Short communication: differential immunoglobulin transfer during mastitis challenge by pathogen-specific components

Mastitis induced by Escherichia coli is often characterized by severe clinical signs, indicating a more powerful combat of the immune system against the pathogen compared with Staphylococcus aureus infections, which are often represented by chronic and subclinical diseases. The aim of this study was to test the major pathogenic component lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Staph. aureus for their effects on blood-milk barrier integrity and the related transfer of immunoglobulins and lactate from blood into milk. A similar somatic cell count (SCC) increase was achieved by intramammary challenge of 1 quarter of 5 cows with 20 µg of LTA, and 8 cows with 0.2 µg of LPS (maximum log SCC/mL: 7). Milk IgG(1) concentrations increased in LPS- but not in LTA-challenged quarters. Milk IgG(2) concentrations were increased in treated quarters at 3h after LPS, and 6h after LTA challenge. Higher maximum levels of IgG(2) were reached in milk of LPS-treated quarters (173 ± 58 μg/mL) than of LTA-challenged quarters (62 ± 13 μg/mL). Immunoglobulin G(1) and IgG(2) levels did not change in control quarters. l-Lactate concentrations in milk increased 4h after LPS and 5h after LTA challenge and reached higher maximum levels in LPS- (221 ± 48 mg/L) than in LTA-treated quarters (77 ± 18 mg/L). In conclusion, a mammary inflammation on a quantitatively similar level based on SCC increase achieves a more efficient transfer of blood components such as IgG(2) via the blood-milk barrier if induced by LPS from E. coli than by LTA from Staph. aureus. This pathogen-specific difference may play an important role in the cure rate of the respective intramammary infection, which is usually lower in Staph. aureus- than in E. coli-induced mastitis.

Occupational role stress is associated with higher cortisol reactivity to acute stress

We investigated whether occupational role stress is associated with differential levels of the stress hormone cortisol in response to acute psychosocial stress. Forty-three medication-free nonsmoking men aged between 22 and 65 years (mean ± SEM: 44.5 ± 2) underwent an acute standardized psychosocial stress task combining public speaking and mental arithmetic in front of an audience. We assessed occupational role stress in terms of role conflict and role ambiguity (combined into a measure of role uncertainty) as well as further work characteristics and psychological control variables including time pressure, overcommitment, perfectionism, and stress appraisal. Moreover, we repeatedly measured salivary cortisol and blood pressure levels before and after stress exposure, and several times up to 60 min thereafter. Higher role uncertainty was associated with a more pronounced cortisol stress reactivity (p = .016), even when controlling for the full set of potential confounders (p < .001). Blood pressure stress reactivity was not associated with role uncertainty. Our findings suggest that occupational role stress in terms of role uncertainty acts as a background stressor that is associated with increased HPA-axis reactivity to acute stress. This finding may represent a potential mechanism regarding how occupational role stress may precipitate adverse health outcomes.

A reduced model clarifies the role of feedback loops and time delays in the Drosophila circadian oscillator.

Although several detailed models of molecular processes essential for circadian oscillations have been developed, their complexity makes intuitive understanding of the oscillation mechanism difficult. The goal of the present study was to reduce a previously developed, detailed model to a minimal representation of the transcriptional regulation essential for circadian rhythmicity in Drosophila. The reduced model contains only two differential equations, each with time delays. A negative feedback loop is included, in which PER protein represses per transcription by binding the dCLOCK transcription factor. A positive feedback loop is also included, in which dCLOCK indirectly enhances its own formation. The model simulated circadian oscillations, light entrainment, and a phase-response curve with qualitative similarities to experiment. Time delays were found to be essential for simulation of circadian oscillations with this model. To examine the robustness of the simplified model to fluctuations in molecule numbers, a stochastic variant was constructed. Robust circadian oscillations and entrainment to light pulses were simulated with fewer than 80 molecules of each gene product present on average. Circadian oscillations persisted when the positive feedback loop was removed. Moreover, elimination of positive feedback did not decrease the robustness of oscillations to stochastic fluctuations or to variations in parameter values. Such reduced models can aid understanding of the oscillation mechanisms in Drosophila and in other organisms in which feedback regulation of transcription may play an important role.

Differential dynamic properties of scleroderma fibroblasts in response to perturbation of environmental stimuli.

Diseases are believed to arise from dysregulation of biological systems (pathways) perturbed by environmental triggers. Biological systems as a whole are not just the sum of their components, rather ever-changing, complex and dynamic systems over time in response to internal and external perturbation. In the past, biologists have mainly focused on studying either functions of isolated genes or steady-states of small biological pathways. However, it is systems dynamics that play an essential role in giving rise to cellular function/dysfunction which cause diseases, such as growth, differentiation, division and apoptosis. Biological phenomena of the entire organism are not only determined by steady-state characteristics of the biological systems, but also by intrinsic dynamic properties of biological systems, including stability, transient-response, and controllability, which determine how the systems maintain their functions and performance under a broad range of random internal and external perturbations. As a proof of principle, we examine signal transduction pathways and genetic regulatory pathways as biological systems. We employ widely used state-space equations in systems science to model biological systems, and use expectation-maximization (EM) algorithms and Kalman filter to estimate the parameters in the models. We apply the developed state-space models to human fibroblasts obtained from the autoimmune fibrosing disease, scleroderma, and then perform dynamic analysis of partial TGF-beta pathway in both normal and scleroderma fibroblasts stimulated by silica. We find that TGF-beta pathway under perturbation of silica shows significant differences in dynamic properties between normal and scleroderma fibroblasts. Our findings may open a new avenue in exploring the functions of cells and mechanism operative in disease development.

THE ROLE OF TYROSINE PHOSPHORYLATION IN THE FUNCTIONS OF THE TUMOR SUPPRESSOR GPRC5A

The retinoic acid inducible G protein coupled receptor family C group 5 type A (GPRC5A) is expressed preferentially in normal lung tissue but its expression is suppressed in the majority of human non-small cell lung cancer cell lines and tissues. This differential expression has led to the idea that GPRC5A is a potential tumor suppressor. This notion was supported by the finding that mice with a deletion of the Gprc5a gene develop spontaneous lung tumors. However, there are various tumor cell lines and tissue samples, including lung, that exhibit higher GPRC5A expression than normal tissues and some reports by other groups that GPRC5A transfection increased cell growth and colony formation. Obviously, GPRC5A has failed to suppress the development of the tumors and the growth of the cell lines where its expression is not suppressed. Since no mutations were detected in the coding sequence of GPRC5A in 20 NSCLC cell lines, it’s possible that GPRC5A acts as a tumor suppressor in the context of some cells but not in others. Alternatively, we raised the hypothesis that the GPRC5A protein may be inactivated by posttranslational modification(s) such as phosphorylation. It is well established that Serine/Threonine phosphorylation of G protein coupled receptors leads to their desensitization and in a few cases Tyrosine phosphorylation of GPCRs has been linked to internalization. Others reported that GPRC5A can undergo tyrosine phosphorylation in the cytoplasmic domain after treatment of normal human mammary epithelial cells (HMECs) with epidermal growth factor (EGF) or Heregulin. This suggested that GPRC5A is a substrate of EGFR. Therefore, we hypothesized that tyrosine phosphorylation of GPRC5A by activation of EGFR signaling may lead to its inactivation. To test this hypothesis, we transfected human embryo kidney (HEK) 293 cells with GPRC5A and EGFR expression vectors and confirmed that GPRC5A can be tyrosine phosphorylated after activation of EGFR by EGF. Further, we found that EGFR and GPRC5A can interact either directly or through other proteins and that inhibition of the EGFR kinase activity decreased the phosphorylation of GPRA5A and the interaction between GPRC5A and EGFR. In c-terminal of GPRC5A, There are four tyrosine residues Y317, Y320, Y347, Y350. We prepared GPRC5A mutants in which all four tyrosine residues had been replaced by phenylalanine (mutant 4F) or each individual Tyr residue was replaced by Phe and found that Y317 is the major site for EGFR mediated phosphorylation in the HEK293T cell line. We also found that EGF can induce GPRC5A internalization both in H1792 transient and stable cell lines. EGF also partially inactivates the suppressive function of GPRC5A on cell invasion activity and anchorage-independent growth ability of H1792 stable cell lines. These finding support our hypothesis that GPRC5A may be inactivated by posttranslational modification- tyrosine phosphorylation.

THE ROLE OF MAP KINASES ON THE FUNCTIONAL HETEROGENEITY OF HUMAN CD8+ T CELL MATURATION SUBSETS

While prior studies have focused on naïve (CD45RA+CD27+) and early stage memory (CD45RA-CD27+) CD8+ T cells, late memory CD8+ T cells (CD45RA+CD27) have received less interest because this subset of T cells is generally recognized as effectors, which produce IFNγ (but no IL-2) and perforin. However, multiple studies suggest that late memory CD8+ T cells may provide inadequate protection in infectious diseases and cancer models. To better understand the unique function of late memory CD8+ T cells, I optimized multi-color flow cytometry techniques to assess the cytokine production of each human CD8+ T cell maturation subset. I demonstrated that late memory CD8+ T cells are the predominant producer of CC chemokines (e.g. MIP-1β), but rarely produce IL-2; therefore they do not co-produce IL-2/IFNγ (polyfunctionality), which has been shown to be critical for protective immunity against chronic viral infection. These data suggest that late memory CD8+ T cells are not just cytotoxic effectors, but may have unique functional properties. Determining the molecular signature of each CD8+ T cell maturation subset will help characterize the role of late memory CD8+ T cells. Prior studies suggest that ERK1 and ERK2 play a role in cytokine production including IL-2 in T cells. Therefore, I tested whether differential expression of ERK1 and ERK2 in CD8+ T cell maturation subsets contributes to their functional signature by a novel flow cytometry technique. I found that the expression of total ERK1, but not ERK2, is significantly diminished in late memory CD8+ T cells and that ERK1 expression is strongly associated with IL-2 production and CD28 expression. I also found that IL-2 production is increased in late memory CD8+ T cells by over-expressing ERK1. Collectively, these data suggest that ERK1 is required for IL-2 production in human CD8+ T cells. In summary, this dissertation demonstrated that ERK1 is down-regulated in human late memory CD8+ T cells, leading to decreased production of IL-2. The data in this dissertation also suggested that the functional heterogeneity in human CD8+ T cell maturation subsets results from their differential ERK1 expression.

Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

Differential dynamic properties of scleroderma fibroblasts in response to perturbation of environmental stimuli.

Diseases are believed to arise from dysregulation of biological systems (pathways) perturbed by environmental triggers. Biological systems as a whole are not just the sum of their components, rather ever-changing, complex and dynamic systems over time in response to internal and external perturbation. In the past, biologists have mainly focused on studying either functions of isolated genes or steady-states of small biological pathways. However, it is systems dynamics that play an essential role in giving rise to cellular function/dysfunction which cause diseases, such as growth, differentiation, division and apoptosis. Biological phenomena of the entire organism are not only determined by steady-state characteristics of the biological systems, but also by intrinsic dynamic properties of biological systems, including stability, transient-response, and controllability, which determine how the systems maintain their functions and performance under a broad range of random internal and external perturbations. As a proof of principle, we examine signal transduction pathways and genetic regulatory pathways as biological systems. We employ widely used state-space equations in systems science to model biological systems, and use expectation-maximization (EM) algorithms and Kalman filter to estimate the parameters in the models. We apply the developed state-space models to human fibroblasts obtained from the autoimmune fibrosing disease, scleroderma, and then perform dynamic analysis of partial TGF-beta pathway in both normal and scleroderma fibroblasts stimulated by silica. We find that TGF-beta pathway under perturbation of silica shows significant differences in dynamic properties between normal and scleroderma fibroblasts. Our findings may open a new avenue in exploring the functions of cells and mechanism operative in disease development.

Differential gene expression in multistep tumorigenesis using an {\it in vitro\/} human cell model

Using a human terato-carcinoma cell line, PA-1, the functional role of the oncogenes and tumor suppressor gene involved in the multistep process of carcinogenesis have been analyzed. The expression of AP-2 was strongly correlated with the susceptibility to ras transformation. The differential responsiveness to growth factors between stage 1 ras resistant cells and stage 2 ras susceptible cells was observed, indicating that the ability of stage 2 cells to respond to the mutated ras oncogenes in transformation correlated with the ability to be stimulated by certain growth factors. Using differential screening of cDNA libraries, a number of differentially expressed cDNA clones was isolated. One of those, clone 12, is overexpressed in ras transformed stage 3 cells. The amino acid sequence of clone 12 is almost identical to a mouse LLrep3 gene that was growth-regulated, and 78% similar to a yeast ribosomal protein S4. These results suggest that the S4 gene may be involved in regulation of growth. Clone 9 is expressed in stage 1 ras resistant cells (3.5-kb and 3.0-kb transcripts) but the expression of this clone in stage 2 ras susceptible cells and stage 3 ras-transformed cells is greatly diminished. The expression of this cDNA clone was increased to at least five fold in ras resistant cells and nontumorigenic hybrids treated with retinoic acid but not increased in retinoic acid treated ras susceptible cells, ras transformed cells and the tumorigenic segregants. Partial sequence of this clone showed no homology to the sequences in Genbank. These findings suggest that clone 9 could be a suppressor gene or the genes that are involved in the biochemical pathway of tumor suppression or neurogenic differentiation. The apparent pleiotropic effect of the loss of this suppressor gene function support Harris' proposal that tumor suppressor genes regulate differentiation. The tumor suppressor gene may act as negative regulator of tumor growth by controlling gene expression in differentiation. ^

Differential effect of p47phox and gp91phox deficiency on the course of Pneumococcal Meningitis.

Bacterial meningitis is a severe inflammatory disease of the central nervous system and is characterized by massive infiltration of granulocytes into the cerebrospinal fluid (CSF). To assess the role of NADPH oxidase-derived reactive oxygen species (ROS) in pneumococcal meningitis, mice deficient in either the gp91 subunit (essential for functioning of the phagocyte enzyme) or the p47 subunit (essential for functioning of homologous enzymes in nonphagocytic cells) were intracisternally infected with live Streptococcus pneumoniae, and defined disease parameters were measured during the acute stage of infection. While none of the parameters measured (including CSF bacterial titers) were significantly different in gp91(-/-) and wild-type mice, the infection in p47(-/-) mice was associated with significantly increased inflammation of the subarachnoid and ventricular space, disruption of the blood-brain barrier, and the presence of interleukin-1 beta, tumor necrosis factor alpha, and matrix metalloproteinase 9 in the cortex. These changes were associated with approximately 10-fold-higher CSF bacterial titers in p47(-/-) mice than in wild-type mice (P < 0.001). In contrast to infection with live bacteria, the inflammatory response, including CSF leukocytosis, was significantly attenuated in p47(-/-) mice (but not gp91(-/-) mice) challenged with a fixed number of heat-inactivated pneumococci. Impairment of the host defense appeared to be responsible for the higher bacterial titers in p47(-/-) mice. Therefore, these results indicate that ROS generated by a gp91-independent NADPH oxidase(s) are important for establishing an adequate inflammatory response to pneumococcal CSF infection.

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K-m = 0.1-0.3 mM), lower for Pro (K-m = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K-m = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.

Marital split-up and widowhood in old age: Differential impact on psychological and social well-being

Marital split-up and spousal loss are among the most stressful critical life events. Numerous studies have documented their detrimental effects on well-being, yet the large individual differences in psychological adaptation are still not well understood. Whereas in old age bereavement is normative and can be anticipated, divorce is an “off-time” transition for this age group. In contrast to bereavement which has been amply studied, research on later life divorce is still missing despite the increasing relevance of the topic due to the significant increase of divorces in older age. Based on a modified and extended view of Amato’s divorce-stress-adjustment model (2000), the aim of this contribution is to explore the differential impact of marital split-up and widowhood in older age on psychological (life satisfaction) and social well-being (social loneliness), and the adaptation to these critical life events. Our analyses are based on data gathered in a questionnaire study, which is part of the Swiss National Centre of Competence in Research LIVES. In a first step we compared three groups of individuals aged 60 to 75 years: a sample of 251 persons with a marital split-up (127 women; 123 men), a sample of 270 widowed persons (170 women; 100 men), and a group of 221 continuously married people (110 women; 111 men), which served as control group. In a second step, we investigated the role of socio-demographic variables, intrapersonal and interpersonal resources and variables of the context of loss as predictors for the psychological adaptation to a marital break-up and loss in old age. First results by ANCOVA indicate significant differences with regard to life satisfaction among the three groups, with divorced persons with the lowest scores, followed by the bereaved ones, and the married controls with the highest. Regarding social loneliness, divorced individuals report higher social loneliness than the bereaved group and the married controls (no significant difference between widowed and the married). In both loss groups, financial and intrapersonal resources, as well as the emotional valence of the loss are the most important predictors for the psychological and social adaptation. However, happiness in the past relationship is an important resource regarding the indicators for adaptation for the widowers, but not for individuals with a marital dissolution.

Wanting to Get More or Protecting One's Assets: Age-Differential Effects of Gain Versus Loss Perceptions on the Willingness to Engage in Collective Action

OBJECTIVES: The present research examined motivational differences across adulthood that might contribute to age-related differences in the willingness to engage in collective action. Two experiments addressed the role of gain and loss orientation for age-related differences in the willingness to engage in collective action across adulthood. METHOD: In Experiment 1, N = 169 adults (20-85 years) were confronted with a hypothetical scenario that involved either an impending increase or decrease of health insurance costs for their respective age group. In Experiment 2, N = 231 adults (18-83 years) were asked to list an advantage or disadvantage they perceived in being a member of their age group. Subsequently, participants indicated their willingness to engage in collective action on behalf of their age group. RESULTS: Both experiments suggest that, with increasing age, people are more willing to engage in collective action when they are confronted with the prospect of loss or a disadvantage. DISCUSSION: The findings highlight the role of motivational processes for involvement in collective action across adulthood. With increasing age, (anticipated) loss or perceived disadvantages become more important for the willingness to participate in collective action.

Cost-effectiveness of peer role play and standardized patients in undergraduate communication training.

BACKGROUND The few studies directly comparing the methodological approach of peer role play (RP) and standardized patients (SP) for the delivery of communication skills all suggest that both methods are effective. In this study we calculated the costs of both methods (given comparable outcomes) and are the first to generate a differential cost-effectiveness analysis of both methods. METHODS Medical students in their prefinal year were randomly assigned to one of two groups receiving communication training in Pediatrics either with RP (N = 34) or 19 individually trained SP (N = 35). In an OSCE with standardized patients using the Calgary-Cambridge Referenced Observation Guide both groups achieved comparable high scores (results published). In this study, corresponding costs were assessed as man-hours resulting from hours of work of SP and tutors. A cost-effectiveness analysis was performed. RESULTS Cost-effectiveness analysis revealed a major advantage for RP as compared to SP (112 vs. 172 man hours; cost effectiveness ratio .74 vs. .45) at comparable performance levels after training with both methods. CONCLUSIONS While both peer role play and training with standardized patients have their value in medical curricula, RP has a major advantage in terms of cost-effectiveness. This could be taken into account in future decisions.