Alzheimer's disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (Aß) peptide in the brain and in the cerebral vessels forming cerebral amyloid angiopathy (CAA). As previously reported, an active immunization strategy of mice with Aß1-42 peptide results in decreased Th1 and increased Th2 cytokine responses as well as an effectively clearance of CNS Aß. This approach has also yielded favorable results for many patients, unfortunately, a small percentage of these study participants developed severe aseptic meningoencephalitis likely secondary to CNS invasion of activated T-cells. We have previously demonstrated that disruption of CD40-40L pathway reduces Aß plaque load, promotes Th2 response, and rescues from cognitive impairments. However, direct blockage of the CD40 pathway by passive vaccination with anti-CD40L antibody leads to immunosupression. Therefore, in its current form this therapeutic strategy poses an unacceptable risk to the recipient of treatment, aged individual. For those reasons, the identification and characterization of alternative modulators/inhibitors of CD40 signaling may be necessary for the development of safe and effective AD immunotherapy. This proposal introduces novel immunomodulatory therapies that are based on previous vaccination strategies or cell based therapies across medial field. We showed that transcutaneous vaccination can both be efficacious and safe, thus clearly demonstrating that the right combination of the antigens, adjuvants, and the routes of administration are crucial for the right vaccine. Furthermore, we demonstrated that the effects of current Aß vaccine strategies could be enhanced by a simultaneous blockade of CD40-40L signaling. As an alternative approach, we explored the possibility of cell-based therapies and showed that human umbilical cord blood cells, which are currently used as a treatment for systemic lupus erythematosus and leukemia, and currently investigated against stroke, amyotropic lateral sclerosis, age-related macular degeneration, multiple sclerosis, and Parkinson's disease, and showed that not just they improved the AD like pathology in transgenic animals but altered both the brain and peripheral inflammation levels. Lastly, we discussed the involvement of microglia, one of the key players in both AD pathogenesis and Aß clearance and suggesed that microglia in actuality has a continuum of physiological activation states that contribute to proinflammation, antiinflammation, and phagocytosis.

Dementia is the top global public health threat of the twenty first century. Within the dementia spectrum, Alzheimer’s disease (AD) is the most common type of dementia that occurs with aging and accounts for about 60% - 80% of diagnosed cases. But currently available discoveries failed to develop disease-modifying therapies for all patients living with AD. Recent discoveries can only partially slow down cognitive decline in a small subset of patients with limited effectiveness. The heterogeneity and complexity of the pathophysiology of AD indicate that a single drug approach may not be sufficient to prevent disease onset and progression. Human umbilical cord blood cells (HUCBC) and lithium treatment have shown promise against numerous neurological conditions, including AD. Yet, they also show significant unwanted, adverse effects. To address this barrier to yield successful treatments, we employed two key modifications to these two treatment strategy. We used human umbilical cord blood derived serum (HUCBS, also labeled as CBS) rather than HUCBC. We also utilized ionic cocrystal of lithium salicylate l-proline (LISPRO, also labeled as LP) instead of usual lithium salt. Both HUCBS and LISPRO have been shown to have strong neuroprotective, anti-inflammatory properties in separate studies conducted in transgenic AD mouse models. The studies detailed herein independently investigated the effectiveness of biological (HUCBS) and pharmacological (LISPRO) approaches in modulating the pathology and cognitive impairments in AD mouse models (e.g., 5XFAD, 3XTg-AD, APPswe/PS1dE9, and Tg2576). While administration of HUCBC stimulate anti-inflammatory pathways shown in previous studies, we found that HUCBS markedly promoted neurotrophic soluble amyloid precursor protein alpha (sAPPα) through non-amyloidogenic amyloid precursor protein (APP) processing pathway compared to adult (ABS) and aged blood serum (AgBS) in Chinese hamster ovary cells expressing wild type APP (CHO/APPwt). Using chromatographic fractionation, mass-spectrometry, and targeting complement proteins in cord blood serum fraction (αCBSF), we discovered the source of sAPPα in HUCBS as C1 complement protein. Further, intraperitoneal administration of αCBSF via osmotic minipump for 6 weeks showed prevention of cognitive impairment in 5XFAD mice assessed by novel object recognition, and Y-maze test. A series of recent studies have shown that lithium can prevent both AD- and age-associated cognitive decline. But, current United States Food and Drug Administration-approved lithium pharmaceutics (carbonate and citrate forms) have a narrow therapeutic window and unstable pharmaceutics that can cause toxicity without monitoring. Here we investigated the safety, pharmacokinetics, and therapeutic efficacy of LISPRO (LP), lithium salicylate (LS), and lithium carbonate (LC) in cell culture and mouse (B6129SF2/J, Tg2576, and 3xTg-AD) models. Cytokine profiles from the brain, plasma and splenocytes demonstrated that 8-week oral treatment with LISPRO downregulates pro-inflammatory cytokines, upregulates anti-inflammatory cytokines and suppress renal cyclooxygenase 2 (COX2) expression in Tg2576 mice. Pharmacokinetic studies indicated that LISPRO provides significantly higher brain and more steady plasma lithium levels in both B6129SF2/J and transgenic Tg2576 mice compared with lithium carbonate. Oral administration of LISPRO for 28 weeks significantly reduced β-amyloid plaques and tau phosphorylation. In addition, LISPRO significantly elevated pre-synaptic (synaptophysin) and post-synaptic protein (post synaptic density protein 95) expression in brains from transgenic 3XTg-AD mice. Moreover, female APPswe/PS1dE9 mice at 4 months of age were orally treated with LP, LS, or LC for 8- to 9- months at 2.25 mmol lithium/kg/day followed by measuring body weight, internal organs’ growth, and cognitive and non-cognitive function. LISPRO treatment prevented cognitive decline compared with transgenic APPswe/PS1dE9 cohort, as shown by shorter escape latency during training and probe trials in the Morris water maze and longer contextual freezing time during fear conditioning. As expected, LISPRO treatment also reduced depression assayed by tail suspension test and irritability assessed with the touch escape test. But, lithium treatment did not alter anxiety or locomotor activity as assessed by open field, elevated plus maze or accelerated rotarod tests. Taken together, these data indicate that both biological HUCBS and pharmacological LISPRO treatment may prove to be viable effective strategy for ameliorating Alzheimer’s like pathology and cognitive impairment in preclinical models.

Human umbilical cord blood, an alternate source of haematopoietic stem cells (HSC), has been successfully used to reconstitute haematopoiesis in both related and unrelated transplant recipients. However, because CB has fewer total cells (and as a consequence fewer HSC and progenitor cells) CB transplant recipients often experience delayed engraftment as compared with that seen in bone marrow or mobilized peripheral blood transplant recipients. Delayed engraftment exposes patients to an increased risk of infection and bleeding. Cytokine-mediated expansion has been investigated to improve engraftment after CB HSC transplantation as a means to expand the total cell number and both the HSC and progenitors populations. However, its effect on HSC function remains controversial. We hypothesise that if cytokine-mediated expansion promotes divisional recruitment and multilineage differentiation it causes changes in phenotype and cell cycle related gene expression which may be detrimental to the engraftment capacity of haematopoietic cells. Therefore we investigated the relationship between cell division, phenotype and engraftment potential of CB CD34+ cells following cytokine-mediated expansion. High resolution cell division tracking using the fluorescent dye CFSE was used to monitor changes as a consequence of cytokine-mediated expansion in phenotype and function in CB CD34+ cells. Cytokine-mediated expansion caused upregulation of lineage and proliferation markers and adhesion molecules and downregulation of putative stem cell markers with concomitant cell division. However, these changes in phenotype as a consequence of cytokine-mediated expansion may not reflect or be predictive of a functional change in the expanded population. Cytokine-mediated expansion of CB CD34+ also caused changes in cell cycle related gene expression of G1 phase regulators. CB CD34+ cells exhibited expression of all D cyclins, albeit at different levels and p21WAF1 was differentially expressed across CB samples. The effect of cell division on the engraftment potential as a consequence of cytokine-mediated expansion was examined in CB CD34+. Cytokine-mediated expansion of CB CD34+ cells reduced, but did not completely eliminate engraftment potential, as a proportion of the expanded and divided cell populations retained their ability to engraft the NOD-SCID mouse. Overall, this study confirms reports in the literature that cytokine-mediated expansion induces changes in the phenotype of HSC and compromises their in vivo function.

Background: The reasons why some individuals become IgE-sensitised and allergic are largely unknown, though genetic- and early life environmental factors seem to be of importance. Objective: The overall aim of this thesis was to investigate the relationship between IgE-sensitisation and allergic disease, viral infections, genetic markers and early life cytokines. Results: IgE-sensitised children were found to have reduced numbers of IL-12 producing cord blood mononuclear cells (CBMC), whereas children diagnosed with eczema were found to have reduced numbers of IFN-γ producing CBMC. When dividing the children into early onset of IgE-sensitisation and late onset of IgE-sensitisation we found that the children with an early onset had low numbers of PHA-induced IL-4, IL-12 and IFN-γ secreting CBMC. At the age of two there was a general exacerbation of cytokine responses in the IgE-sensitised children, and the results were similar for the children with early onset IgE-sensitisation. Children with a late onset IgE-sensitisation were more similar to the non-sensitised children, but with a specific increase in the response to cat allergen (IL-4 and IFN-γ). The mothers of IgE-sensitised children, were just as their children, found to have an exaggerated cytokine response as compared to mothers of non-sensitised children. Maternal responses correlated well to the responses seen in the child, though the samples were taken two years after delivery. Cytomegalovirus (CMV) infection in early life was associated to reduced numbers of IL-4, and increased numbers of IFN-γ producing cells at the age of two. No association between CMV seropositivity and IgE-sensitisation was seen. Epstein-Barr virus (EBV) infection, on the other hand, was inversely correlated with IgE –sensitisation, whereas no statistically significant association to cytokine production could be seen. We also showed that the IL12B 1188 C-allele was associated to having a positive skin prick test at the age of two. The rare alleles of the three SNPs investigated (IL12B 1188C, IL12RB1132C and IRF1 1688A) were all associated to low IL-12 production at birth. Conclusions: Our results indicate that allergic diseases are complex traits, and that both the genetic and the cytokine background differ between the different allergic diseases. We can also conclude that the time of onset seem to play a role when investigating IgE-sensitisation, and that perhaps early and late onset IgE-sensitisation have partly different causes. CMV and EBV infection early in life are associated to a protective cytokine profile and to protection from IgE-sensitisation, respectively, again indicating the heterogeneity and the complexity of allergic diseases.

Exposure to a strong T-helper 2 (Th2)-like environment during fetal development may promote allergy development. Increased cord blood (CB) levels of the Th2-associated chemokine CCL22 were associated with allergy development during the first 2 y of life. The aim of the present study was to determine whether CB Th1- and Th2-associated chemokine levels are associated with allergy development during the first 6 y of life, allowing assessment of respiratory allergic symptoms usually developing in this period. The CB levels of cytokines, chemokines, and total IgE were determined in 56 children of 20 women with allergic symptoms and 36 women without allergic symptoms. Total IgE and allergen-specific IgE antibody levels were quantified at 6, 12, 24 mo, and 6 y of age. Increased CB CCL22 levels were associated with development of allergic sensitization and asthma and increased CCL17 levels with development of allergic symptoms, including asthma. Sensitized children with allergic symptoms showed higher CB CCL17 and CCL22 levels and higher ratios between these Th2-associated chemokines and the Th1-associated chemokine CXCL10 than nonsensitized children without allergic symptoms. A pronounced Th2 deviation at birth, reflected by increased CB CCL17 and CCL22 levels, and increased CCL22/CXCL10 and CCL17/CXCL10 ratios might promote allergy development later in life.

! BACKGROUND: Exposure to Environmental Tobacco Smoke (ETS) is elevating blood levels of inflammatory mediators and chemoattractants which seem to play an important role in the development of several diseases (e.g. Chronic Obstructive Pulmonary Disease). First evidences showed that men and women might differ in their proneness for these diseases. The aim of this study was to investigate whether there are effects of ETS during pregnancy on inflammatory cytokines in cord blood and in mother’s blood and if there are any differences between male and female newborns. METHODS: Within the LiNA (Lifestyle and environmental factors and their influence on Newborn Allergy Risk) study, whole blood samples of 460 mother-child pairs were analyzed for the concentrations of IL-6, IL-8, IL-10, IL-12, IL-4, IL-5, INF-gamma, TNF-alpha and MCP-1 using cytrometic bead asseys. The association between ETS exposure and cytokines was calculated using the Mann-Whitney-U-test and adjusted with a multiple regression model for parental atopy, parental education status and cat ownership. The exposure assessment is based on questionnaire data on smoking behaviour of the parents and measurement of indoor benzene concentration. RESULTS: Female newborn, being exposed in utero to 10 cigarettes a day or more, had significantly higher blood concentrations of IL-8, IL-6 and MCP-1 whereas there have been no elevations in male newborn being exposed to the same amount of cigarettes. Furthermore a significantly decreased amount of INF-gamma was found in cord blood of male newborns but not in female newborns. General increasing levels of TNF-alpha in cord blood where found for daily smoke exposure without relating it to the exact number of cigarettes. CONCLUSION: The data of this study refer to gender-specific differences in the susceptibility to ETS exposure. The induction of inflammatory signals in cord blood in response to cigarette smoke exposure is stronger in female than in male newborn. / Die vorliegende Arbeit ist Teil einer umweltepidemiologischen Kohortenstudie (LiNA), in der der Einfluss von Umwelt- und Lebensbedingungen auf die Entwicklung von Immunsystem und Allergien bei Neugeborenen unter Einbezug der vorgeburtlichen Zeit untersucht wird. In welchem Maße sich eine Rauchbelastung während der Schwangerschaft auf die Zytokinmuster der Neugeborenen im Nabelschnurblut auswirkt und inwiefern dies mit dem Zytokinmuster der Mutter korreliert, sollte das Ziel dieser Dissertation sein. Dafür wurden Daten von insgesamt 629 Mutter-Kind-Paaren erhoben, Zytokin- und Chemokinbestimmungen, sowie die des Gesamt-IgE aus den Blutproben der 34. SSW und denen der Nabelschnur vorgenommen. Interessanterweise konnten geschlechterspezifische Unterschiede im Zytokinspektrum der Neugeborenen gefunden werden. Bei den weiblichen Neugeborenen zeigte sich eine deutliche Erhöhung proinflammatorischer Marker, wenn deren Mütter dem Rauch von mehr als 10 Zigaretten pro Tag ausgesetzt waren. Dieser Anstieg war weder im Blut der männlichen Neugeborenen noch im Blut der Schwangeren in der 34. SSW zu beobachten. Zusätzlich konnte beobachtet werden, dass auch einzig die männlichen Neugeborenen stark negativ mit ihrer IFN-gamma-Produktion auf die passive Rauchbelastung reagieren. Die mit dieser Arbeit ermittelten Daten, dass das Immunsystem beim Neugeborenen geschlechterspezifisch unterschiedlich auf Tabakrauch zu reagieren scheint, sind erstmals in der Literatur zu finden. Die Erforschung des Immunsystems und dessen Beteiligung an zahlreichen Erkrankungen, besonders den chronisch Inflammatorischen, ist durchaus relevant im medizinischen Alltag. Diese Arbeit trägt einen weiteren Baustein dazu bei und gibt Anstoß für weitere Studien.

Prevalence of childhood asthma in Taiwan has increased 9 times from 1.3% to 10-14% in the past 4 decades. Many studies worldwide have demonstrated that many genes in different chromosomes are implicated in childhood asthma in different ethnic populations. A growing body of evidence suggests that allergic sensitization could occur in perinatal stage and correlate to the development of childhood asthma. Epidemiological studies, however, indicate that prevalence of childhood asthma is much higher in developed countries than that in developing countries; and prevalence of childhood asthma in metropolitan area is higher than that in country sites. This suggests that certain genes can interact with the environmental factors in developed countries to promote the development of childhood atopic disorders. Interests are now increasing on what is (are) the real pathogenic gene-gene interaction(s) for childhood atopic disorders under influence of age, gender and environmental factors? In a large perinatal cohort study with 1,211 pregnant women and their offspring from the obstetrics and pediatrics of Kaohsiung Chang Gung Memorial Hospital, we analyzed 159 allergy candidate genes with 384 single nucleotide polymorphisms and showed that 14 genes over 22 somatic and X chromosomes risk to or protective from cord blood immunoglobulin E (CBIgE) elevation are different from those genes associated with IgE elevation in children under 1.5, 3 and 6 years of age (12, 15 and 12 genes, respectively). CX3CL1, IL13, PDGFRA and FGF1 polymorphisms were associated with elevated IgE at earlier ages (newborn, 1.5 and 3 years); HLA-DPA1, HLA-DQA1, CCR5 and IL5RA polymorphisms were associated with IgE production at 6 years of age. Further analysis by multifactor dimensionality reduction (MDR) developed from data reduction strategy, we found that there are interactions among innate immunity, adaptive immunity, and response and remodeling genes on IgE production begin in prenatal stage. For example, The gene-gene interaction among IL13, rs1800925, CYFIP2, rs767007 and PDE2A, rs755933 was significantly associated with IgE production at 3 years of age. This suggests that different genotypes of genes interact one another on the IgE production contributing to the development of allergic diseases. Since the concentration of IgE is an important indicator of atopic disorders and allergic sensitization, we believe after clarifying the natural course of the genomic profiles on IgE elevation, certain early predictor(s) and preventive regimens for allergic sensitization or atopic disorders may be made possible.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Understanding the molecular networks that maintain articular cartilage and regulate chondrogenic differentiation of mesenchymal stromal cells (MSCs) are important prerequisites for the improvement of cartilage repair strategies. The first study within this thesis demonstrates that equine cord blood-derived MSCs induced towards a chondrogenic phenotype showed significantly increased miR-140 expression from day 0 to day 14, which was accompanied by decreased expression of previously identified miR-140 targets; ADAMTS-5 and CXCL12. The second study shows that in vitro chondrogenesis on fibronectin coated-PTFE inserts results in more homogeneous hyaline-like cartilage with an increased number of differentiated cells compared with pellet cultures. Finally, the expression of miR-140, miR-9, miR-155 and miR-146a was investigated in an in vitro model of osteoarthritis and suggests a possible role for miR-146a. These results suggest that microRNAs may be useful for directing or enhancing eCB-MSC chondrogenic differentiation and for developing novel biomarker panels of in vivo joint health. / Danish Agency for Science, Technology and Innovation; Equine Guelph; Grayson-Jockey Club Research Foundation; BioE.