Pre- and postnatal nutrition – target for allergy risk reduction

A rapid increase in allergic diseases in Western societies has led to the conclusion that our modern lifestyle is a risk factor for immune dysregulation. Potential culprits and benefactors are searched among early dietary and microbial exposures, which may act to program later allergic disease. The aim of this thesis was to investigate the role of early maternal and child nutrition in reducing the risk of child allergy. The study population comprised of 256 mother – child pairs from families with a history of allergy participating in a randomized controlled dietary counseling and probiotic intervention (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12) study from early pregnancy onwards. The dietary counseling aimed for a diet complying with dietary recommendations for pregnant and lactating women, with special attention to fat quality. Maternal dietary counseling was reflected in cord blood fatty acids suggesting better essential fatty acid status in infants in the counseling group. Dietary counseling with probiotics or placebo had no effect on child allergy risk, but associations between maternal diet during pregnancy and breastfeeding and child allergic outcomes were found in secondary analyses. During pregnancy, milk intake was related to decreased and cheese intake to increased risk of child atopic eczema. During breastfeeding, intake of vitamin C was related to increased risk of asthma and intake of egg was related to decreased risk of atopic eczema. The timing of introduction of complementary foods to infant’s diet was not associated with risk of atopic eczema, when adjusted with parental opinion of child allergic symptoms (i.e., potential reverse causality). In conclusion, the results demonstrate that infant fatty acid supply can be modified via maternal dietary changes. In addition, interesting associations of maternal diet with child allergy risk were discovered. However, no difference in the incidence of allergic diseases with dietary counseling was observed. This suggests that more potent dietary interventions might be necessitated to induce clinical risk reduction of allergy. Highrisk families can safely adhere to dietary recommendations for pregnant and lactating women, and the results support the current conception that no additional benefit is gained with delaying introduction of complementary feeding.

Gene Regulation in The Human Immune System. Gene Expression Signatures of Th2 Cell Differentiation, Type 1 Diabetes, and Intrauterine Immune Adaptation

The human immune system is constantly interacting with the surrounding stimuli and microorganisms. However, when directed against self or harmless antigens, these vital defense mechanisms can cause great damage. In addition, the understanding the underlying mechanism of several human diseases caused by aberrant immune cell functions, for instance type 1 diabetes and allergies, remains far from being complete. In this Ph.D. study these questions were addressed using genome-wide transcriptomic analyses. Asthma and allergies are characterized by a hyperactive response of the T helper 2 (Th2) immune cells. In this study, the target genes of the STAT6 transcription factor in naïve human T cells were identified with RNAi for the first time. STAT6 was shown to act as a central activator of the genes expression upon IL-4 signaling, with both direct and indirect effects on Th2 cell transcriptome. The core transcription factor network induced by IL-4 was identified from a kinetic analysis of the transcriptome. Type 1 diabetes is an autoimmune disease influenced by both the genetic susceptibility of an individual and the disease-triggering environmental factors. To improve understanding of the autoimmune processes driving pathogenesis in the prediabetic phase in humans, a unique series of prospective whole-blood RNA samples collected from HLA-susceptible children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study was studied. Changes in different timewindows of the pathogenesis process were identified, and especially the type 1 interferon response was activated early and throughout the preclinical T1D. The hygiene hypothesis states that allergic diseases, and lately also autoimmune diseases, could be prevented by infections and other microbial contacts acquired in early childhood, or even prenatally. To study the effects of the standard of hygiene on the development of neonatal immune system, cord blood samples from children born in Finland (high standard of living), Estonia (rapid economic growth) and Russian Karelia (low standard of living) were compared. Children born in Russian Karelia deviated from Finnish and Estonian children in many aspects of the neonatal immune system, which was developmentally more mature in Karelia, resembling that of older infants. The results of this thesis offer significant new information on the regulatory networks associated with immune-mediated diseases in human. The results will facilitate understanding and further research on the role of the identified target genes and mechanisms driving the allergic inflammation and type 1 diabetes, hopefully leading to a new era of drug development.

Expression of Fos protein in the rat central nervous system in response to noxious stimulation: effects of chronic inflammation of the superior cervical ganglion

The aim of this study was to investigate the possible interactions between the nociceptive system, the sympathetic system and the inflammatory process. Thus, the superior cervical ganglion of rats was submitted to chronic inflammation and Fos expression was used as a marker for neuronal activity throughout central neurons following painful peripheral stimulation. The painful stimulus consisted of subcutaneously injected formalin applied to the supra-ocular region. Fos-positive neurons were identified by conventional immunohistochemical techniques, and analyzed from the obex through the cervical levels of the spinal cord. In the caudal sub-nucleus of the spinal trigeminal nuclear complex, the number of Fos-positive neurons was much higher in rats with inflammation of the superior cervical ganglion than in control rats, either sham-operated or with saline applied to the ganglion. There was a highly significant difference in the density of Fos-positive neurons between the inflamed and control groups. No significant difference was found between control groups. These results suggest that the inflammation of the superior cervical ganglion generated an increased responsiveness to painful stimuli, which may have been due to a diminished sympathetic influence upon the sensory peripheral innervation.

Modulation of fibronectin expression in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis

Fibronectin (FN), a large family of plasma and extracellular matrix (ECM) glycoproteins, plays an important role in leukocyte migration. In normal central nervous system (CNS), a fine and delicate mesh of FN is virtually restricted to the basal membrane of cerebral blood vessels and to the glial limitans externa. Experimental autoimmune encephalomyelitis (EAE), an inflammatory CNS demyelinating disease, was induced in Lewis rats with a spinal cord homogenate. During the preclinical phase and the onset of the disease, marked immunolabelling was observed on the endothelial luminal surface and basal lamina of spinal cord and brainstem microvasculature. In the paralytic phase, a discrete labelling was evident in blood vessels of spinal cord and brainstem associated or not with an inflammatory infiltrate. Conversely, intense immunolabelling was present in cerebral and cerebellar blood vessels, which were still free from inflammatory cuffs. Shortly after clinical recovery minimal labelling was observed in a few blood vessels. Brainstem and spinal cord returned to normal, but numerous inflammatory foci and demyelination were still evident near the ventricle walls, in the cerebral cortex and in the cerebellum. Intense expression of FN in brain vessels ascending from the spinal cord towards the encephalon preceded the appearance of inflammatory cells but faded away after the establishment of the inflammatory cuff. These results indicate an important role for FN in the pathogenesis of CNS inflammatory demyelinating events occurring during EAE.

An update on the physiology of two- and three-toed sloths

Physiological and pharmacological research undertaken on sloths during the past 30 years is comprehensively reviewed. This includes the numerous studies carried out upon the respiratory and cardiovascular systems, anesthesia, blood chemistry, neuromuscular responses, the brain and spinal cord, vision, sleeping and waking, water balance and kidney function and reproduction. Similarities and differences between the physiology of sloths and that of other mammals are discussed in detail.

Biology and clinical utilization of mesenchymal progenitor cells

Within the complex cellular arrangement found in the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal progenitor cells (MPC). These cells can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into at least seven types of cells: osteocytes, chondrocytes, adipocytes, tenocytes, myotubes, astrocytes and hematopoietic-supporting stroma. This broad multipotentiality, the feasibility to obtain MPC from bone marrow, cord and peripheral blood and their transplantability support the impact that the use of MPC will have in clinical settings. However, a number of fundamental questions about the cellular and molecular biology of MPC still need to be resolved before these cells can be used for safe and effective cell and gene therapies intended to replace, repair or enhance the physiological function of the mesenchymal and/or hematopoietic systems.

Catecholamine response to exercise in individuals with different levels of paraplegia

The purpose of this study was to investigate the effect of the level of injury on the serum level of norepinephrine (Nor) and epinephrine (Epi) at rest and after maximal exercise in individuals with paraplegia. Twenty-six male spinal cord-injured subjects with complete paraplegia for at least 9 months were divided into two groups of 13 subjects each according to the level of injury, i.e., T1-T6 and T7-T12. Serum Nor and Epi concentrations were measured by HPLC-ECD, at rest (PRE) and immediately after a maximal ergospirometric test (POST). Statistical analysis was performed using parametric and non-parametric tests. Maximal heart rate, peak oxygen uptake, and PRE and POST Nor were lower in the T1-T6 than in the T7-T12 group (166 ± 28 vs 188 ± 10 bpm; 18.0 ± 6.0 vs 25.8 ± 4.1 ml kg-1 min-1; 0.54 ± 0.26 vs 0.99 ± 0.47 nM; 1.48 ± 1.65 vs 3.07 ± 1.44 nM). Both groups presented a significant increase in Nor level after exercise, while only the T7-T12 group showed a significant increase in Epi after exercise (T1-T6: 0.98 ± 0.72 vs 1.11 ± 1.19 nM; T7-T12: 1.24 ± 1.02 vs 1.89 ± 1.57 nM). These data show that individuals with paraplegia above T6 have an attentuated catecholamine release at rest and response to exercise as compared to subjects with injuries below T6, which might prevent a better exercise performance in the former group.

Chronic intrathecal cannulation enhances nociceptive responses in rats

The influence of a chronically implanted spinal cannula on the nociceptive response induced by mechanical, chemical or thermal stimuli was evaluated. The hyperalgesia in response to mechanical stimulation induced by carrageenin or prostaglandin E2 (PGE2) was significantly increased in cannulated (Cn) rats, compared with naive (Nv) or sham-operated (Sh) rats. Only Cn animals presented an enhanced nociceptive response in the first phase of the formalin test when low doses were used (0.3 and 1%). The withdrawal latency to thermal stimulation of a paw inflamed by carrageenin was significantly reduced in Cn rats but not in Nv or Sh rats. In contrast to Nv and Sh rats, injection in Cn animals of a standard non-steroid anti-inflammatory drug, indomethacin, either intraperitoneally or into the spinal cord via an implanted cannula or by direct puncture of the intrathecal space significantly blocked the intensity of the hyperalgesia induced by PGE2. Cannulated animals treated with indomethacin also showed a significant inhibition of second phase formalin-induced paw flinches. Histopathological analysis of the spinal cord showed an increased frequency of mononuclear inflammatory cells in the Cn groups. Thus, the presence of a chronically implanted cannula seems to cause nociceptive spinal sensitization to mechanical, chemical and thermal stimulation, which can be blocked by indomethacin, thus suggesting that it may result from the spinal release of prostaglandins due to an ongoing mild inflammation.

The pathogenesis of tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy

Tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy (TSP/HAM) is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis with sphincter disturbances, no/mild sensory loss, the absence of spinal cord compression and seropositivity for HTLV-I antibodies. The pathogenesis of this entity is not completely known and involves a multivariable phenomenon of immune system activation against the presence of HTLV-I antigens, leading to an inflammatory process and demyelination, mainly in the thoracic spinal cord. The current hypothesis about the pathogenesis of TSP/HAM is: 1) presence of HTLV-I antigens in the lumbar spinal cord, noted by an increased DNA HTLV-I load; 2) CTL either with their lytic functions or release/production of soluble factors, such as CC-chemokines, cytokines, and adhesion molecules; 3) the presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord; 4) the presence of B cells with neutralizing antibody production, or complement activation by an immune complex phenomenon, and 5) lower IL-2 and IFN-gamma production and increased IL-10, indicating drive to a cytokine type 2 pattern in the TSP/HAM subjects and the existence of a genetic background such as some HLA haplotypes. All of these factors should be implicated in TSP/HAM and further studies are necessary to investigate their role in the development of TSP/HAM.

The extracellular matrix in multiple sclerosis: an update

Extracellular matrix (ECM) molecules play important roles in the pathobiology of the major human central nervous system (CNS) inflammatory/demyelinating disease multiple sclerosis (MS). This mini-review highlights some recent work on CNS endothelial cell interactions with vascular basement membrane ECM as part of the cellular immune response, and roles for white matter ECM molecules in demyelination and remyelination in MS lesions. Recent basic and clinical investigations of MS emphasize axonal injury, not only in chronic MS plaques, but also in acute lesions; progressive axonal degeneration in normal-appearing white matter also may contribute to brain and spinal cord atrophy in MS patients. Remodeling of the interstitial white matter ECM molecules that affect axon regeneration, however, is incompletely characterized. Our ongoing immunohistochemical studies demonstrate enhanced ECM versican, a neurite and axon growth-inhibiting white matter ECM proteoglycan, and dermatan sulfate proteoglycans at the edges of inflammatory MS lesions. This suggests that enhanced proteoglycan deposition in the ECM and axonal growth inhibition may occur early and are involved in expansion of active lesions. Decreased ECM proteoglycans and their phagocytosis by macrophages along with myelin in plaque centers imply that there is "injury" to the ECM itself. These results indicate that white matter ECM proteoglycan alterations are integral to MS pathology at all disease stages and that they contribute to a CNS ECM that is inhospitable to axon regrowth/regeneration.

Axonal pathfinding mechanisms at the cortical midline and in the development of the corpus callosum

The corpus callosum is a large fiber tract that connects neurons in the right and left cerebral hemispheres. Agenesis of the corpus callosum (ACC) is associated with a large number of human syndromes but little is known about why ACC occurs. In most cases of ACC, callosal axons are able to grow toward the midline but are unable to cross it, continuing to grow into large swirls of axons known as Probst bundles. This phenotype suggests that in some cases ACC may be due to defects in axonal guidance at the midline. General guidance mechanisms that influence the development of axons include chemoattraction and chemorepulsion, presented by either membrane-bound or diffusible molecules. These molecules are not only expressed by the final target but by intermediate targets along the pathway, and by pioneering axons that act as guides for later arriving axons. Midline glial populations are important intermediate targets for commissural axons in the spinal cord and brain, including the corpus callosum. The role of midline glial populations and pioneering axons in the formation of the corpus callosum are discussed. Finally the differential guidance of the ipsilaterally projecting perforating pathway and the contralaterally projecting corpus callosum is addressed. Development of the corpus callosum involves the coordination of a number of different guidance mechanisms and the probable involvement of a large number of molecules.

JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.

Isolation and culture of umbilical vein mesenchymal stem cells

Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

Effect of strenuous maternal exercise before and during pregnancy on rat progeny renal function

The effects of strenuous exercise before and during pregnancy on the renal function and morphological alterations of the progeny were determined in a study on female Wistar rats. This research was done based on a previous study carried out in our laboratory, which showed morphological alterations in rats submitted to this kind of exercise. As the form is related to the function, the physiological relevance of submitting a pregnant female to a high-intensity exercise training regimen could be explained by the fact that morphological alterations can influence kidney function. The animals were assigned to one of two groups: control animals that did not exercise during pregnancy and trained animals that swam for 120 min 5 days a week for 8 weeks before pregnancy and daily for 60 min over a period of 8 weeks starting on the second day of pregnancy. Seven rats of each group were analyzed for morphological alterations and for renal function. The progeny of the rats used for morphological evaluation were born by cesarean section and the progeny of the animals used to evaluate renal function were born normally. The progeny were two months old when renal function was evaluated. Fertility and morbidity were the same for both groups. Strenuous maternal exercise had no significant influence on glomerular filtration rate (GFR) but renal plasma flow was lower in the progeny of the trained group (mean ± SD, 16.65 ± 3.77 ml min-1 kg-1) compared to the progeny of the control group (33.42 ± 2.56 ml min-1 kg-1). Antidiuretic and antinatriuretic effects on the progeny of the trained group were observed, since urine flow as percentage of GFR and the fraction of urinary sodium excretion were lower in this group (1.38 ± 0.10 and 0.60 ± 0.04%, respectively) compared to the progeny of the control group (2.36 ± 0.11 and 1.55 ± 0.20%, respectively). Moreover, in this exercise program, fetuses from trained animals were small-sized (2.45 ± 0.19 vs 4.66 ± 2.45 g for control animals) and showed lower differentiation compared to fetuses from the control group. These effects were probably caused by caloric restriction, hypoxia and reduction of umbilical cord length.