The age-dependent association of risk factors with pancreatic cancer.

BACKGROUND: Pancreatic cancer presents as advanced disease in >80% of patients; yet, appropriate ages to consider prevention and early detection strategies are poorly defined. We investigated age-specific associations and attributable risks of pancreatic cancer for established modifiable and non-modifiable risk factors. PATIENTS AND METHODS: We included 167 483 participants from two prospective US cohort studies with 1190 incident cases of pancreatic cancer during >30 years of follow-up; 5107 pancreatic cancer cases and 8845 control participants of European ancestry from a completed multicenter genome-wide association study (GWAS); and 248 893 pancreatic cancer cases documented in the US Surveillance, Epidemiology, and End Results (SEER) Program. Across different age categories, we investigated cigarette smoking, obesity, diabetes, height, and non-O blood group in the prospective cohorts; weighted polygenic risk score of 22 previously identified single nucleotide polymorphisms in the GWAS; and male sex and black race in the SEER Program. RESULTS: In the prospective cohorts, all five risk factors were more strongly associated with pancreatic cancer risk among younger participants, with associations attenuated among those aged >70 years. The hazard ratios comparing participants with three to five risk factors with those with no risk factors were 9.24 [95% confidence interval (CI) 4.11-20.77] among those aged ≤60 years, 3.00 (95% CI 1.85-4.86) among those aged 61-70 years, and 1.46 (95% CI 1.10-1.94) among those aged >70 years (Pheterogeneity = 3×10-5). These factors together were related to 65.6%, 49.7%, and 17.2% of incident pancreatic cancers in these age groups, respectively. In the GWAS and the SEER Program, the associations with the polygenic risk score, male sex, and black race were all stronger among younger individuals (Pheterogeneity ≤0.01). CONCLUSIONS: Established risk factors are more strongly associated with earlier-onset pancreatic cancer, emphasizing the importance of age at initiation for cancer prevention and control programs targeting this highly lethal malignancy.

Dissecting the self-assembly kinetics of multimeric pore-forming toxins.

Pore-forming toxins are ubiquitous cytotoxins that are exploited by both bacteria and the immune response of eukaryotes. These toxins kill cells by assembling large multimeric pores on the cell membrane. However, a quantitative understanding of the mechanism and kinetics of this self-assembly process is lacking. We propose an analytically solvable kinetic model for stepwise, reversible oligomerization. In biologically relevant limits, we obtain simple algebraic expressions for the rate of pore formation, as well as for the concentration of pores as a function of time. Quantitative agreement is obtained between our model and time-resolved kinetic experiments of Bacillus thuringiensis Cry1Ac (tetrameric pore), aerolysin, Staphylococcus aureus α-haemolysin (heptameric pores) and Escherichia coli cytolysin A (dodecameric pore). Furthermore, our model explains how rapid self-assembly can take place with low concentrations of oligomeric intermediates, as observed in recent single-molecule fluorescence experiments of α-haemolysin self-assembly. We propose that suppressing the concentration of oligomeric intermediates may be the key to reliable, error-free, self-assembly of pores.

Rac1 is required for Prkar1a-mediated Nf2 suppression in Schwann cell tumors.

Schwannomas are peripheral nerve sheath tumors that often occur in the setting of an inherited tumor predisposition syndrome, including neurofibromatosis types 1 (NF1) and 2 (NF2), familial schwannomatosis and Carney complex. Loss of the NF2 tumor suppressor (encoding NF2, or Merlin) is associated with upregulation of the Rac1 small GTPase, which is thought to have a key role in mediating tumor formation. In prior studies, we generated a mouse model of schwannomas by performing tissue-specific knockout (KO) of the Carney complex gene Prkar1a, which encodes the type 1A regulatory subunit of protein kinase A. These tumors exhibited down-regulation of Nf2 protein and an increase in activated Rac1. To assess the requirement for Rac1 in schwannoma formation, we generated a double KO (DKO) of Prkar1a and Rac1 in Schwann cells and monitored tumor formation. Loss of Rac1 reduced tumor formation by reducing proliferation and enhancing apoptosis. Surprisingly, the reduction of tumor formation was accompanied by re-expression of the Nf2 protein. Furthermore, activated Rac1 was able to downregulate Nf2 in vitro in a Pak-dependent manner. These in vivo data indicate that activation of Rac1 is responsible for suppression of Nf2 protein production; deficiency of Nf2 in Schwann cells leads to loss of cellular growth control and tumor formation. Further, PKA activation through mutation in Prkar1a is sufficient to initiate Rac1 signaling, with subsequent reduction of Nf2 and schwannomagenesis. Although in vitro evidence has shown that loss of Nf2 activates Rac1, our data indicate that signaling between Nf2 and Rac1 occurs in a bidirectional fashion, and these interactions are modulated by PKA.

The effect of brimonidine tartrate on retinal blood flow in patients with ocular hypertension.

PURPOSE: To study the effects of topical brimonidine tartrate 0.2%, an alpha(2)-agonist ocular hypotensive drug, on retinal capillary blood flow in patients with ocular hypertension. METHODS: The study was a double-masked, randomized, placebo-controlled trial set in a tertiary eye center. Ocular hypertensive patients with repeatable intraocular pressures greater than 21 mm Hg and normal visual fields and optic disks were consecutively recruited. After an eye examination, baseline retinal blood flow measurements were made with confocal scanning laser Doppler flowmetry in one study eye. Patients were then randomly assigned to receive either brimonidine or placebo (saline) twice daily for 8 weeks. Blood flow and intraocular pressure measurements were then repeated after 4 and 8 weeks. RESULTS: Seventeen patients were randomly assigned to receive brimonidine, and 14 received placebo. One patient in each group failed to complete the study. The mean group differences in baseline age and intraocular pressure were not statistically significant (59. 23 [+/-10.24] and 52.23 [+/-16.46] years, respectively, and 24.84 [+/-2.08] and 24.56 [+/-2.85] mm Hg, respectively). Brimonidine reduced intraocular pressure by 17.90% and 16.17% at 4 and 8 weeks, respectively, with a significant difference in treatment effect compared with the placebo group (P <.007). The group difference in treatment effect in any of the three hemodynamic parameters velocity, volume, and flow was within 8% and not significantly different at 4 or 8 weeks (P.360). Based on a type I error of 0.05, our study had a power greater than or equal to 75% to detect group differences in treatment effect of greater than or equal to 15% to 20%. CONCLUSIONS: Brimonidine reduces intraocular pressure without altering retinal capillary blood flow in patients with ocular hypertension.

The effect of brimonidine tartrate on retinal blood flow in patients with ocular hypertension [corrected].

PURPOSE: To study the effects of topical brimonidine tartrate 0.2%, an alpha2-agonist ocular hypotensive drug, on retinal capillary blood flow in patients with ocular hypertension. METHODS: The study was a double-masked, randomized, placebo-controlled trial set in a tertiary eye center. Ocular hypertensive patients with repeatable intraocular pressures greater than 21 mm Hg and normal visual fields and optic disks were consecutively recruited. After an eye examination, baseline retinal blood flow measurements were made with confocal scanning laser Doppler flowmetry in one study eye. Patients were then randomly assigned to receive either brimonidine or placebo (saline) twice daily for 8 weeks. Blood flow and intraocular pressure measurements were then repeated after 4 and 8 weeks. RESULTS: Seventeen patients were randomly assigned to receive brimonidine, and 14 received placebo. One patient in each group failed to complete the study. The mean group differences in baseline age and intraocular pressure were not statistically significant (59.23 [+/-10.24] and 52.23 [+/-16.46] years, respectively, and 24.84 [+/-2.08] and 24.56 [+/-2.85] mm Hg, respectively). Brimonidine reduced intraocular pressure by 17.90% and 16.17% at 4 and 8 weeks, respectively, with a significant difference in treatment effect compared with the placebo group (P < .007). The group difference in treatment effect in any of the three hemodynamic parameters velocity, volume, and flow was within 8% and not significantly different at 4 or 8 weeks (P > .360). Based on a type I error of 0.05, our study had a power greater than or equal to 75% to detect group differences in treatment effect of greater than or equal to 15% to 20%. CONCLUSIONS: Brimonidine reduces intraocular pressure without altering retinal capillary blood flow in patients with ocular hypertension.

Differential responses of adult cardiac fibroblasts to in vitro biaxial strain patterns.

Different patterns of extracellular matrix (ECM) remodeling in the heart are thought to be dependent on altered mechanical and chemical conditions and can contribute to cardiac dysfunction. Cardiac fibroblasts are the primary regulators of the ECM and may respond to mechanical factors in vitro. We hypothesized that different types of in vitro strains, e.g. tensile or compressive, can stimulate different functional responses in cultured adult rat cardiac fibroblasts. In this study, we first showed that a single step in strain applied by a uniaxial stretch system stimulated collagen III and fibronectin mRNA levels and transforming growth factor-beta(1) (TGF-beta(1)) activity in the adult phenotype of rat cardiac fibroblasts. Two-dimensional deformations were measured by tracking fluorescent microspheres attached to the substrate and cultured cells. For 10% uniaxial strain, mean principal strains were 0. 104 +/- 0.018 in the direction of stretch and -0.042 +/- 0.013 in the perpendicular direction, verifying that the fibroblasts were simultaneously subjected to tensile (positive) and compressive (negative) strains. Furthermore, these cells were also subjected to area change and to shear. In order to examine the distinct effects of different types of deformation on cardiac fibroblasts, an equibiaxial stretch system was used to apply either pure tensile or compressive area strains, in the absence of shear. Magnitudes of equibiaxial strain were selected to apply local cell area changes identical to those applied in the uniaxial system. Results showed that pure tensile and compressive area strains induced divergent responses in ECM mRNA levels. TGF-beta(1) activity was dependent on the magnitude of applied area strain regardless of the mode of deformation. These findings demonstrate that adult cardiac fibroblasts may respond differently to varied types of mechanical loading, suggesting that ECM remodeling may be locally regulated by specific mechanical stimuli in the heart.

Equibiaxial strain and strain rate stimulate early activation of G proteins in cardiac fibroblasts.

Cardiac fibroblasts are responsible for the production of the extracellular matrix of the heart, with alterations of fibroblast function implicated in myocardial infarction and cardiac hypertrophy. Here the role of heterotrimeric GTP-binding proteins (G proteins) in the mechanotransduction of strain in rat cardiac fibroblasts was investigated. Cells in an equibiaxial stretch device were incubated with the photoreactive GTP analog azidoanalido [alpha-32P]GTP (AAGTP) and were subjected to various regimens of strain. Autoradiographic analysis showed a 42-kDa protein labeled for cells exposed to 12 cycles of 3% strain or 6 cycles of 6% strain over 60 s (strain rate of 1.2%/s), whereas 6 cycles of 3% strain (0.6%/s) elicited no measurable response. To further investigate the role of strain rate, a single 6% cycle over 10 or 60 s (1.2% and 0.2%/s, respectively) was applied, with the more rapid cycle stimulating AAGTP binding, whereas the lower strain rate showed no response. In cells subjected to a single 6% cycle/10 s, immunoprecipitation identified the AAGTP-labeled 42-kDa band as the G protein subunits G alpha q and G alpha i1. These results demonstrate that G protein activation represents one of the early mechanotransduction events in cardiac fibroblasts subjected to mechanical strain, with the rate at which the strain is applied modulating this response.

The perceived life stressors among elderly Chinese immigrants: are they different from those of other elderly Americans?

This is a descriptive study examining the perceived life stressors among elderly Chinese immigrants and comparing their stressors to those experienced by other elderly Americans. Lazarus and Folkman's stress theory and Roy's adaptation model were used as the theoretical basis for this study. Based on this framework, it was predicted that elderly Chinese immigrants would report more life stressors than elderly Americans, because they experience a changing cultural environment along with the aging process. The sample was a convenience sample of 30 elderly people from two Chinese churches in one northeastern metropolitan city. Participants were asked to describe a stressful event that they had experienced within the past month. Data were collected by a bilingual (Chinese and English) interviewer using open-ended questions. The research design was based on Manfredi and Pickett's (1987) research exploring the stressors among elderly Americans, and those research results provide the comparison data for this study. The findings suggest that the amount and sources of stress reported by elderly Chinese immigrants are different from those reported by other elderly Americans. Additional studies are needed to identify the coping strategies used by elderly Chinese immigrants. These findings have implications for gerontologists, policy makers, community healthcare providers, and the Chinese immigrant population.

Regulation of the Salmonella typhimurium pepT gene by cyclic AMP receptor protein (CRP) and FNR acting at a hybrid CRP-FNR site.

The Salmonella typhimurium pepT gene is induced nearly 30-fold in response to anaerobiosis. Anaerobic expression is dependent on the transcriptional regulator encoded by fnr (previously oxrA). Primer extension analysis and site-directed mutagenesis experiments show that pepT is transcribed from two sigma 70 promoters. One promoter (P1) is FNR dependent and anaerobically induced, while the other (P2) appears to be constitutive. The potABCD operon is divergently transcribed from a promoter near pepT P2. Sequence analysis of pepT promoter mutations which either elevate anaerobic expression or confer constitutive expression revealed that these mutations affect the -10 region of the P1 or P2 promoter, respectively. The pepT200 mutation, which changes the -10 region of the FNR-dependent P1 promoter to the consensus, has the surprising effect of allowing five- to sevenfold anaerobic induction in the absence of FNR. We have shown that the anaerobic induction of pepT-lacZ in a pepT200 fnr strain is dependent on wild-type alleles of both crp and cya. In a pepT200 pepT-lacZ strain, beta-galactosidase activity was elevated aerobically in the presence of exogenous cyclic AMP (cAMP) and was elevated also in succinate minimal medium relative to its level in glucose minimal medium. Primer extension analysis confirmed that P1 is the cAMP receptor protein (CRP)-dependent promoter. Site-directed mutagenesis experiments indicated that a hybrid CRP-FNR binding site positioned at -41 of the P1 promoter is utilized by both FNR and CRP. CRP-cAMP also appeared to repress FNR-dependent transcription of pepT under anaerobic conditions in both the pepT+ and pepT200 backgrounds. Although both CRP and FNR are capable of binding the hybrid site and activating transcription of pepT, CRP requires the consensus -10 sequence for efficient activation.

Multiaxial myocardial mechanics and extracellular matrix remodeling: mechanochemical regulation of cardiac fibroblast function.

Substantial evidence suggests that not only does the structure of the cardiac extracellular matrix affect the mechanical properties of myocardium, but that mechanical loading affects the synthesis of the extracellular matrix. However, loading conditions in vivo are nonhomogeneous and multiaxial. An experimental approach that combines mechanics and cell biology is used to examine the mechanisms of extracellular matrix remodeling in the heart. The results indicate that differential biological responses in adult cardiac fibroblasts can be correlated with specific physical signals, such as the magnitude and two dimensional (2D) pattern of strain. Some effects of flow-function relations are discussed.

Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: oafA is a member of a family of integral membrane trans-acylases.

Lipopolysaccharide (LPS) coats the surface of gram-negative bacteria and serves to protect the cell from its environment. The O-antigen is the outermost part of LPS and is highly variable among gram-negative bacteria. Strains of Salmonella are partly distinguished by serotypic differences in their O-antigen. In Salmonella typhimurium, the O-antigen is acetylated, conferring the 05 serotype. We have previously provided evidence that this modification significantly alters the structure of the O-antigen and creates or destroys a series of conformational epitopes. Here we report the detailed mapping, cloning, and DNA sequence of the oafA gene. The locus contains one open reading frame that is predicted to encode an inner membrane protein, consistent with its role in modification of the O-antigen subunit. The OafA protein shows homology to proteins in a number of prokaryotic and one eukaryotic species, and this defines a family of membrane proteins involved in the acylation of exported carbohydrate moieties. In many of these instances, acylation defines serotype or host range and thus has a profound effect on microbe-host interaction.

An equibiaxial strain system for cultured cells.

We developed a device that applies homogeneous equibiaxial strains of 0-10% to a cell culture substrate and quantitatively verified transmission of substrate deformation to cultured cardiac cells. Clamped elastic membranes in both single-well and multiwell versions of the device are uniformly stretched by indentation with a plastic ring, resulting in strain that is directly proportional to the pitch-to-radius ratio. Two-dimensional deformations were measured by tracking fluorescent microspheres attached to the substrate and to cultured adult rat cardiac fibroblasts. For nominal stretches up to 18%, strains along circumferential and radial axes were equal in magnitude and homogeneously distributed with negligible shear. For 5% stretch, circumferential and radial strains in the substrate were 0.046 +/- 0.005 and 0.048 +/- 0.004 [not significant (NS)], respectively, and shear strain was 0.001 +/- 0.003 (NS). Calibration of both single-well and multiwell versions permits strain selection by device rotation. The reproducible application and quantification of homogeneous equibiaxial strain in cultured cells provides a quantitative approach for correlating mechanical stimuli to cellular transduction mechanisms.

Adenovirus-mediated overexpression of human transforming growth factor-beta 1 in rat cardiac fibroblasts, myocytes and smooth muscle cells.

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cardiac cell function and its overexpression in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis. We wished to develop a high efficiency gene transfer method that could be used both in vitro and in vivo and result in the overexpression of TGF-beta 1. For this purpose, we constructed a replication-deficient human adenovirus 5 vector encoding for human TGF-beta 1 and used for control purposes an adenovirus lacZ vector. The adenovirus 5 construct was capable of infecting neonatal rat cardiac myocytes, fibroblasts and VSMCs. Of the three cell types, cardiac myocytes appear more susceptible to infection by the adenovirus 5 construct as assessed through beta-galactosidase staining. Infection of cardiac fibroblasts, myocytes and VSMCs with the hTGF-beta 1 adenovirus leads to the expression of hTGF-beta 1 mRNA and enhanced levels of bioactive and total TGF-beta 1 protein. Infection with hTGF-beta 1 adenovirus also results in enhanced levels of collagen type III gene expression in VSMCs and fibroblasts whereas in cardiac myocytes it leads to increased levels for sarcomeric and beta-actin. Thus, this adenoviral vector might be used for the exploration of in vivo effects of altered levels of cardiac TGF-beta 1.

Angiotensin II stimulates the autocrine production of transforming growth factor-beta 1 in adult rat cardiac fibroblasts.

Angiotensin II (Ang II) has been implicated in the development of cardiac hypertrophy and myocardial fibrosis. While recent in vivo and in vitro studies performed in cultured cardiac myocytes and fibroblasts support this role for Ang II, the mechanisms of Ang II action at the cellular level remain unclear. In the present study, we postulated that Ang II action in adult cardiac fibroblasts may stimulate the autocrine production and release of transforming growth factor-beta 1 (TGF-beta 1), a known regulator of cardiac fibroblast and myocyte function. We examined the ability of Ang II to regulate the gene expression, biological activity, and protein production of TGF-beta 1 in cultured adult rat cardiac fibroblasts. Treatment of fibroblast cultures with Ang II (10(-9) M) induced a two-fold increase in TGF-beta 1 mRNA levels within 4 h that was sustained through 24 h (P < 0.01). TGF-beta 1-like activity in Ang II-treated cultures was significantly increased compared with control as measured by bioassay (P < 0.001). Specificity for TGF-beta 1-like activity was confirmed through its neutralization with a TGF-beta 1 specific antibody (100 micrograms/ml). Total concentration of TGF-beta 1 (latent plus active forms) in conditioned media from Ang II-treated cardiac fibroblasts was also found to be greater than control (P < 0.01). These findings suggest that the effects of Ang II in the adult myocardium may be mediated in part by autocrine/paracrine mechanisms, including the production and release of TGF-beta 1 by cardiac fibroblasts.

Trophic effects of angiotensin II on neonatal rat cardiac myocytes are mediated by cardiac fibroblasts.

Cultured neonatal rat cardiac fibroblasts (NF) and myocytes (NM) were used to examine the distribution of angiotensin II (ANG II) receptors and the potential role of NF in mediating the trophic response to ANG II in the heart. In NM preparations cultured for 2-5 days, specific binding to 125I-ANG II was < 10% of the specific binding in cultured NF. Binding assays, immunocytochemistry, and autoradiography in NM cultured for > 5 days identified two populations of cells, one with fibroblast-like morphology and high density of ANG II receptors and another with low binding, comparable to NM cultures at day 5 or earlier. Conditioned medium (CM) from untreated NF increased cell surface area and net [3H]leucine (Leu) incorporation 1.4-fold in NM. CM from ANG II-treated NF enhanced [3H]Leu incorporation 2.2-fold in NM. This potentiating effect of ANG II was inhibited by losartan and was absent when ANG II was added directly to NM. In addition, studies using antibodies and bioassay for transforming growth factor-beta 1 (TGF-beta 1) suggested that TGF-beta 1 does not mediate the trophic effects of ANG II on NM. We conclude that ANG II receptors are localized predominantly on NF and that ANG II can indirectly stimulate hypertrophy of NM by stimulating NF to produce a transferrable factor(s). These data suggest that cardiac fibroblasts may play a critical role in mediating the hypertrophic response to ANG II in the rat heart.

Animated visualization of a high resolution color three dimensional digital computer model of the whole human head.

The interactive visualization of animated images through a computerized three dimensional (3D) full color model of an unstained cadaveric human head is presented. Serial full color images were taken of the blockface of a cryomicrotomed frozen human head every 200 microns. From this series of images a three dimensional digital model with a resultant pixel resolution of 200 microns3 was created on a UNIX workstation. Using this database, resampled images were computed along orthogonal axes and written sequentially to a write-once-read-many times (WORM) videodisc unit. Playback of this customized videodisc dataset provides animations of the digitally reconstructed slices and 3D reconstructed surface models. An interactive interface to the animated sequences is provided through a PC based tutorial package. This tutorial program is able to access videodisc frames to display animations and labeled still images in a software window to illustrate various neuroanatomic topics. The technique of animation as applied to this high resolution 3D model provides insight into complex spatial relationships and has great potential in research and as a teaching tool in the neurosciences.

Duplex carotid sonography. Peak systolic velocity in quantifying internal carotid artery stenosis.

Duplex ultrasonography combining high-resolution imaging and Doppler spectrum analysis was performed in 92 consecutive patients (total, 180 vessels) and compared with the findings of conventional arteriography. All duplex studies were categorized into four groups based upon the maximum internal carotid artery (ICA) velocity: group 1: less than 125 cm/sec; group 2: 125 to 224 cm/sec; group 3: greater than 225 cm/sec; and group 4: no flow. Sensitivities and specificities were highest when peak ICA velocity was used as one of several criteria in quantifying the degree of ICA stenosis. These additional criteria were: (1) the presence of extensive sonographically visible plaque within the ICA; (2) an abnormal spectral waveform with elevated diastolic velocity (greater than 100 cm/sec); (3) resistive pattern ("externalization") of the common carotid artery (CCA) waveform; and (4) the ratio of the right CCA peak velocity to the left of less than 0.7 or greater than 1.3. The overall accuracy for the combined groups using all criteria was 94%.

The relationship between selected student characteristics and activity patterns in a required high school physical education class.

This study described the activity patterns of students in a high school fitness class and explored the structural relationships between particular student characteristics and their systematically coded exercise behavior. Although percent of time spent jogging was low (18%), with no gains made in cardiovascular fitness, the amount of time spent jogging, the distance covered, and fitness level were all significantly correlated. A LISREL VI computer program was used to test a structural equation model representing the Fishbein Behavioral-Intention Model. In support of the model, results showed the prediction of exercise behavior by attitude and subjective norm was significantly mediated by intention. Although not significant, it is worth noting that subjective norm was found to be the stronger predictor of intention over attitude. Background variables were found to indirectly influence intention through their significant influence on attitude and subjective norm. For this sample of 9th and 10th graders, significant others, particularly their peers and teachers, had a stronger impact on behavior than personal attitudes about activity.

Parents' accuracy in estimating child weight status.

Mothers of 107 preschool children estimated their child's weight status, and the accuracy of these estimates was examined. The majority of mothers (72%) were accurate. Of those who were inaccurate, 83% had underestimated the child's weight status, whereas only 17% had overestimated. Mothers of heavier children were more likely to underestimate their child's weight status.