide were reported to be under the influence of alcohol at the time. In the Udmurt Republic, where data on both offender and victim were available, victims were also commonly intoxicated.The nature of homicide in Russia has changed considerably in less than a decade, with many more now involving aggravating circumstances, such as murder to conceal another crime, in association with robbery or rape, or by a group of people. Although still a small proportion of the total convicted, the number of murders by hired killers is also on the rise.The characteristics of those convicted of homicide have also changed during the 1990 s. They are now younger, less likely to have previous convictions, and to have a more diverse range of educational levels. The previous urban-rural gap, with higher levels in rural areas, has also narrowed.     

The influence of conditioned medium from mouse intestinal epithelial cells on the proliferative activity of crypt cells: role of granulocyte-macrophage colony-stimulating factor

Background: The aim of this work was to study the influence of soluble factors produced by native mouse intestinal epithelial cells (IECs) on the proliferative activity of freshly isolated intestinal crypt cells. Methods: The crypt cells were cultured with either conditioned medium and its ultrafiltrates or recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of neutralizing anti-GM-CSF antibodies. GM-CSF in culture medium was identified by the electrochemiluminescence method. Results: It was demonstrated that the IEC conditioned medium contained GM-CSF. This cytokine led to both the upregulation and downregulation of crypt cell proliferative activity, depending on its concentration in the culture medium. The effect of native GM-CSF was reproduced with recombinant mouse GM-CSF: 25 and 5 ng/ml inhibited the proliferative activity, whereas 1 ng/ml led to its significant stimulation. Conclusions: Freshly isolated murine IECs produce GM-CSF, which plays a critical role in crypt cell proliferative activity in vitro. These results suggest the involvement of this factor in the regulation of the crypt proliferative zone, in an autocrine and/or paracrine manner. Received: February 25, 2002 / Accepted: July 26, 2002     

Generation and characterization of monoclonal antibodies to protein kinase 2 (CK2) beta subunit

Protein kinase 2 (CK2) is a ubiquitous and constitutively active serine/threonine protein kinase with various cell functions. It typically forms tetrameric complexes consisting of two catalytic (alpha and/or (alpha') and two regulatory (beta) subunits. The aim of this study was to produce monoclonal antibodies (MAbs) against the CK2beta subunit and to characterize their suitability for Western blotting, immunoprecipitation, and immunohistochemical applications. Bacterially expressed CK2beta-6His-GST recombinant protein has been used as an antigen. Balb/c mice were immunized and given a final boost, and their spleen cells were collected and fused with SP2/0 myeloma cells using PEG 2000. The fused cells were then selected in the HAT-RPMI medium. Anti- CK2beta high-titer antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in HT-RPMI medium supplemented with 20% fetal bovine serum (FBS). A total of 10 IgG-producing cell lines were selected and further tested for their reactivity with the CK2beta subunit using ELISA, Western blots, immunoprecipitation, and immunostaining of formaldehyde-fixed paraffin-embedded tissue sections. The results obtained clearly indicate that several clones produce antibodies that recognize specifically recombinant and endogenous CK2beta subunit in Western blotting and immunoprecipitation, and are suitable for immunohistochemical analysis. In summary, the produced antibodies will be useful for researchers investigating signaling pathways involving CK2 kinase and their deregulation in human pathologies.