Regulatory T cells (Tregs), interleukin 17-producing T helper cells (Th17 cells), and other immune cells play important roles in the maintenance of pregnancy, and their function is impacted by HIV infection. I hypothesized that FIV-infection may likewise alter placental T cell gene expression causing aberrant immune function and compromised pregnancy. The purpose of this project was to evaluate the expression placental immunomodulators related to Treg and Th17 cell function in the FIV-infected cat model. Gene expression was quantified in placenta and serum using quantitative PCR and ELISA, respectively. Abnormal expression of cytokines was frequently associated with infection and fetal nonviability, resulting in discordant relationships between cytokine pairs and the nuclear transcriptional regulators FoxP3 and RORã. The expression of IL-6 in the periphery mirrored that of the placenta, indicating a potential serological means to predict pregnancy outcome. In conclusion, aberrant placental immunomodulation accompanied failed pregnancy in the FIV-infected cat model.

Endometriosis is a steroid-dependent common gynecological condition characterized by the growth of endometrial epithelial and stromal cells outside of its cavity. Estrogen dependence, progesterone resistance, in situ estrogen production, increased inflammation, resistance to apoptosis, active cell growth and proliferation, and overall disease’s heterogeneity makes it challenging to treat patients without compromising fertility. BDNF and its high affinity receptor TrkB has been shown to be dysregulated in women with endometriosis suggesting their role in disease progression. Immunocompromised mice (Rag2γc) (n=25) underwent an implantation surgery during which a cell suspension consisting of human endometrioma cells was injected into the animals’ peritoneal cavity. Upon lesion stablishment all animals were divided into five groups and treated with either high (7.5mg/kg/day), medium (5mg/kg/day) or low (2.5mg/kg/day) dose of TrkB inhibitor cyclotraxin-b, negative control (saline) or 0.04mg/kg/day letrozole. After four weeks of treatment all animals were sacrificed, all major organs were collected and assessed with routine histology for potential adverse effects of the treatment, all endometriotic implants were analyzed with routine histology and IHC for a panel of markers: BDNF, TrkB, anti-human mitochondrial protein, CD31, VEGFa, VEGFR1, VEGFR2. Treatment did not cause any significant side effects. There was a dose-dependent trend in reduction of endometriotic implants’ volume and number. IHC confirmed expression of the angiogenic markers within endometriotic implants. A larger study may be required to replicate results and advance the search for novel non-hormonal endometriosis treatment. / Thesis / Master of Science in Medical Sciences (MSMS) / Endometriosis is a chronic pain condition affecting more than 176 million women worldwide. Despite its commonality and severity its gold standard diagnosis is performed through invasive laparoscopy and there is no cure resulting in low quality of both physical and mental health of the affected individuals. Levels of a neurotrophic factor - BDNF and its high affinity receptor TrkB have been found increased in women with endometriosis and thus, BDNF-TrkB signaling cascade appears to be a promising therapeutic target. This study investigated an effect of TrkB inhibitor – cyclotraxin-b on reduction of endometriotic lesion number and volume within an immunocompromised model. Replication of results and increase of sample size may provide more details about cyclotraxin-b clinical relevance for treatment of endometriosis.

Alcohol-use disorders affect 15 million people nationwide, 4% of which are adolescents (12-17). Clinical data indicate that adolescents who binge drink greatly increase their likelihood of developing an alcohol-use disorder later in life. Moreover, research indicates that binge-drinking during adolescence produces long-lasting alterations in brain circuitry that underlie the processing of rewarding stimuli. The current study sought to determine the effect of adolescent exposure to chronic intermittent ethanol (AIE) on the consumption of, and motivation to obtain, sucrose solution in adulthood. Alcohol naïve, male Wistar rats arrived at the laboratory on post-natal day (PND) 25 and were randomly divided into two exposure groups (AIE and Air). Animals were provided 3 days to allow for acclimation to the animal colony, prior to the start or experimental procedure (PND 28). The AIE procedure involves inducing alcohol dependence by placing rats, in their home cage, into an alcohol vapor chamber for 14 consecutive days, with each exposure day consisting of 12 hrs of exposure in the chambers (8 am to 8 pm) and 12 hrs out of the chambers. The control rats (Air) are treated the same as AIE rats but without exposure to ethanol vapors. Following the AIE paradigm, all rats remained in their home-cage until adulthood (>PND 70) at which time they started operant training/testing in standard operant chambers equipped with two sipper tubes connected via tubing to liquid delivery solenoids. When the response requirement (# of licks) was met, animals received a delivery of 0.1 ml of 5% sucrose solution. All animals were instrumented to the operant procedure on a fixed-ratio (FR) 2 schedule which increased to an FR4 then FR8. Finally, all rats underwent a progressive ratio test in which response requirement increased exponentially for each liquid delivery. On lower schedules (FR2 and FR4) animals did not exhibit a significant difference in licks or reinforcers earned. However, for higher schedules (FR8 and PR) animals in the Air group exhibited a significantly higher level of behavior (licks) and received a significantly greater number of reinforcers than the AIE group. Overall, the data suggest that exposure to AIE, which approximates binge-like EtOH intake and dependence, differentially affects consumatory and appetitive behavior in response to a non-drug reward in adulthood.

Autism is a neurodevelopmental disorder characterized by impairments in social communication and interaction and by repetitive patterns of behaviour, interests and activities. It is perhaps the most common and handicapping neurological disorder of childhood and as such represents a significant public health problem and a huge burden for education and social service systems. Currently there is no diagnostic test or cure available for autism and the molecular mechanisms underlying autistic behaviour remain to be elucidated. Mutations in genes linked to autism adversely affect molecules involved in synapse development and plasticity including brain-derived neurotrophic factor receptor (TrkB) and its downstream effector mammalian target of rapamycin (mTOR), which is increased in several forms of syndromic autism. Here, we investigated whether TrkB, mTOR and their signaling pathways are disrupted in postmortem brain tissue from subjects with idiopathic autism, that is, cases of autism without a known genetic cause and thought to be of environmental/epigenetic origin. We next further examined the contribution of defective TrkB signaling to autistic behaviour in mice exposed to the histone deacetylase inhibitor valproic acid (VPA), a well-established model of environmental/epigenetic origin of autism. We found that TrkB signaling pathways were reduced in idiopathic autism and that these disruptions were associated with decreased excitatory postsynaptic marker PSD-95, suggesting fewer excitatory synapses. Moreover, we showed that similar molecular deficits were present in VPA-exposed mice that lacked sociability and displayed increased repetitive, stereotyped behaviour. We also determined that behavioural deficits in these mice were rescued by administration of the partial TrkB agonist LM22A-4 but not by treatment with the active tripeptide fragment of the insulin-like growth factor-1, (1-3)IGF-1. Lastly, reduced TrkB signaling in VPA-exposed mice was normalized by LM22A-4 administration combined with behavioural enrichment. The present work provides a better understanding of the molecular mechanisms that contribute to autistic behaviour and implicates TrkB signaling in autism pathogenesis. Furthermore, these data demonstrate that molecular changes observed in brains of patients with idiopathic autism differ from syndromic forms and highlight that both too much and too little signaling can be equally disruptive. The present work also shows that maternal challenge with VPA resulted in social deficits, increased repetitive, restrictive behaviour and reduced TrkB signaling in mice, pointing to epigenetic modifications as a potential underlying mechanism of molecular and behavioural disruptions in autism. Lastly, these findings suggest that pharmacological activation of TrkB using compounds such as the partial TrkB agonist LM22A-4 might play a role in treating sociability and repetitive, perseverative behaviour in autism. / Thesis / Doctor of Philosophy (PhD)

Indiana University-Purdue University Indianapolis (IUPUI) / Methamphetamine (METH) is a psychostimulant with high abuse potential. Currently there are no pharmacological treatments specific for relapse to METH use disorder. Chronic METH abuse has been associated with changes to the dopamine and glutamate neurotransmitter systems, as well as inflammation. Phosphodiesterase-4 inhibitors are known to affect cAMP involved in dopaminergic and glutamatergic neurotransmission, as well as having anti-inflammatory action. In pre-clinical models, phosphodiesterase inhibitors can reduce behaviors associated with the self-administration of drugs of abuse if given directly before tests of relapse-like behavior. However, they have not been examined in the more clinically relevant context as a treatment for use during abstinence from drugs of abuse. To address this gap, a METH self-administration model in the rat was used in which roflumilast, a phosphodiesterase 4 inhibitor, was administered during the abstinence period before a relapse test. The overarching hypothesis was that roflumilast inhibited inflammation associated with METH self-administration abstinence to reduce subsequent relapse-like behaviors. A detailed behavioral analysis showed that the chronic treatment with roflumilast during 7 days of forced abstinence reduced relapse-like behavior to METH seeking and METH taking. Roflumilast treatment during 7 days of forced abstinence did not affect subsequent sucrose seeking and sucrose taking behaviors. Biochemical analyses of proteins related to dopamine and glutamate neurotransmission did not reveal changes in these neurotransmitter systems, nor was there evidence of overt inflammation. These data suggest that roflumilast may be a treatment for METH use disorder that is effective when taken during abstinence, but further studies related to the mechanism of action of roflumilast are needed.

Traditional animal models of severe asthma do not recapitulate defining asthma characteristics, including persistent airway hyper-responsiveness, and chronic neutrophilic inflammation. This is problematic because moderating airway hyper-responsiveness decreases asthma frequency and severity, making it a paramount pharmacological goal in asthma research. Employing a spontaneous equine asthma model (equine pasture asthma, EPA), we first confirmed reversible airway obstruction in eight diseased horses during asthma exacerbations in response to ß2renergic agonist stimulation. Next, non-specific airway hyper-responsiveness was confirmed using methacholine bronchoprovocation to identify the provocative concentration causing a 40% increase in baseline lung resistance (PC40RL)- a threshold similarly employed in evaluating human asthmatics unable to mount forced expiration. The PC40RL of ten EPA horses was consistently <1mg/ml of methacholine, which is a cutoff that has been used to diagnose severe human asthma. Like non-asthmatic humans, ten control horses did not respond to methacholine doses up to 16 mg/ml. Finally, persistence of AHR was documented during absence of seasonal aeroallergen triggers in five horses that were evaluated between 3 and 31 months following the initial methacholine bronchoprovocation. This unique ability of EPA horses to model AHR attributes that are not addressed by other animal models points to the suitability of EPA horses to decipher the mechanistic basis of airway hyper-responsiveness. Building on knowledge that 2renergic receptor (AR) signaling is required to develop the asthma phenotype in a murine model, differentially expressed genes from serial lung biopsies of two EPA affected and two controls were filtered to identify genes that interact with the 2-AR. Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 4 (HCN4) was prioritized because of its interactions with the 2-AR. Relative to control horses, HCN4 was constitutively expressed in airway smooth muscle of EPA horses during remission and increased during seasonal disease exacerbation. Agonism of airway contraction by HCN4 was proven using the specific HCN4 antagonist, ivabradine, which caused dose dependent decreases in carbachol induced contractile responses in both EPA and control bronchi in vitro. These findings highlight utility of EPA as a model of severe asthma and HCN4 as a mediator of airway contraction that warrants further investigation in severe human asthma.

No description available.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Juergen A. Richt / Rift Valley fever virus (RVFV) is a vector-borne zoonotic pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that causes severe disease in ruminants and humans. RVFV is a significant threat to US livestock and public health due to a lack of licensed, efficacious vaccines and its ability to become established in non-endemic areas. Subunit vaccine candidates based on RVFV N- and C-terminal glycoproteins (Gn and Gc) are a viable option for use in ruminants due to their ease of production, safety, and ability to induce immune responses that offer differentiation between infected and vaccinated animals (DIVA). Importantly, subunit Gn+Gc vaccine candidates have demonstrated efficacy in sheep. However, despite the efficacy of a dual glycoprotein vaccine, no studies have directly compared protective efficacies of the individual glycoproteins. Furthermore, although RVFV demonstrates 2.1% maximum pairwise amino acid strain divergence within Gn/Gc ectodomains, it remains unclear how this may affect cross-protective vaccine efficacy. In this study, we used a BALB/c mouse model to determine the median lethal dose (LD₅₀) of 3 wildtype RVFV strains and used this information to standardize challenge doses in subsequent vaccine efficacy studies using baculovirus-expressed Gn/Gc antigens derived from RVFV strain Zagazig Hostpital 1977 (ZH548). Strains Kenya 2006 (Ken06) and Saudi Arabia 2001 (SA01) demonstrated equally high virulence (LD₅₀= 7.9pfu), while recombinant strain South Africa 1951 (rSA51) was less virulent (LD₅₀=150pfu). Following prime-boost vaccination, 100% (10/10) of the Gn+Gc vaccinated mice survived challenge with x1000 LD₅₀ Ken06 and SA01, while only 50% (5/10) of Gn+Gc vaccinated mice survived challenge with rSA51. Additionally, 90% (9/10) of Gn-only vaccinated and 40% (4/10) of Gc-only vaccinated mice survived challenge with Ken06. These data suggest that a Gn-only subunit vaccine is an efficacious alternative to dual glycoprotein vaccine candidates and that our ZH548-derived Gn+Gc vaccine has the potential to cross-protect against divergent RVFV strains. Results from this study can be used to optimize current vaccine formulations and inform future vaccine efficacy and licensure studies in ruminants.

Sepsis and septic shock are conditions, with severe outcome or in many cases death. Sepsis is a systemic inflammatory response trigger by bacteraemia but systemic inflammatory response can also be triggered by major trauma, major surgery, pancreatitis, severe burns etc. The systemic inflammatory reaction initiating the evolvement of septic organ dysfunction can be modelled using endotoxin, a Gram-negative bacterial lipopolysaccharide. This thesis used a porcine experimental sepsis model to examine timing of the inflammatory response due to endotoxin infusion (Paper I) and the influence of steroid treatment on the inflammatory response in endotoxemic pigs (Paper II). Timing of steroid treatment and the role of neutrophil granulocyte activation was evaluated with pig specific NGAL assessing neutrophil activation (Paper III). A clinical observational study was performed with the aim to differentiate between sepsis and other inflammatory conditions (e.g. trauma due to major surgery) evaluated by calprotectin as a marker of neutrophil activation (Paper IV). There was a dose-dependency in endotoxin tolerance which was measured with TNF-a. Pre-exposure to endotoxin did not reduce the pulmonary response to endotoxemic challenge. In fact, both PaO2 / FiO2 and static pulmonary compliance were reduced in this group when pre-treated with endotoxin at low dose. Endotoxemic animals treated with hydrocortisone were more stable in circulatory variables than those without such treatment. This was not explained by an ability of steroids to modulate the production of NO (Nitric oxide), which has been suggested to be a mechanism of steroids in this aspect. Pre-treatment with hydrocortisone attenuated the neutrophil granulocyte response and consequently diminished the release of NGAL in plasma. Circulatory derangement was associated with high plasma NGAL levels. Urine NGAL levels did not differ among the four groups. Plasma calprotectin levels on ICU admission is a sensitive marker of systemic inflammation and are markedly increased in patients with sepsis and patients with systemic inflammatory response. Plasma Calprotectin performed better than any of the other inflammatory variables in predicting mortality at 30 days, except from the composite mortality prediction score, SAPS 3.

Com o aumento da expectativa de vida, surgem indagações em relação à qualidade desses anos que serão vividos a mais. Durante o processo de envelhecimento, ocorrem alterações naturais que podem resultar em disfunções fisiológicas. Da mesma forma, o cérebro se altera e se adapta ao longo desse processo, podendo ocorrer danos à funcionalidade desse órgão. Entre os agravos mais comuns ao sistema nervoso central está a doença de Alzheimer (DA), que compromete a função cognitiva, aprendizado e memória. A abordagem terapêutica utilizada no tratamento da DA até o momento não é capaz de reverter as perdas funcionais no cérebro, além de causar alguns efeitos colaterais indesejados. Diante disso, uma alternativa ou adjuvante no tratamento da perda de memória na DA faz-se necessário. Nosso grupo de pesquisa demonstrou o efeito do carbonato de lítio em microdose na estabilização da memória em estudos prévios. No entanto, ainda existe muito a ser avaliado a respeito de aspectos moleculares relacionados ao tratamento com esse íon. O presente estudo buscou avaliar os aspectos comportamentais do efeito da microdose desse íon na memória e ansiedade de camundongos com envelhecimento acelerado (SAMP-8). Foram utilizados camundongos fêmeas da linhagem SAMP-8 (\"senescence-accelerated mouse prone\") que possuem envelhecimento acelerado. Os animais SAMP-8 foram divididos em 2 grupos: um grupo que recebeu somente água filtrada por via oral durante todo o processo de envelhecimento (n = 12) e um grupo que recebeu carbonato de lítio na dose de 0,25 mg/kg por via oral (n = 12). Como controle dos animais SAMP-8, foram utilizados os animais resistentes ao envelhecimento acelerado SAMR-1 (\"senescence-accelerated mice-resistant\", n = 10). Foram aplicados testes de comportamento para avaliar a memória espacial e aversiva, bem como o comportamento de ansiedade desses animais. Foi verificado que tratamento com lítio em microdose promoveu a manutenção da memória espacial e aversiva ao longo do envelhecimento. Os testes de avaliação de ansiedade mostraram um efeito positivo da microdose do lítio na redução do estado ansioso / Due to an increase in life expectancy, questions arise about the quality of those years people will live more. During the aging process, natural changes occur that can result in physiological dysfunctions. The brain can change and adapt during this process and that can compromise its effectiveness. Among the most common dysfunction to the central nervous system is Alzheimer\'s disease (AD), which compromises cognitive function, learning and memory. The current therapeutic approach used in the treatment of AD is not able to reverse functional losses in the brain. Besides, it can cause some unwanted side effects. Therefore, an alternative or adjuvant in the treatment of memory loss in AD is necessary. Our research group demonstrated the effect of microdose lithium carbonate in stabilizing memory in previous studies. However, there is still much to be discover regarding molecular aspects related to treatment with this ion. The present study aimed to evaluate the behavioral aspects of the microdose lithium effect on memory and anxiety in Senescence accelerated mouse prone (SAMP-8). For that purpose, we used female SAMP-8 mice (senescence-accelerated mouse prone). SAMP-8 mice were divided into 2 groups: one group that received only oral filtered water during the whole aging process (n = 12) and one group that received 0.25 mg / kg lithium carbonate (n = 12). SAMR-1 (senescence-accelerated mice-resistant, n = 10) were used as a control. Behavior tests were applied to evaluate the aversive and spatial memory, as well as animals\' anxiety behavior. Treatment with microdose lithium promoted the maintenance of the spatial and aversive memory in SAMP-8 MICE along the aging process. Anxiety testing showed a positive effect of microdose lithium on reducing anxiety in SAMP-8