Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry

Cancer cells, including leukemia cells, reside in a complex microenvironment, which influences biology and activity of the cells. The protective role of bone marrow stromal cells is already commonly recognized. Remodeling of stroma cell functions by leukemia cells is also well documented. In this respect, different routes of interactions were defined, such as direct cell-cell interactions or indirect cross talk, by release of soluble factors or vesicular particles containing proteins, RNAs and other molecules. Since intercellular communication seems to play a role in various biological processes, it might be important to conduct studies in co-culture systems, which at least mimic partially more physiological conditions, and enables this intercellular exchange to occur. Thus, it is crucial to improve analytical methods of investigation of co-cultured cells, to study their interactions and so to understand biology of leukemia in order to understand molecular mechanisms and offer novel therapeutic strategies. The present chapter outlines the importance of modern, multiparameter flow cytometry methods, which allow to analyze interactions between different types of cells within the leukemia microenvironment. Importantly, the proposed experimental setups can be easily transformed to study different cell types and different biological systems

Dietary macronutrients and fluid intakes in a sample of pregnant women with either gestational diabetes or type 1 diabetes mellitus, assessed in comparison with Polish nutritional guidelines

Objectives: Pregnancy is a critical period during which environmental factors such as nutrition can affect development. Maintaining proper nutrition becomes even more significant when pregnant women have diabetes. The aim of this study was to measure changes in energy and macronutrient intakes among pregnant women and patients diagnosed either with gestational diabetes mellitus (GDM) during pregnancy, or, type 1 diabetes mellitus (T1DM) before pregnancy, and to assess the pregnant women’s dietary intakes in comparison with Polish Institute of Food and Nutrition nutritional guidelines.Material and methods: The analysis was conducted among 83 pregnant women (29 GDM patients, 26 T1DM patients and 28 normal pregnancy patients — the control group) from whom we gathered nutritional data during the second part of their pregnancies. Data on each woman’s diet during pregnancy was collected is self-completed dietary records during seven consecutive 24-hour periods.Results: The mean macronutrient intake of the GDM patients was 32.1% fat, 19.5% protein, and 48.3% carbohydrates; in the T1DM group the results were 34.2%, 19.4% and 46.4% respectively; and in control group they were 31.8%, 17.6% and 50.5% respectively. This study showed that many of the pregnant women did not reach the recommended level of energy intake during pregnancy. Moreover, most of the women exceeded their fat requirements, and fat intake as a proportion of energy intakes also exceeded the guidelines in more than 60% of the women across all groups.Conclusions: The implications and possible causes of excessive fat intake during pregnancy and pregnancies complicated by diabetes are underestimated and undertreated by obstetricians and warrant further investigation, especially in association with gestational weight gain, maternal and fetal perinatal complications, and post-gestational diabetes

Dietary vitamin and mineral intakes in a sample of pregnant women with either gestational diabetes or type 1 diabetes mellitus, assessed in comparison with Polish nutritional guidelines

Objectives: Maintaining proper nutrition during pregnancy is crucial for pregnant women and especially for who havebeen diagnosed with type 1 diabetes mellitus (T1DM) or who develop gestational diabetes mellitus (GDM).Material and methods: To measure differences in vitamin and mineral intakes among women with normal pregnancies,pregnant women with GDM, and pregnant women with pre-gestational T1DM; and to assess the women’s dietary intakes in comparison with Polish nutritional guidelines. The analysis was conducted among 83 pregnant women (29 GDM patients, 26 T1DM patients and 28 normal pregnancy participants) from whom we collected seven-day 24-hour dietary records during the second part of their pregnancies.Results: There were no statistically significant differences observed for most of the vitamin and mineral intakes across the three groups. However, we did observe a significant difference in the vitamin C and calcium intakes between groups. The mean vitamin C and calcium intakes were significantly higher in the control group than among the diabetic patients. Insufficient dietary calcium intakes were found among 52.3% of the GDM patients and 61.6% of the T1DM participants, while only 28.6% of the normal pregnancy patients experienced a calcium deficiency. The highest incidence of inadequate intake in each of the GDM, T1DM and control groups was observed for vitamin D (100%, 100%, 100%), folate (97.7%, 100%, 100%), iron (97.7%, 100%, 100%), and iodine (97.7%, 92.4%, 85.7%), respectively.Conclusions: Diet alone may not be enough to provide adequate levels of vitamins and minerals for most micronutrients. Supplement use reduces the risk of inadequate intake for many micronutrients, but diet-related issues during pregnancy and pregnancy diagnosed with diabetes remain, and they deserve to be addressed during public health interventions

A designer hyper interleukin 11 (H11) is a biologically active cytokine

<p>Abstract</p> <p>Background</p> <p>Interleukin 11 (IL-11) is a pleiotropic cytokine with anti-apoptotic, anti-inflammatory and hematopoietic potential. The IL-11 activity is determined by the expression of the IL-11R receptor alpha (IL-11Rα) and the signal transducing subunit β (gp130) on the cell membrane. A recombinant soluble form of the IL-11Rα (sIL-11Rα) in combination with IL-11 acts as an agonist on cells expressing the gp130 molecule. We constructed a designer cytokine Hyper IL-11 (H11), which is exclusively composed of naturally existing components. It contains the full length sIL-11Rα connected with the mature IL-11 protein using their natural sequences only. Such a construct has two major advantages: (i) its components are as close as possible to the natural forms of both proteins and (ii) it lacks an artificial linker what should avoid induction of antibody production.</p> <p>Results</p> <p>The H11 construct was generated, the protein was produced in a baculovirus expression system and was then purified by using ion exchange chromatography. The H11 protein displayed activity in three independent bioassays, (i) it induced acute phase proteins production in HepG2 cells expressing IL-11, IL-11Rα and gp130, (ii) it stimulated the proliferation of B9 cells (cells expressing IL-11Rα and gp130) and (iii) proliferation of Baf/3-gp130 cells (cells not expressing IL-11 and IL-11Rα but gp130). Moreover, the preliminary data indicated that H11 was functionally distinct from Hyper-IL-6, a molecule which utilizes the same homodimer of signal transducing receptor (gp130).</p> <p>Conclusions</p> <p>The biologically active H11 may be potentially useful for treatment of thrombocytopenia, infertility, multiple sclerosis, cardiovascular diseases or inflammatory disorders.</p

Gender-specific association of body composition with inflammatory and adipose-related markers in healthy elderly Europeans from the NU-AGE study

Objectives: The aim of this work was to examine the cross-sectional relationship between body composition (BC) markers for adipose and lean tissue and bone mass, and a wide range of specific inflammatory and adipose-related markers in healthy elderly Europeans. Methods: A whole-body dual-energy X-ray absorptiometry (DXA) scan was made in 1121 healthy (65–79 years) women and men from five European countries of the “New dietary strategies addressing the specific needs of elderly population for a healthy aging in Europe” project (NCT01754012) cohort to measure markers of adipose and lean tissue and bone mass. Pro-inflammatory (IL-6, IL-6Rα, TNF-α, TNF-R1, TNF-R2, pentraxin 3, CRP, alpha-1-acid glycoprotein, albumin) and anti-inflammatory (IL-10, TGF-β1) molecules as well as adipose-related markers such as leptin, adiponectin, ghrelin, and resistin were measured by magnetic bead-based multiplex-specific immunoassays and biochemical assays. Results: BC characteristics were different in elderly women and men, and more favorable BC markers were associated with a better adipose-related inflammatory profile, with the exception of skeletal muscle mass index. No correlation was found with the body composition markers and circulating levels of some standard pro- and anti-inflammatory markers like IL-6, pentraxin 3, IL-10, TGF-β1, TNF-α, IL-6Rα, glycoprotein 130, TNF-α-R1, and TNF-α-R2. Conclusions: The association between BC and inflammatory and adipose-related biomarkers is crucial in decoding aging and pathophysiological processes, such as sarcopenia. DXA can help in understanding how the measurement of fat and muscle is important, making the way from research to clinical practice. Key Points: • Body composition markers concordantly associated positively or negatively with adipose-related and inflammatory markers, with the exception of skeletal muscle mass index. • No correlation was found with the body composition markers and circulating levels of some standard pro- and anti-inflammatory markers like IL-6, pentraxin 3, IL-10, TGF-β1, TNF-α, IL-6Rα, gp130, TNF-α-R1, and TNF-α-R2. • Skeletal muscle mass index (SMI) shows a good correlation with inflammatory profile in age-related sarcopenia

Emotion Processing In Children with Conversion Disorders: A Developmental and Relational Perspective

Objectives: Historically, conversion symptoms have been understood to emerge within situations that generate strong emotions. From the modern perspective of integrative neuroscience, conversion symptoms can be conceptualised more specifically as involving motor-sensory/autonomic responses to situations that present a serious threat to self and that, in turn, elicit strong negative emotions. Like other responses to threat, they may be primed and fine tuned in the context of repeating, stressful interactions with attachment figures—the child’s environmental context—and automatically triggered when the individual is highly aroused and when other mechanisms for managing threat and danger (for example, verbal negotiation or running away) have failed or been blocked. This theoretical framework predicts that children and adolescents presenting with conversion disorders ought to process emotional information differently to healthy controls and that differences in emotion processing ought to be evident on standardized measures of emotion processing. To study this hypothesis, a research program was developed, involving multiple levels of analysis—psychological (cognitive and behavioural), physiological, and neural. The material presented in this thesis includes (a) articulation of the ideas (theory) that underpin the individual studies and the broader research program; (b) a detailed description of the methodology for the research program as a whole, and of the Dynamic-Maturational-Model of attachment (DMM) methodology used to assess emotion processing on the cognitive-psychological level (as investigated in the studies comprising this thesis); (c) presentation of the pilot attachment data; and (d) presentation of the final attachment data. The remaining components of the broader research program—analyses on the behavioural-psychological, physiological, and neural levels—are still in progress and will be carried through after completion of the thesis. Methods: Consecutive conversion subjects and age- and sex-matched controls were recruited over four years from the candidate’s consultation-liaison service at The Children’s Hospital at Westmead, Australia. Seventy-six subjects participated in structured interviews about attachment relationships and autobiographical events. Fifty-seven of these subjects completed a computerized battery of psychological and physiological tests in the laboratory (the broader research program). Recruitment for a modified version of this test battery in the functional magnetic resonance imaging machine (fMRI) is still ongoing. Publications Being Presented for Examination (Articles 1-4): Kozlowska, K. (2007). The developmental origins of conversion disorders. Clinical Child Psychology and Psychiatry, 12(4), 487–510. Kozlowska, K., & Williams, L. M. (2010). Self-protective Organization in Children with Conversion Symptoms: A Cross-Sectional Study Looking at Psychological and Biological Correlates. Mind & Brain, The Journal of Psychiatry, 1(2), 43–57. Kozlowska, K., & Williams, L. M. (2009). Self-protective organization in children with conversion and somatoform disorders. Journal of Psychosomatic Research, 67, 223–233. Kozlowska, K., Scher, S. et al. (2011). Patterns of emotional-cognitive functioning in pediatric conversion patients: implications for the conceptualization of conversion disorders, Psychosomatic Medicine 73(9): 775–788. Summary of Results: On a cognitive level of analysis—using the DMM—children and adolescents with conversion disorders show differences in emotion processing versus controls. Children and adolescents with conversion disorders were classified as falling into non-normative patterns of attachment, implying a chronic disruption of what are normally safe and nurturing attachments. Their attachment narratives were characterised by distortions in information processing: discrepancies in information about emotional events held in different memory systems; imbalance of information about affect and temporal order; imbalance of self and other perspectives; imbalance of self-responsibility and blame on others; a lack linguistic coherence and a high frequency of linguistic markers for unresolved loss and trauma. Unlike controls who fell largely into normative patterns of attachment (Type A1-2, Type B1-5, Type C1-2), children with conversion disorders showed two distinct patterns of emotion processing. Psychological inhibition (Type 3-6 or Type A+) was associated with conversion symptoms defined by discrete motor-sensory loss and positive motor symptoms (tremors and tics). Psychological coercion-preoccupation (Type 3-6 or Type C+) was associated with all other motor symptoms—bizarre gaits and postures, whole-body floppiness, and refusal to move. Non-epileptic seizures occurred across both groups. Additional publications articulating the application of this research to clinical practice. The following articles describe the integration of theoretical ideas and research findings from this thesis into clinical practice. The articles were written by the candidate in collaboration with her clinical team during the period of the thesis and are included in appendix 3 (articles B-E). Kozlowska, K. and R. Khan (2011). A developmental, body-oriented intervention for children and adolescents with medically unexplained chronic pain. Clinical Child Psychology and Psychiatry, 16(4): 575–598. Kozlowska, K., English, M., & Savage, B. (in press). Connecting body and mind: The first interview with somatizing patients and their families. Clinical Child Psychology and Psychiatry. Kozlowska, K., English, M., Savage, B., & Chudleigh, C. (in press). Multimodal rehabilitation: A mind-body, family-based intervention for children and adolescents impaired by medically unexplained symptoms. Part 1: The program. American Journal of Family Therapy. Kozlowska, K., English, M., Savage, B., Chudleigh, C., Davies, F., Paull, M., et al. (in press). Multimodal rehabilitation: A mind-body, family-based intervention for children and adolescents impaired by medically unexplained symptoms. Part 2: Case studies and outcomes. American Journal of Family Therapy. Conclusions: Contrary to the classic understanding of conversion disorder as a unified diagnostic entity with diverse symptoms, the studies comprising this thesis identified two distinct subgroups of conversion patients—those using psychological inhibition and those using psychological coercion-preoccupation—whose symptoms fell into discrete clusters. Psychological inhibition was associated with conversion symptoms reflecting motor-sensory inhibition or failure of inhibition, and psychological coercion-preoccupation was associated with all other motor-sensory conversion symptoms. Non-epileptic seizures occurred across both groups. These results suggest functional linkages between cognitive and motor-sensory processes in patients with conversion disorders. Motor-sensory conversion symptoms appear to be subcomponents or the unwanted consequences of broader self-protective patterns of response to threats—integrated sets of reactions involving psychological (cognitive and behavioural), physiological, and neural components. By contrast, non-epileptic seizures appear to be underpinned by an independent mechanism. Further research is needed to determine the neural mechanisms underlying these various processes

Riemann-Hilbert Problems and their applications in mathematical physics

The aim of this thesis is to present the reader with the very effective and rigorous Riemann-Hilbert approach of solving asymptotic problems. We consider a transition problem for a Toeplitz determinant; its symbol depends on an additional parameter t. When t > 0, the symbol has one Fisher-Hartwig singularity at an arbitrary point z1 6= 1 on the unit circle (with associated α1, β1 ∈ C strengths) and as t → 0, a new Fisher-Hartwig singularity emerges at the point z0 = 1 (with α0, β0 ∈ C strengths). The asymptotics we present for the determinant are uniform for sufficiently small t. The location of the β-parameters leads to the consideration of two cases, both of which are addressed in this thesis. In the first case, when | Re β0 − Re β1| < 1 we see a transition between two asymptotic regimes, both given by the same result by Ehrhardt, but with different parameters, thus producing different asymptotics. In the second case, when | Re β0 − Re β1| = 1 the symbol has Fisher-Hartwig representations at t = 0, and the asymptotics are given the Tracy-Basor conjecture. These double scaling limits are used to explain transition in the theory of XY spin chains between different regions in the phase diagram across critical lines

Biological Fluids as a Source of Information on the Exposure of Man to Environmental Chemical Agents

The human body is exposed to a variety of environmental pollutants. Many chemical compounds, including volatile organochlorine compounds and metals, have a harmful effect on the tissues and organs of the human body, in many cases causing irreversible damage and illnesses. In order to assess the degree of occupational hazard, it has become more and more common to determine the concentrations of chemical substances or their metabolites in biological fluids (i.e., in urine, blood, or less often, in human milk, bile, saliva, and sperm). However, in order to determine concentrations levels of trace components in biological fluids such as urine, blood, milk, bile, saliva, or sperm, the samples collected have to be prepared for the final analysis because their matrix is so complex as to preclude direct determination of the analytes by any analytical method available. This article contains the literature data on: analyte isolation and/or enrichment techniques from samples of biological fluids prior to the final determination step; and final determination techniques for a variety of compounds, both organic and inorganic, being determined in the above samples. The data include both the primary pollutants that found their way into the human body as a result of environmental and occupational exposure and the products of their conversion (metabolism) in the organism

Investigation of stability of aqueous solutions containing trace amounts of volatile organic analytes

When determining volatile organohalogen compounds in an aqueous matrix, specific properties of these compounds should be taken into consideration, when preparing aqueous standard solutions used for calibration and during model investigations, namely:•low boiling points;•high vapor pressure; and,•relatively low solubility in water.Because of the significance of this problem and the lack of practical information available in the literature, we carried out model investigations of stability of selected volatile analytes in aqueous solutions. The experimental results were used to estimate the time after which the result of determination may not reflect the true concentration of the analyte. The investigations provided information on:•the method of preparation of model solutions (in appropriate containers);•the method of storage and the storage time of these solutions; and,•number of repetitions needed in the final determination (repeated sampling of the solution from a volumetric flask, which requires the flask to be opened for each sampling). This parameter is especially important when determining the repeatability and the reproducibility in validating the procedure for TLHS-DAI-GC-ECD