Comparative characterisation of extracellular vesicles from canine and human plasma: a necessary step in biomarker discovery.

Extracellular Vesicles (EV) have become an interesting focus as novel biomarkers of disease and are increasingly reported upon in humans and other species. The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines were published to improve rigor and standardisation within the EV field and provide a framework for the reliable isolation and characterisation of EV populations. However, this rigor and standardisation has been challenging in the area of comparative medicine. Herein we present the successful isolation of EVs from human and canine plasma using Size Exclusion Chromatography and characterise these EVs according to best international practice. This study provides evidence for the reliable comparison of human and canine EVs isolated by this approach, and a baseline description of the EVs from healthy dogs to inform future biomarker studies. This work also demonstrates that the MISEV2018 guidelines can be successfully applied to EVs isolated from canine plasma.

Parental attributions of controllability as a moderator of the relationship between developmental disability and behaviour problems.

BACKGROUND: Children with developmental disabilities present behaviour problems to a greater extent than do typically developing children. Psychosocial models of child development suggest that parental attributions of child and adult controllability could moderate this relationship between child disability status and behaviour. METHODS: The influence of parental attributions of adult and child controllability on the relationship between problem behaviours and disability was explored in mothers of children with developmental disabilities (DD) (N = 20) with a mean age of 9 years 3 months (SD 24.6 months), and in mothers of typically developing (TD) children (N = 26) with a mean age of 9 years 4 months (standard deviation 23.7 months). The DD group comprised 11 children with autistic spectrum disorders or other communication impairments, three children with Down Syndrome, one with cerebral palsy, one with attentional problems, and four with specific or complex developmental problems. Child behaviour was measured by the Child Behaviour Checklist. Parental attributions were measured using a modified version of the Parent Attribution Test and mothers were divided into higher and lower controllability groups on the basis of their responses on this test. RESULTS: Multivariate analysis of variance found significant group × adult controllability interaction effects for 'aggressive behaviour', 'rule-breaking behaviour', as well as borderline significant effects for 'social problems' and 'other problems'. Simple effects analysis suggested that when mothers had lower attributions of adult controllability, there were indeed significantly more problem behaviours in the DD group, but when mothers had attributions of higher adult controllability there was no longer any significant difference in problematic behaviour between the two groups. CONCLUSIONS: Parental attributions of controllability may moderate the well-established effect of disability on problem behaviour. Implications for parent intervention programmes are discussed.

Reducing the Risk of Death From Pneumocystis jirovecii Pneumonia After Radical Radiation Therapy to the Lung.

AIMS: Lung cancer is the leading cause of cancer death. Radiotherapy given in the curative setting is associated with a 3% risk of death from Pneumocystis jirovecii pneumonia (PJP). Prolonged courses of high-dose steroids also increase the risk of PJP. International guidelines recommend the use of chemoprophylaxis with trimethoprim-sulfamethoxazole for patients at high risk. We assessed the effect of an intervention designed to reduce the impact of PJP. MATERIALS AND METHODS: Prophylaxis guidelines were introduced in 2016. Case records of patients treated with radical radiotherapy were examined for the periods 2014 to 2015 (pre-intervention) and 2017 to 2018 (post-intervention). In total, 247 patients were treated pre-intervention and 334 post-intervention. RESULTS: Freedom from PJP death at 1 year was 96% before intervention and 99% after (hazard ratio 0.3, 95% confidence interval 0.1-0.9, P = 0.029). Although the rate of use of chemoprophylaxis according to the guideline rose from 1% to 13% (P = 0.003), the use of high-dose steroids also fell from 35% to 16% (P < 0.00001). CONCLUSIONS: Reducing radiotherapy-associated infections is an important component of radical treatment in lung cancer. Highlighting chemoprophylaxis guidelines reduced the death rate from PJP, with an associated more judicious use of steroids. Advocating prophylaxis in patients with lymphocyte count <0.6 × 109/l is the next intervention to be studied.

What Can Proton Beam Therapy Achieve for Patients with Pectus Excavatum Requiring Left Breast, Axilla and Internal Mammary Nodal Radiotherapy?

AIMS: Exposure of the heart to radiation increases the risk of ischaemic heart disease, proportionate to the mean heart dose (MHD). Radiotherapy techniques including proton beam therapy (PBT) can reduce MHD. The aims of this study were to quantify the MHD reduction achievable by PBT compared with volumetric modulated arc therapy in breath hold (VMAT-BH) in patients with pectus excavatum (PEx), to identify an anatomical metric from a computed tomography scan that might indicate which patients will achieve the greatest MHD reductions from PBT. MATERIALS AND METHODS: Sixteen patients with PEx (Haller Index ≥2.7) were identified from radiotherapy planning computed tomography images. Left breast/chest wall, axilla (I-IV) and internal mammary node (IMN) volumes were delineated. VMAT and PBT plans were prepared, all satisfying target coverage constraints. Signed-rank comparisons of techniques were undertaken for the mean dose to the heart, ipsilateral lung and contralateral breast. Spearman's rho correlations were calculated for anatomical metrics against MHD reduction achieved by PBT. RESULTS: The mean MHD for VMAT-BH plans was 4.1 Gy compared with 0.7 Gy for PBT plans. PBT reduced MHD by an average of 3.4 Gy (range 2.8-4.4 Gy) compared with VMAT-BH (P < 0.001). PBT significantly reduced the mean dose to the ipsilateral lung (4.7 Gy, P < 0.001) and contralateral breast (2.7 Gy, P < 0.001). The distance (mm) at the most inferomedial extent of IMN volume (IMN to heart distance) negatively correlated with MHD reduction achieved by PBT (Spearman's rho -0.88 (95% confidence interval -0.96 to -0.67, P < 0.001)). CONCLUSION: For patients with PEx requiring left-sided breast and IMN radiotherapy, a clinically significant MHD reduction is achievable using PBT, compared with the optimal photon technique (VMAT-BH). This is a patient group in whom PBT could have the greatest benefit.

Young people's views of mental health education in secondary schools: a Scottish study.

BACKGROUND: This exploratory study used mixed methods to investigate young people's preferences in the delivery of mental health education and to investigate possible age and gender differences. METHOD: Information was gathered about the delivery of mental health education in three secondary schools. Nine pupil focus groups were carried out to identify key themes which were then further developed and administered through questionnaires to a larger sample of 773 pupils. RESULTS: Gender and age differences were found in young people's preferences about who should deliver mental health education, and what, when, where and how this should be delivered. CONCLUSION: Mental health education should reflect the needs of young people. Age and gender preferences should be considered when designing these programmes.

A case-control study of community-associated Clostridium difficile infection.

OBJECTIVES: The aim of this study was to determine the incidence of and risk factors for community-associated Clostridium difficile infection (CDI). METHODS: Prospective surveillance of community-derived faecal samples for C. difficile cytotoxin, followed by a questionnaire-based case-control study in two distinct patient cohorts (one semi-rural and the other urban). RESULTS: The proportion of randomly selected faecal samples positive for C. difficile cytotoxin was 2.1% in both patient cohorts (median ages 73 and 45 years for the urban and semi-rural cohorts, respectively). Exposure to antibiotics in the previous 4 weeks, particularly multiple agents (P < 0.001), aminopenicillins (P < 0.05) and oral cephalosporins (P < 0.05), was significantly more frequent among cases than controls. Hospitalization in the preceding 6 months was significantly associated with CDI (45% versus 23%; P = 0.022). However, almost half the cases had not received antibiotic therapy in the month before C. difficile detection, and approximately one-third neither had exposure to antibiotics nor recent hospitalization. Contact with infants aged < or =2 years was significantly associated with CDI (14% versus 2%; P = 0.02). Prior exposure to gastrointestinal-acting drugs (proton pump inhibitor, H2 antagonist or non-steroidal anti-inflammatory) was not significantly more common in CDI cases. C. difficile PCR ribotype 001 caused 60% and 13% of urban and semi-rural community-associated CDI cases, respectively. CONCLUSIONS: Reliance on antibiotic history and age (> or =65 years) will contribute to missed diagnoses of community-associated CDI. Potential risk factors for community-associated CDI should be explored further to explain the large proportion of cases not linked to recent antibiotic therapy or hospitalization.

Manuscript evolution.

Frequently, letters, words and sentences are used in undergraduate textbooks and the popular press as an analogy for the coding, transfer and corruption of information in DNA. We discuss here how the converse can be exploited, by using programs designed for biological analysis of sequence evolution to uncover the relationships between different manuscript versions of a text. We point out similarities between the evolution of DNA and the evolution of texts.

Candidate gene analysis of 21q22: support for S100B as a susceptibility gene for bipolar affective disorder with psychosis.

A genome-wide scan in 60 bipolar affective disorder (BPAD) affected sib-pairs (ASPs) identified linkage on chromosome 21 at 21q22 (D21S1446, NPL = 1.42, P = 0.08), a BPAD susceptibility locus supported by multiple studies. Although this linkage only approaches significance, the peak marker is located 12 Kb upstream of S100B, a neurotrophic factor implicated in the pathology of psychiatric disorders, including BPAD and schizophrenia. We hypothesized that the linkage signal at 21q22 may result from pathogenic disease variants within S100B and performed an association analysis of this gene in a collection of 125 BPAD type I trios. S100B single nucleotide polymorphisms (SNPs) rs2839350 (P = 0.022) and rs3788266 (P = 0.031) were significantly associated with BPAD. Since variants within S100B have also been associated with schizophrenia susceptibility, we reanalyzed the data in trios with a history of psychosis, a phenotype in common between the two disorders. SNPs rs2339350 (P = 0.016) and rs3788266 (P = 0.009) were more significantly associated in the psychotic subset. Increased significance was also obtained at the haplotype level. Interestingly, SNP rs3788266 is located within a consensus-binding site for Six-family transcription factors suggesting that this variant may directly affect S100B gene expression. Fine-mapping analyses of 21q22 have previously identified transient receptor potential gene melastatin 2 (TRPM2), which is 2 Mb upstream of S100B, as a possible BPAD susceptibility gene at 21q22. We also performed a family-based association analysis of TRPM2 which did not reveal any evidence for association of this gene with BPAD. Overall, our findings suggest that variants within the S100B gene predispose to a psychotic subtype of BPAD, possibly via alteration of gene expression.

Association between carcinogen-DNA adducts in white blood cells and lung cancer risk in the physicians health study.

In this matched case-control study nested within the prospective Physicians' Health Study, we evaluated whether DNA damage in blood samples collected at enrollment significantly predicted risk, consistent with our hypothesis that cases have greater biological susceptibility to polycyclic aromatic hydrocarbons and other aromatic tobacco carcinogens. The subjects were 89 cases of primary lung cancer and 173 controls, all males, matched on smoking, age, and duration of follow-up. Aromatic-DNA adducts were measured in WBCs by the nuclease P1-enhanced (32)P-postlabeling method that primarily detects smoking-related adducts. Among current smokers, but not former or nonsmokers, there was a significant increase in mean adduct levels of cases compared with controls (11.04 versus 5.63; P = 0.03). "Healthy" current smokers who had elevated levels of aromatic DNA adducts in WBCs were approximately three times more likely to be diagnosed with lung cancer 1-13 years later than current smokers with lower adduct concentrations (odds ratio, 2.98; 95% confidence interval, 1.05-8.42; P = 0.04). We were not able to discern case-control differences in former smokers and nonsmokers. The findings are of interest because they suggest that individuals who become cases have greater biological susceptibility to tobacco carcinogens, a biological difference, which manifests most clearly while exposure is ongoing.

The effect of the native kinematics of the knee on the outcome following total knee arthroplasty.

AIMS: The aim of this study was to investigate differences in pain, range of movement function and satisfaction at three months and one year after total knee arthroplasty (TKA) in patients with an oblique pattern of kinematic graph of the knee and those with a varus pattern. PATIENTS AND METHODS: A total of 91 patients who underwent TKA were included in this retrospective study. Patients (59 women and 32 men with mean age of 68.7 years; 38.6 to 88.4) were grouped according to kinematic graphs which were generated during navigated TKA and the outcomes between the groups were compared. RESULTS: The graphs were varus in 50 patients (55%), oblique in 19 (21%), neutral in 17 (18.5%) and valgus in five (5.5%). After adjustment for pre-operative scores and gender, compared with patients with varus knee kinematics, patients with an oblique kinematic graph had a poorer outcome with lower Knee Society scores at three months (9.2 points, p = 0.038). CONCLUSION: We found four distinct kinematic graphs in knees and that patients with an oblique graph have a poorer outcome in the short-term after TKA. Cite this article: Bone Joint J 2016;98-B:1471-8.

Effects of acute and chronic sunitinib treatment on cardiac function and calcium/calmodulin-dependent protein kinase II.

BACKGROUND AND PURPOSE: Calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac contractile function and dysfunction and may be an unwanted secondary target for anti-cancer drugs such as sunitinib and imatinib that have been reported to alter cardiac performance. This study aimed to determine whether anti-cancer kinase inhibitors may affect CaMKII activity and expression when administered in vivo. EXPERIMENTAL APPROACH: Cardiovascular haemodynamics in response to acute and chronic sunitinib treatment, and chronic imatinib treatment, were assessed in guinea pigs and the effects compared with those of the known positive and negative inotropes, isoprenaline and verapamil. Parallel studies from the same animals assessed CaMKIIδ expression and CaMKII activity following drug treatments. KEY RESULTS: Acute administration of sunitinib decreased left ventricular (LV) dP/dtmax. Acute administration of isoprenaline increased LVdP/dtmax dose-dependently, while LVdP/dtmax was decreased by verapamil. CaMKII activity was decreased by acute administration of sunitinib and was increased by acute administration of isoprenaline, and decreased by acute administration of verapamil. CaMKIIδ expression following all acute treatments remained unchanged. Chronic imatinib and sunitinib treatments did not alter fractional shortening; however, both CaMKIIδ expression and CaMKII activity were significantly increased. Chronic administration of isoprenaline and verapamil decreased LV fractional shortening with parallel increases in CaMKIIδ expression and CaMKII activity. CONCLUSIONS AND IMPLICATIONS: Chronic sunitinib and imatinib treatment increased CaMKIIδ expression and CaMKII activity. As these compounds are associated with cardiac dysfunction, increased CaMKII expression could be an early indication of cellular cardiotoxicity marking potential progression of cardiac contractile dysfunction.

Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses.

Estimates of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers. Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk.

Gender differences in autoantibodies to oxidative DNA base damage in cigarette smokers.

Oxidative DNA damage and antibodies to that damage have been implicated in lung, breast, and colorectal cancer. In this observational validation study, the relationship between anti-5-hydroxymethyl-2'-deoxyuridine (HMdU) autoantibody (aAb) and plasma micronutrients was assessed in 140 heavy smokers by ELISA. Anti-HMdU aAbs were 50% higher in women after adjustment for cigarettes/day (CPD; P = 0.002), although men smoked more and had higher plasma cotinine levels. The women reported taking more vitamin C (P < 0.005) and had higher plasma levels of alpha-carotene and beta-carotene (P < 0.001) and cryptoxanthin (P < 0.01) than men. Neither CPD nor cotinine was associated with aAb titers. Anti-HMdU aAbs were associated inversely with alpha-tocopherol (P = 0.10), retinol (P = 0.06), and age (P = 0.04) in women but not in men. In contrast to the men, women 50 years of age (P = 0.05). Given the same duration of exposure, women had higher anti-HMdU aAbs and also reached peak levels at a lower cumulative smoking exposure (30 years) compared with male smokers (40 years). Subjects smoked an average of 28.9 +/- 0.81 CPD and initiated smoking at 17.2 +/- 0.33 (SE) years of age. Therefore, smokers who reported smoking for 30 years were typically <50 years old. Women

Serum autoantibodies recognizing 5-hydroxymethyl-2'-deoxyuridine, an oxidized DNA base, as biomarkers of cancer risk in women.

Human sera contain anti-5-hydroxymethyl-2'-deoxyuridine (HMdU; an oxidized thymidine) autoantibodies (aAbs), which are significantly higher in chronic inflammatory diseases. The intent of this study was to establish whether anti-HMdU aAbs can serve as predictors of breast and colorectal cancer risk. Sera of 169 women were analyzed by ELISA. Women healthy at blood donation but who were diagnosed 0.5-6 years later with breast or colorectal cancer exhibited significantly increased anti-HMdU aAbs over the age-matched controls (P = 0.028 and P < 0.001, respectively). Subjects diagnosed with rectal cancer had the highest levels of anti-HMdU aAbs (44.80 +/- 11.50; n = 6) in comparison to colon (29.03 +/- 2.49; n = 33) and breast (35.86 +/- 8.55; n = 9) cancers. Individuals with benign breast disease also had elevated anti-HMdU aAb (35.12 +/- 8.77; n = 10), with a borderline statistical significance (P = 0.095), whereas those with benign gastrointestinal tract diseases had those titers (30.95 +/- 3.64; n = 8) significantly increased (P < 0.02). Anti-HMdU aAb levels in subjects with a family history of any cancer (23.57 +/- 2.86; n = 55) did not significantly differ from those of the controls (19.41 +/- 2.90; n = 48), but women with a family history of breast cancer (two primary relatives or one with a bilateral disease) showed increased levels (34.48 +/- 8.16; n = 8; P = 0.024). Ps for linear trend of age-adjusted odds ratios were 0.049 for breast and < 0.001 for colorectal cancers. Anti-HMdU aAb titers showed a remarkable stability over a period of 6 years, with a low (14%) intraindividual variance. Thus, elevated anti-HMdU aAb titers may be an early signal of cancer risk, because they were significantly increased in otherwise healthy women who had a family history of breast cancer; in those who had benign breast disease or benign gastrointestinal tract diseases; and, most importantly, in those who at 0.5-6 years after the initial blood donation developed breast or colorectal cancer.

Carcinogen-DNA adducts in human breast tissue.

Breast cancer is the second leading cause of cancer death among American women. Known risk factors account for only approximately one-third of the 182,000 new cases diagnosed each year in the United States. There is both concern and debate over the contribution of environmental exposures related to lifestyle, occupation, and ambient pollution, particularly in high risk areas such as Long Island, NY and the rest of the northeastern United States. Biomarkers such as carcinogen-DNA adducts can help to explore the role of environmental risk factors for breast cancer by documenting DNA damage from specific carcinogens directly in human tissue. In this pilot study, a total of 31 breast tissue samples were analyzed by the 32P-postlabeling method for carcinogen-DNA adducts characteristic of complex mixtures of aromatic compounds (such as polycyclic aromatic hydrocarbons) and tobacco smoke. The samples included tumor and tumor-adjacent tissues from 15 women with breast cancer and normal tissue samples from 4 women undergoing breast reduction. Among the breast cancer cases, the mean aromatic/hydrophobic-DNA adduct level in all tissues assayed was 5.3 +/- 2.4 (SD) adducts/10(8) nucleotides compared to 2.3 +/- 1.5 among the samples from the noncancer patients. Breast tissue (tumor and/or nontumor) from 30% (5 of 15) of women with breast cancer displayed a pattern of adducts (referred to as a diagonal zone of radioactivity) associated previously, in studies of other tissues, with exposure to tobacco smoke. The 5 positive samples were from current smokers; tissue samples from the 8 nonsmoking cases did not show this characteristic pattern (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Organochlorine compounds (DDE and PCB) in plasma and breast cyst fluid of women with benign breast disease.

The organochlorines, dichloro-diphenyl-trichloroethane and polychlorinated biphenyl (PCB) are pervasive environmental contaminants. Results from previous studies have been conflicting regarding the relationship between the internal dose of these organochlorine residues and breast cancer risk. To determine whether these compounds are present in breast cyst fluids and whether cyst fluid and plasma concentrations are correlated, we analyzed organochlorines in paired cyst fluid and plasma samples from 24 subjects using gas chromatography and electron capture detection. All but one of the women had a history of multiple cysts, suggesting that they were at elevated risk for future breast cancer. DDE (a metabolite of dichloro-diphenyl-trichloroethane) was present in 22 of the cyst samples and PCB was detected in 19 of the cyst samples. Organochlorine levels were more concentrated in the plasma than in breast cyst fluids. Levels of DDE in plasma were significantly correlated with those in cyst fluid (r = 0.73; P < 0.001); in contrast to PCB levels in cyst and plasma (r = 0.37; P = 0.12). Congener specific analysis of the PCBs showed that some individual congeners were preferentially excluded from or concentrated in the cyst fluid. To our knowledge, this study is the first to demonstrate that PCB and DDE are present in cyst fluids and thus in contact with the ductal epithelium of the breast. These results support the use of plasma DDE as a proxy for DDE in the target tissue in research on the role of environmental factors in breast cancer.

Decline of DNA damage and other biomarkers in peripheral blood following smoking cessation.

Serial samples from 40 heavy smokers ( > or = pack/day for > or = 1 year) enrolled in a smoking cessation program were assayed for cotinine, polycyclic aromatic hydrocarbon (PAH)-DNA, 4-aminobiphenyl-hemoglobin (4-ABP-Hb) adducts, and glycophorin A (GPA) mutations. Blood samples were taken while subjects were smoking, and 10 weeks and 8 and 14 months after quitting. Cotinine was used to assess compliance with the cessation protocol. A significant reduction in mean PAH-DNA and 4-ABP-Hb adducts was observed after cessation in all persons who were cotinine-verified quitters ( < or = 25 ng/ml) for > or = 8 months (P < 0.05). Neither the GPA N/phi nor the GPA N/N mutation Vf was significantly reduced after smoking cessation, but results are limited by the small number (n = 18) of heterozygous individuals studied. The substantial reduction (50-75%) in PAH-DNA and 4-ABP-Hb adduct levels after quitting indicates these carcinogen adducts are reflective of smoking. Passive exposure to smoke at home was significantly associated with PAH-DNA adducts in active smokers and in ex-smokers 10 weeks after quitting (P < 0.01). The estimated half-life of the PAH-DNA adducts in leukocytes is 9-13 weeks by inspection of the mean biomarker levels from baseline and 10 weeks sample and 23 (95% confidence interval, 10-36 weeks) using a linear regression model that adjusted for background.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible role for protein kinase B in the anti-apoptotic effect of prolactin in rat Nb2 lymphoma cells.

Prolactin (PRL) is a mitogen for a number of cell types and its action as a survival factor has recently been demonstrated in Nb2 lymphoma cells. However, the intracellular signalling pathways by which PRL promotes the survival of Nb2 cells is unknown. In previous studies, we have shown that PRL caused the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its association with tyrosine phosphorylated fyn. Protein kinase B (PKB), a serine/threonine kinase, is now known to be a downstream component of the PI3-kinase pathway. The aim of the present study was to examine the effect of PRL on the activation of PKB and to find out whether this has any role on the PRL-induced survival of Nb2 cells. Our studies have revealed the phosphorylation and activation of PKB in PRL-stimulated Nb2 cells. We have also observed, using confocal microscopy, translocation of PKB to the membrane of Nb2 cells in response to PRL. These effects were blocked by the PI3-kinase inhibitor, LY294002 (10 microgram/ml). Apoptosis was induced by the general protein kinase inhibitor, staurosporine (STS; 0.1-1 microM), the synthetic glucocorticoid, dexamethasone (Dex; 100 nM) or ionising radiation by exposing Nb2 cells to X-irradiation (IR; 10 Gy). PRL had no effect on STS-induced apoptosis. On the other hand, PRL (100 ng/ml) inhibited apoptosis induced by Dex or IR; this effect of PRL was reversed by the addition of LY294002 (10 microgram/ml). Furthermore, Western blot analysis using phosphospecific PKB antibody on lysates from PRL-treated Nb2 cells showed that phosphorylation of PKB in response to PRL was inhibited by STS (0.5 microM), but not by Dex (100 nM). These results suggest that the PI3-kinase/PKB pathway may mediate the anti-apoptotic effect of PRL in Nb2 cells.

Molecular epidemiology in environmental carcinogenesis.

Molecular epidemiology has significant potential in preventing cancer and other diseases caused by environmental exposures (related to lifestyle, occupation, or ambient pollution). This approach attempts to prevent cancer by incorporating laboratory methods to document the molecular dose and preclinical effects of carcinogens, as well as factors that increases individual susceptibility to carcinogens. Recently we have carried out validation studies of biologic markers such as carcinogen--DNA and carcinogen--protein adducts, gene and chromosomal mutations, alterations in target oncogenes or tumor suppressor genes, polymorphisms in putative susceptibility genes (individual P450s, glutathione transferase M1), and serum levels of micronutrients. This research involves adults, infants, and children exposed to varying levels of carcinogens, as well as cancer cases and controls. On a group level, dose-response relationships have frequently been seen between various biomarkers and environmental exposures such as polycyclic aromatic hydrocarbons, cigarette smoke (active and passive), and ambient indoor and workplace air pollution. However, there is significant interindividual variation in biomarkers that appears to reflect a modulating effect on biomarkers (hence potential risk) by genetic and acquired susceptibility factors. Ongoing retrospective and nested case-control studies of lung and breast cancer are examining the association between biomarkers and cancer risk. Results of these studies are encouraging; they suggest that biomarkers, once validated, can be useful in identifying populations and individuals at risk in time to intervene effectively.