Unfractionated spleen cells, B cells from normal mice, and nu/nu spleen cells respond to the addition of bacterial lipopolysaccharide (LPS) and T-cell-replacing factor (TRF) by production of plaque-forming cells (PFC) in excess of the number expected from the addition of LPS and TRF separately. This synergistic activity is dependent on the presence of the antigen, SRBC. Supernatants of both allogeneic spleen cell mixtures and spleen cells cultured with Con A are effective and synergize best at concentrations suboptimal for their ability to act as TRF alone. Culture supernatants of unstimulated normal or fractionated cell populations are ineffective. Synergy is not dependent on the presence of macrophages in the cultures. Purified LPS free from active contaminants, as well as commercially available LPS, show synergy with TRF. Synergy was seen when TRF was added at initiation of culture or 24 hr later. It is suggested that synergy is the equivalent of LPS adjuvant activity, that the role of T cells in LPS adjuvanticity is that of a conventional cooperating cell, and the LPS acts as an adjuvant by inducing B cells to become more sensitive to T cell helper factors.

Previous studies in anesthetized animals reported that the primary auditory cortex (A1) showed homogenous phasic responses to FM tones, namely a transient response to a particular instantaneous frequency when FM sweeps traversed a neuron's tone-evoked receptive field (TRF). Here, in awake cats, we report that A1 cells exhibit heterogeneous FM responses, consisting of three patterns. The first is continuous firing when a slow FM sweep traverses the receptive field of a cell with a sustained tonal response. The duration and amplitude of FM response decrease with increasing sweep speed. The second pattern is transient firing corresponding to the cell's phasic tonal response. This response could be evoked only by a fast FM sweep through the cell's TRF, suggesting a preference for fast FM. The third pattern was associated with the off response to pure tones and was composed of several discrete response peaks during slow FM stimulus. These peaks were not predictable from the cell's tonal response but reliably reflected the time when FM swept across specific frequencies. Our A1 samples often exhibited a complex response pattern, combining two or three of the basic patterns above, resulting in a heterogeneous response population. The diversity of FM responses suggests that A1 use multiple mechanisms to fully represent the whole range of FM parameters, including frequency extent, sweep speed, and direction.

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.

OBJECTIVE

To investigate the effect of the radiographic and clinical quality of coronal restorations on the composition of the root canal flora of teeth with necrotic pulps and teeth with root fillings associated with apical periodontitis.

METHODS

Twenty-eight necrotic pulps and 35 root filled canals with signs of apical periodontitis were studied. Both the coronal filling (presence of radiographically or clinically deficient margins and/or secondary caries) and the root filling (homogeneity and length) were scored. Bacterial root canal samples were taken with sterile paper points under rubber dam and using measures to prevent contamination. A DNA-based nonculture bacterial identification technique was used, namely terminal restriction fragment length polymorphism (T-RFLP) analysis.

RESULTS

Twelve samples were negative for bacterial DNA. A total of 33 different terminal restriction fragments (TRFs) were detected. The Fusobacterium nucleatum/Streptococcus mitis group was the most frequently encountered TRF. The mean number of TRFs per necrotic pulp was 6.2 and 5.8 for the groups with acceptable and unacceptable coronal restorations, respectively. This difference was not significant. In the root filled group, these values (respectively, 5.2 and 8.6) were statistically significantly different (P < 0.05). The following parameters in root filled teeth had no significant influence on the mean numbers of TRFs detected: the length and homogeneity of the root filling and the type of tooth (anterior-premolar-molar).

CONCLUSIONS

T-RFLP allowed the rapid assessment of bacterial biodiversity in root canal samples. The technique revealed the presence of bacteria that have rarely been described in the root canals of teeth with apical periodontitis. Biodiversity in the root filled group was high, as compared with culture-dependent studies where monoinfections were more frequently reported. Only in root filled teeth did defective coronal restorations have a statistically significant influence on the mean numbers of detected TRFs per sample.

OBJECTIVE

: To develop suitable strategies for quantification of longitudinal relaxation time (T1) by means of ultrashort echo time (UTE) sequences and the variable flip-angle approach in materials and tissues with extremely fast signal decay.

METHODS

: A recently published modified Ernst equation, which correctly accounts for in-pulse relaxation of transverse magnetization, was used to numerically determine optimal flip angles for reliable assessment of T1 in case of extremely short effective transverse relaxation time (T2*). Various ratios of repetition time (TR) to T1 and radiofrequency (RF) pulse duration (TRF) to T2* were evaluated. Theoretical considerations were applied to solid polymeric material (T2* = 0.295 milliseconds), and T1 quantification was performed using various optimized flip-angle approaches at different RF pulse durations (TRF = 0.1-0.4 milliseconds). Furthermore, in vivo measurement of T1 in cortical bone was exemplarily performed in 3 healthy volunteers to test the applicability of the proposed method in vivo. For in vitro and in vivo studies, MR imaging was performed on a 3 T whole-body MR system using a 3D UTE sequence with a rectangular excitation pulse and centric radial readout.

RESULTS

: Optimal flip angles were shown to be strongly dependent on TR/T1 and TRF/T2* ratios. Exemplarily, longitudinal relaxation time of the investigated solid polymeric material was determined to T1 = 223.1 ± 3.1 milliseconds with RF pulse duration of TRF = 0.2 milliseconds, and 12 acquired flip angles ranging from 5 to 60 degrees. Using only 2 optimized flip angles (8 degrees, 44 degrees), T1 of the same material was determined to T1 = 223.8 ± 4.2 milliseconds in a markedly less acquisition time. In vivo evaluation of cortical bone was feasible and showed T1 values of 80.4 ± 25.1 milliseconds, exemplarily.

CONCLUSIONS

: Using the modified Ernst equation, it seems possible to rapidly evaluate 3D distribution of longitudinal relaxation time in materials and tissues with extremely fast signal decay by means of UTE sequences and only 2 measurements with optimized flip angles.

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.

BACKGROUND

Chronic myeloid leukemia (CML) is a clonal disease with specific cytogenetic changes involving the Philadelphia (Ph) translocation. The authors examined the relationship between telomere length (terminal restriction fragment [TRF]) and therapy-associated cytogenetic responses in CML patients.

METHODS

The authors examined the telomere length and telomerase activity in 44 patients with Ph-positive CML in the chronic phase. TRF was determined by Southern blot analysis using the (TTAGGG)4 probe and telomerase activity was assessed by the telomeric repeat amplification protocol and fluorescent-labeled primers.

RESULTS

At the time of CML diagnosis, 19 patients had TRFs within the age-matched normal range (mean +/- 2 x standard deviation [SD]) and the remaining 25 patients had TRFs shorter than the age-matched normal range (< mean +/- 2 x SD). Hematologic findings, including leukocyte count, hemoglobin level, platelet count, and percentage of bone marrow blasts at the time of diagnosis did not significantly differ between patients with normal and shortened TRFs; however, those with shortened TRFs had high levels of telomerase activity (P = 0.045). In a group of patients treated with alpha-interferon (n = 32), those with normal TRFs had a significantly lower frequency of blast crises (P = 0.0328), a significantly higher incidence of cytogenetic responses (P = 0.0185), and a favorable prognosis (P < 0.01) compared with those with shortened TRFs.

CONCLUSIONS

These findings suggest that normal TRFs in a small number of CML patients at the time of diagnosis may have a significant amount of normal stem cells remaining. The authors suggest that normal TRFs at the time of diagnosis indicate a subset of CML patients who may respond favorably to alpha-interferon therapy.

There are two models for CD4+ T-cell depletion leading to AIDS: a kinetic model and an immune suppression model. In the kinetic model, direct cell killing due to viral replication results in a continuous demand for CD4+ T-cells, which eventually exhausts their capacity for renewal by proliferative mechanisms. In the immune suppression model, CD4+ T-cell decline is due to an indirect global inhibitory effect of the virus on uninfected immune cell function. In order to address differences in the two models, we investigated proliferative history and thymic output in PBMC from the GRIV cohort of fast (FP) and slow/non-progressors (S/NP), and uninfected controls. Proliferative history and thymic output were assessed by measurement of mean telomeric restriction fragment (TRF) length and T-cell receptor Rearrangement Excision Circles (TREC) levels, respectively. Mean TRF lengths were significantly shorter in S/NP (n = 93, 7.59 +/- 0.11 kb) and FP (n = 42, 7.25 +/- 0.15 kb) compared to controls (n = 35, 9.17 +/- 0.19 kb). Mean TRF length in PBMC (n = 9, 7.32 +/- 0.31 kb) and CD4+ enriched fractions (n = 9, 7.41 +/- 0.30 kb) from a subset of non-GRIV HIV-1 infected samples were also significantly smaller than PBMC (n = 8, 9.77 +/- 0.33 kb) and CD4+ fractions (n = 8, 9.41 +/- 0.32 kb) from uninfected controls. Rates of telomeric shortening, however, were similar among S/NP (n = 93, -45 +/- 20 bp/yr), FP (n = 42, -41 +/- 14 bp/yr) and controls (-29 +/- 17 bp/yr). Paralleling differences observed in mean TRF length, TREC levels were significantly reduced in S/NP (n = 10, 3,433 +/- 843 mol/mu and FP (n = 8, 1,193 +/- 413) compared to controls (n = 15, 22,706 +/- 5,089), indicative of a defect in thymopoiesis in HIV-1 infection. When evaluated in the context of reduced thymopoiesis, the difference in mean TRF length between S/NP and controls (1.58 +/- 0.30 kb) is similar to that observed between memory and naïve T-cells (1.4 +/- 0.1 kb), and may reflect perturbations in the peripheral T-cell population due to a decline in thymic output of naïve T-cells rather than increased turnover. Based on the different clinical criteria used to select S/NP and FP, the sight difference in TREC between these two groups suggests the threshold for pathogenesis as a result of naïve T-cell depletion may be quite low, and incremental increases in thymic output may yield substantial clinical results. Future studies regarding therapeutic vaccination, specifically with HIV-1 Tat targeted anti-immunosuppressive vaccines, should address the defect in thymic output in HIV-1 infection by using TREC analysis as a rapid method for biological evaluation.

Vitamin E has been suggested to modulate age-associated changes by altering the redox balance resulting in altered gene and/or protein expression. Here we have utilized proteomics to determine whether such regulation in protein expression occurs in human lymphocytes from two different age groups stressed with H₂O₂ and then treated with vitamin E in the form of tocotrienol-rich fraction (TRF). In this study, lymphocytes obtained from young (30-49 years old) and old (>50 years old) volunteers were first challenged with 1 mM H₂O₂. They were then treated by exposure to 50, 100 and 200 μg/ml TRF. Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF (matrix-assisted laser desorption/ionization time-of-flight/time-of-flight) tandem mass spectrometry was then performed on whole-cell protein extracts to identify proteins that have changed in expression. A total of 24 proteins were found to be affected by H₂O₂ and/or TRF treatment. These included proteins that were related to metabolism, antioxidants, structural proteins, protein degradation and signal transduction. Of particular interest was the regulation of a number of proteins involved in stress response--peroxiredoxin-2, peroxiredoxin-3 and peroxiredoxin-6-all of which were shown to be down-regulated with H₂O₂ exposure. The effect was reversed following TRF treatment. The expression of peroxiredoxin-2 and peroxiredoxin-6 was confirmed by quantitative reverse transcriptase polymerase chain reaction. These results suggested that TRF directly influenced the expression dynamics of the peroxiredoxin-2, thus improving the cells ability to resist damage caused by oxidative stress.

BACKGROUND

In severe intrauterine growth restriction (IUGR) due to placental insufficiency a haemodynamic adaptation occurs, resulting in preferential blood flow to the fetal brain (brain sparing). With Doppler ultrasound an increased ratio between the umbilical and the cerebral artery pulsatility index (U/C ratio) can be demonstrated. IUGR is associated with impaired neurodevelopmental outcome.

OBJECTIVE

Evaluation of the effect of fetal brain sparing on behavioural problems at eleven years in premature born children.

METHODS

Prospective cohort study in premature children born in 1989, with a gestational age of 26 0/7 to 33 0/7 weeks. An U/C ratio>0.72 was defined as brain sparing. Behavioural problems were assessed with the parent-reported Child Behaviour Check List (CBCL) and the Teacher's Report Form (TRF). T scores >60 for total problem score and subscales of internalizing and externalizing behaviour, were considered abnormal.

RESULTS

Ninety-eight of the 116 survivors were assessed, of which 31 with antenatally established fetal brain sparing. According to the CBCL-total problem score 23.3% of the premature born babies in the brain sparing group had behavioural problems compared with 22.8% of those without brain sparing. According to the <em>TRF</em>-total problem score the percentages were 21.4% and 20.0%, respectively. Logistic regression analysis failed to show a significant association of U/C ratio with behavioural problems. In this model oxygen dependency at 28 days, IQ<85 at five years, cranial ultrasound abnormalities, fetal growth ratio<0.80, Apgar scores<7 after 5 min and birth weight<p10 contributed significantly.

CONCLUSIONS

In this cohort brain sparing itself has no significant association with behavioural problems at eleven years.

This study examined the criterion validity of the Child Behavior Checklist (CBCL) and Teacher's Report Form (TRF) problem scales and items in demographically-matched Singapore samples of referred and non-referred children (840 in each sample for the CBCL and 447 in each sample for the TRF). Internal consistency estimates for both the CBCL and TRF scales were good. Almost all CBCL and TRF problem scales and items significantly discriminated between referred and non-referred children, with referred children scoring higher, as expected. The largest referral status effects were on attention problems scales and their associated items, with the TRF having larger effects than the CBCL. Effect sizes for demographic variables such as age, gender, ethnicity and SES were much smaller than effect sizes for referral status, across both the CBCL and TRF forms and at both the scale and item levels. These findings suggest that teachers can be effective partners in identifying children who need mental health services and those who do not.

OBJECTIVE

To observe the effect of perioperative application of Sijunzi Decoction and enteral nutrition on T-cell subsets and nutritional status in patients with gastric cancer after operation.

METHODS

In this prospective, single-blinded, controlled clinical trial, fifty-nine patients with gastric cancer were randomly divided into three groups: control group (n=20) and two study groups (group A, n=21; group B, n=18). Sjunzi Decoction (100 ml) was administered via nasogastric tube to the patients in the study group B from the second postoperation day to the 9th postoperation day. Patients in the two study groups were given an isocaloric and isonitrogonous enteral diet, which was started on the second day after operation, and continued for eight days. Patients in the control group were given an isocaloric and isonitrogonous parenteral diet for 9 days. All variables of nutritional status such as serum albumin (ALB), prealbumin (PA), transferrin (TRF) and T-cell subsets were measured one day before operation, and one day and 10 days after operation.

RESULTS

All the nutritional variables and the levels of CD3(+), CD4(+), CD4(+)/CD8(+) were decreased significantly after operation. Ten days after operation, T-cell subsets and nutritional variables in the two study groups were increased as compare with the control group. The levels of ALB, TRF and T-cell subsets in the study group B were increased significantly as compared with the study group A (P<0.05).

CONCLUSIONS

Enteral nutrition assisted with Sijunzi Decoction can positively improve and optimize cellular immune function and nutritional status in the patients with gastric cancer after operation.

Vitamin E is important not only for its cellular antioxidant and lipid-lowering properties, but also as an antiproliferating agent. It has also been shown to contribute to immunoregulation, antibody production, and resistance to implanted tumors. It has recently been shown that tocotrienols are the components of vitamin E responsible for growth inhibition in human breast cancer cells in vitro as well as in vivo through estrogen-independent mechanisms. Although tocotrienols act on cell proliferation in a dose-dependent manner and can induce programmed cell death, no specific gene regulation has yet been identified. In order to investigate the molecular basis of the effect of a tocotrienol-rich fraction (TRF) from palm oil, we performed a cDNA array analysis of cancer-related gene expression in estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells. The human breast cancer cells were incubated with or without 8 mug/mL of tocotrienols for 72 h. RNA was subsequently extracted and subjected to reverse transcription before being hybridized onto cancer arrays. Tocotrienol supplementation modulated significantly 46 out of 1200 genes in MDA-MB-231 cells. In MCF-7 cells, tocotrienol administration was associated with a lower number of affected genes. Interestingly, only three were affected in a similar fashion in both cell lines: c-myc binding protein MM-1, 23-kDa highly basic protein, and interferon-inducible protein 9-27 (IFITM-1). These proteins are most likely involved in the cell cycle and can exert inhibitory effects on cell growth and differentiation of the tumor cell lines. These data suggest that tocotrienols are able to affect cell homeostasis, possibly independent of their antioxidant activity.

This study examined the applicability of the Chinese Version of Teacher's Report Form (TRF-CV) and estimated the prevalence of behavioral problems in a general population sample of 2,936 children aged 6 through 11 years in the Shandong Province of China. Teachers completed the TRF-CV and the Conners Hyperkinesis Index (CHI). The TRF-CV total scale showed satisfactory 2-week test-retest reliability (r = .83) and internal consistency (Cronbach's alpha = .94). The TRF-CV Total Problems, Attention Problems, Delinquent Behavior, and Aggressive Behavior had acceptable concurrent validity with the CHI (mean r = .62). With the TRF-CV Total Problems score of 26 as a cutoff, an overall correct classification rate of 90% for clinical sample and nonreferral required children was obtained. Exploratory factor analysis yielded six syndromes: Aggressive/Delinquent Behavior, Withdrawn/Depressed, Somatic Complaints, Attention Problems, Social Problems, and Thought Problems, with significant correlations with corresponding American cross-informant syndromes (mean r = .84). The overall prevalence rate of behavioral problems was 15.5% (95% CI = 14.2-16.8%), with a boy-to-girl ratio of 2.0:1 (chi2 = 59.70, p < .001). Younger boys exhibited more externalizing problems. These findings indicate that the TRF-CV is applicable for Chinese children, and the prevalence of behavioral problems shown by it among Chinese children seems comparable to that found in other countries. Although most of the American syndromes were well replicated, the differences in the present subjects, when submitted to principal components analysis, from American samples from whom the original syndromes were derived, could have prevented the study from replicating distinctions between aggressive vs. delinquent and depressed vs. withdrawn syndromes.

BACKGROUND

Aldosterone accelerates cardiovascular aging by mechanisms that generate reactive oxygen species. Telomere length in white blood cells (WBCs) may be a bioindicator that registers the accruing burden of systemic oxidative stress. The aim of the present study was, therefore, to examine the relationship between plasma aldosterone and telomere length in WBCs.

METHODS

We studied 75 normotensive and never-treated mildly hypertensive men whose blood was drawn for the measurements of plasma aldosterone concentration and the terminal restriction fragment (TRF) length in WBCs.

RESULTS

The slope of the TRF-age relationship in the entire cohort showed a decrease in telomere length of 26 +/- 5 base pairs per year (r = -0.46, p <.001). Age-adjusted TRF length was the longest in the lowest aldosterone quartile (6.74 +/- 0.12 kb) and shortest in the highest aldosterone quartile (6.36 +/- 0.11 kb), with intermediate TRF lengths in the second and third aldosterone quartiles (analysis of variance [ANOVA] trend test, p =.025). In telomeric attrition equivalence, participants in the upper aldosterone quartile were 15 years older than their peers in the lowest quartile.

CONCLUSIONS

The inverse relationship between aldosterone and WBC telomere length suggests not only that aldosterone is pro-oxidant but that elevated concentrations of this hormone might be linked to a higher rate of telomere attrition and perhaps increased biological aging in humans.

Two closely related shrew species, Sorex granarius and Sorex araneus, in which Robertsonian rearrangements have played a primary role in karyotype evolution, present very distinct telomere length patterns. S. granarius displays hyperlong telomeres specifically associated with the short arms of acrocentrics, whereas telomere lengths in S. araneus are rather short and homogenous. Using a combined approach of chromosome and fibre FISH, modified Q-FISH, 3D-FISH, Ag-NOR staining and TRF analysis, we carried out a comparative analysis of telomeric repeats and rDNA distribution on chromosome ends of Sorex granarius. Our results show that rDNA sequences forming active nuclear organizing regions are interspersed with the long telomere tracts of all short arms of acrocentrics. These observations suggest that the major rearrangements that gave rise to today's karyotype in S. granarius were accompanied by a profound reorganization of chromosome ends, which comprised extensive amplification of telomeric and rDNA repeats on the short arms of acrocentrics and finally contributed to the stabilization of telomeres. This is the first time that such telomeric structures have been observed in any mammalian species.

A large community-based sample of Russian youths (n = 841, age M = 13.17 years, SD = 2.51) was assessed with the Child Behavior Checklist (mothers and fathers separately), Teacher's Report Form, and Youth Self-Report. The multiple indicator-version of the correlated trait-correlated method minus one, or CT-C(M - 1), model was applied to analyze (a) the convergent and divergent validity of these instruments in Russia, (b) the degree of trait-specificity of rater biases, and (c) potential predictors of rater-specific effects. As expected, based on the published results from different countries and in different languages, the convergent validity of the instruments was rather high between mother and father reports, but rather low for parent, teacher, and self-reports. For self- and teacher reports, rater-specific effects were related to age and gender of the children for some traits. These results, once again, attest to the importance of incorporating information from multiple observers when psychopathological traits are evaluated in children and adolescents.

The blood-brain barrier (BBB) is a critical extrameningeal site of injury during bacterial meningitis, manifested by enhanced BBB permeability (BBBP). Previous methods to measure altered BBBP during meningitis involve radioactive materials, or are poorly quantified. Europium (EU) is a fluorescent, non-radioactive metal that is a sensitive and stable marker. Europium fluorescence can be measured with a spectrophotometer capable of time-resolved fluorescence (TRF). We used EU-albumin (EU-A) to examine BBBP in experimental lipopolysaccharide (LPS) induced meningitis. The results presented here introduce a simple and accurate method for measuring BBB permeability.

OBJECTIVE

Tocotrienol possess beneficial effects not exhibited by tocopherol. In vitro studies using animal models have suggested that these effects are caused via modulation of gene and protein expression. However, human supplementation studies using tocotrienol-rich isomers are limited. This study aims to identify plasma proteins that changed in expression following tocotrienol-rich fraction (TRF) supplementation within two different age groups.

METHODS

Subjects were divided into two age groups-32 ± 2 (young) and 52 ± 2 (old) years old. Four subjects from each group were assigned with TRF (78% tocotrienol and 22% tocopherol, 150 mg/day) or placebo capsules for 6 months. Fasting plasma were obtained at 0, 3, and 6 months. Plasma tocopherol and tocotrienol levels were determined. Plasma proteome was resolved by 2DE, and differentially expressed proteins identified by MS. The expressions of three proteins were validated by Western blotting.

RESULTS

Six months of TRF supplementation significantly increased plasma levels of tocopherols and tocotrienols. Proteins identified as being differentially expressed were related to cholesterol homeostasis, acute-phase response, protease inhibitor, and immune response. The expressions of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor were validated. The old groups showed more proteins changing in expression.

CONCLUSIONS

TRF appears to not only affect plasma levels of tocopherols and tocotrienols, but also the levels of plasma proteins. The identity of these proteins may provide insights into how TRF exerts its beneficial effects. They may also be potentially developed into biomarkers for the study of the effects and effectiveness of TRF supplementation.

OBJECTIVE

GH deficiency (GHD) is usually associated with a higher incidence of cardiovascular disease (CVD). The aim of this study was to establish whether patients with GHD, like those with CVD, show an increase in fibrinogen (FBG), type-1 plasminogen activator inhibitor (PAI-1), acute phase response proteins (APR), and soluble adhesion molecules. The effect of recombinant human GH (rhGH) replacement, on these parameters was also investigated.

METHODS

Concentrations of PAI-1 antigen (Ag), adhesion molecules sE-selectin, sP-selectin, and intercellular adhesion molecule-1 (sICAM-1), FBG and levels of APR orosomucoid (ORM), and 'negative APR' transferrin (TRF) were established in 11 panhypopituitary (PHP) patients (eight men and three women, age median 39.0 years, body mass index (BMI) 27. 49 +/- 3.89 kg/m2) before and after 12-month replacement with rhGH. Control values were obtained by examination of 33 healthy age and sex matched subjects (24 men and nine women with BMI 24.16 +/- 1.99 kg/m2).

RESULTS

PHP patients had higher concentrations of ORM (0.80 +/- 0.25, vs. 0.61 +/- 0.20 g/l; P = 0.05), FBG (3.22 +/- 0.48, vs. 2.57 +/- 0.47 g/l; P = 0.001), PAI-1 Ag (97.12 +/- 33.23, vs. 44.11 +/- 21.40 microgram/l; P = 0.001), sE-selectin (72.42 +/- 28.35, vs. 42. 80 +/- 12.60 microgram/l; P = 0.004), sP-selectin (221.26 +/- 75.12, vs. 104.79 +/- 26.01 microgram/l; P = 0.001) sICAM-1 (409.75 +/- 137.78, vs. 228.10 +/- 37.54 microgram/l; P = 0.001), and lower levels of TRF (2.14 +/- 0.40, vs. 2.76 +/- 0.39 g/l; P = 0.001) than controls. After 12-month rhGH replacement the patients showed an increase of TRF (2.64 +/- 0.84 g/l, P = 0.037) and decrease of soluble adhesion molecules (sE-selectin 57.98 +/- 27.04 microgram/l, P = 0.01, sP-selectin 121.74 +/- 50.42 microgram/l, P = 0.007; and sICAM-1 279.95 +/- 88.32 microgram/l, P = 0.005), which then, similarly to the ORM (0. 67 +/- 0.12 g/l) and FBG level (2.82 +/- 0.51 g/l), did not statistically differ from the values in the control group.

CONCLUSIONS

rhGH replacement led to modulation of the 'inflammatory response' in panhypopituitary patients. This modulation occurred locally at vascular endothelium level where after rhGH replacement, sE-selectin, sP-selectin and sICAM-1 concentrations decreased, a similar effect as in the systemic inflammatory response, as was also apparent from the changes in acute phase response protein levels.

Age estimation based on evidence found in teeth has received considerable attention within the field of forensic science. We determined the terminal restriction fragment (TRF) length, as telomere length, to estimate age. Using dental pulp DNA we found the average TRF length showed a tendency to shortening with aging. Our findings show that telomere shortening, based on dental pulp DNA is a new and useful approach to estimate age of the subject at the time of death.

Bacteria associated with tissues of metal-hyperaccumulating plants are of great interest due to the multiple roles they may play with respect to plant growth and resistance to heavy metals. The variability of bacterial communities associated with plant tissues of three populations of Alyssum bertolonii, a Ni hyperaccumulator endemic of serpentine outcrops of Central Italy, was investigated. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes was applied to DNA extracted from leaf tissues of 30 individual plants from three geographically separated serpentine outcrops. Moreover, T-RFLP fingerprinting was also performed on DNA extracted from the same soils from which the plants were collected. Fifty-nine unique terminal-restriction fragments (TRFs) were identified, with more than half of the taxonomically interpreted TRFs assigned to Alpha- and Gamma-Proteobacteria and Clostridia. Data were then used to define the extent of variation of bacterial communities due to single plants or to plant populations. Results indicated a very high plant-by-plant variation of leaf-associated community (more than 93% of total variance observed). However, a core (numerically small) of plant-specific TRFs was found. This work demonstrates that plant-associated bacterial communities represent a large reservoir of biodiversity and that the high variability existing between plants, even from the same population, should be taken into account in future studies on association between bacteria and metal-hyperaccumulating plants.

PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.

The present paper describes a novel modification of polymerase chain reaction (PCR) for the detection of Streptococcus pneumoniae DNA in clinical specimens. PCR was based on the detection of a 209-base pair segment of the S. pneumoniae pneumolysin gene. For the demonstration of the amplification product, microwell hybridization with a Europium-labelled oligonucleotide probe complementary to a biotinylated strand of the PCR product was performed, and the presence of the PCR product was monitored by time-resolved fluorescence (TRF) of the Europium chelate. The sensitivity of the assay for purified S. pneumoniae DNA was 50 fg DNA corresponding to 20 genome equivalents of S. pneumoniae DNA. The efficiency of the hybridization step was monitored by using known amounts of synthetic target oligonucleotides as standards. Sensitivity of 3 x 10(8) molecules per individual reaction well was achieved with a 30-min attachment time and a 3-h hybridization time. Detection of PCR-amplified products by the microwell hybridization technique and TRF was compared to agarose gel electrophoresis in 50 middle ear fluid samples obtained from children with acute otitis media. The agarose gel and TRF detection methods identified all culture-positive samples, but both were also positive for 55% of the culture-negative samples. The results suggest that the detection of amplified PCR products by microwell hybridization using Europium-labelled oligonucleotides is a reliable method for the demonstration of the pneumolysin gene fragment. Furthermore, the method is suitable for automation and, thus, for testing high numbers of samples. The clinical significance of the PCR findings remains to be studied.