Severe bacterial sepsis leads to a pro-inflammatory condition that can manifest as septic shock, multiple organ failure, and death. Neutrophils are critical for the rapid elimination of bacteria, however, the role of neutrophil chemoattractant CXCL1, pattern recognition receptors (PRRs)- NLR protein 3 (NLRP3) and alcohol in bacterial clearance during sepsis remains elusive. We demonstrate that CXCL1 plays a pivotal role in mediating host defense to polymicrobial sepsis following cecal ligation and puncture (CLP) in gene-deficient mice. CXCL1 appears to be essential for restricting bacterial outgrowth and preventing multiple organ failure and death in mice. Moreover, CXCL1 is essential for neutrophil migration, expression of pro-inflammatory mediators, Recombinant interleukin 17 (IL-17) rescued impaired host defenses in cxcl1−/− mice. CXCL1 is important for IL-17A production via Th17 differentiation. CXCL1 is essential for reactive oxygen species production and neutrophil extracellular trap (NET) formation. This study reveals a novel role for CXCL1 in neutrophil recruitment via modulating T cell function and neutrophil-related bactericidal functions. These studies suggest that modulation of CXCL1 levels could reduce bacterial burden and excessive inflammatory injury in sepsis. NLRP3-/- mice or mice treated with NLRP3 inhibitor were protected in response to polymicrobial sepsis. NLRP3-/- mice showed reduced bacterial burden and production of proinflammatory cytokines. Intriguingly, neutrophils obtained from NLRP3-/- or NLRP3-inhibited mice display impaired critical functions of neutrophils, including phagocytosis, bacterial killing, NET formation, autophagy, chemotaxis, and cell death. These unique and novel findings position NLRP3 as a critical linker between neutrophil function and bacterial clearance, highlighting NLRP3 as a therapeutic target to control infection in polymicrobial sepsis. Alcoholics are more susceptible to bacterial sepsis and thus have higher mortality rate as compared to non-alcoholics. In this study, acute alcohol intoxication prior to the induction of polymicrobial sepsis show reduced NETosis. Diminished NETosis was consistent with attenuated ROS production and bacterial clearance in alcohol-challenged CLP-induced mice. Our findings demonstrate that alcohol-suppressed NETosis and NET-mediated extracellular killing of bacteria contribute to the pathogenesis of polymicrobial sepsis, and thus, furthers our understanding on alcohol-induced immune defect during bacterial infection.

The outer surface lipoprotein (osp) layer forms an interface between the internal and the external environment of the Lyme disease spirochete, Borrelia burgdorferi. The homeostatic maintenance of the osp layer effectuates adaptation of B. burgdorferi as it gets transmitted from the tick vector to a mammalian host and vice-versa. However, the regulation of the outer surface lipoproteins (osps) is still a conundrum for borrelia scientists. Part of this dissertation inquires about the homeostatic maintenance of the osp layer. We found that the deletion of the dominantly expressed tick phase osp, OspA, induces expression of two other osps. OspD, and BBJ41. Also, increased expression of OspC was seen in borrelia mutants lacking OspA, OspD, and BBJ41. These results suggest constant osp layer maintenance, irrespective of the presence or the absence of the dominant Osps, like OspA and OspC. Furthermore, our conclusive electron microscopic study demonstrates that the overall density of the osp layer remains identical in wild type and mutant B. burgdorferi, lacking either several osps or the dominantly expressed OspA. OspA is abundantly expressed on the borrelial surface as it persists in an unfed tick. A blood meal causes rapid downregulation of OspA as B.burgdorferi prepares to infect the mammalian host. The downregulation of OspA is speculated to be regulated by an unknown repressor protein. The remaining part of this dissertation pertains to the investigation of this unknown repressor protein for ospA. The borrelia oxidative stress regulator protein, BosR, has been attributed with an indirect role in OspA downregulation. However, due to its homolgy with a family of transcriptional repressors, BosR is more likely to cause direct repression of OspA. Therefore, we investigated the direct interaction of BosR and the ospA regulatory region. The DNA binding experiments demonstrated that borrelia oxidative stress regulator, BosR, binds directly to the cisI and cisII regulatory regions of ospA promoter. Thus, conclusively, BosR acts as a repressor protein which causes OspA downregulation in B. burgdorferi.

Dengue virus (DENV) is maintained in a primarily anthroponotic cycle between humans and the mosquito, Aedes aegypti. Investigations into DENV infection of the vertebrate host generally do not account for the contribution of vector saliva, an inherent part of the mosquito-borne viral inoculum. Feeding by mosquitoes on vertebrate hosts is initiated by probing, which results in physical damage to the skin and vasculature, and the simultaneous introduction of DENV and saliva into the skin. Saliva contains many individual proteins with the potential to modulate host hemostasis and immune responses, thereby facilitating blood feeding and virus transmission. As exogenous antigens, both DENV and these salivary proteins encounter the vertebrate host immune system and consequently could have an effect on the immunological environment and response of the bite site during viral establishment, as well as the ensuing viremia. My overarching hypothesis is that mosquito saliva aids in the establishment of DENV infections within the vertebrate, and that distinct immunological alterations involved in this enhancement will be attributable to individual salivary proteins. I therefore conducted investigations into the triad of vector-virus-vertebrate interactions aimed at further characterizing 1) the strain-based impact of DENV infection on salivary protein transcript expression in Ae. aegypti; 2) the probing-based modulation of vertebrate immune responses during DENV infection in the skin of a murine model of transmission; 3) the effect of individual salivary proteins on DENV production in a human hematopoietic cell line; and 4) the influence of the salivary protein aegyptin on DENV infection in the mouse; with emphasis on early, establishment-relevant time points and differences in infection kinetics with the potential to alter transmission success.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous neurotropic alphaherpesvirus transmitted by contact with mucocutaneous surfaces of infected individuals. HSV-1 enters the host by fusion of the viral envelope with the host cell plasma membrane, followed by translocation of the viral capsids to the nucleus where viral DNA is injected into the host cell nucleus to initiate viral replication. To generate infectious virions, newly assembled capsids travel to the cytoplasm and undergo a process called secondary envelopment by budding into cytoplasmic vesicles derived from the trans-Golgi network. Cytoplasmic envelopment is a complex process involving interactions between a multitude of viral membrane and tegument proteins. To investigate the relative importance of a subset of viral membrane and tegument proteins in secondary envelopment, a number of recombinant viruses were constructed in the HSV-1(F) genetic background. A mutant virus unable to express gE, gM and the C-terminus of gD was characterized and compared to additional mutants unable to express both gE and gM or gE and the C-terminus of gD and to mutants lacking expression of just one of these glycoproteins, in addition to mutants lacking expression of both pUL11 and gM, and pUL20 alone. Characterization of all mutant viruses by plaque morphology, viral replication kinetics, electron microscopy and particle-to-PFU ratios revealed a hierarchy of defects in cytoplasmic envelopment and infectious virus production, with deletion of pUL20 having the greatest effect, followed by the deletion of pUL11 and gM. Characterization of additional mutants containing multiple mutations revealed that gE, gM and gD do not function in a redundant manner in cytoplasmic envelopment supporting a preeminent role for the pUL20/gK protein complex in cytoplasmic envelopment and egress. An epitope tag insertion adjacent to the pUL37 Y480 (DC480) exhibited a severe defect in cytoplasmic envelopment similar to gK and pUL20-null viruses. Importantly, this mutant virus was partially complemented when grown on cells expressing pUL20, suggesting an interaction with the pUL20/gK protein complex. This pUL37 interaction with pUL20/gK was verified by co-immunoprecipitation and proximity ligation assays suggesting that it facilitates cytoplasmic virion envelopment.

The immune response to the self-cure phenomenon seen during gastrointestinal nematode (GIN) parasitism of small ruminants was compared between sheep breeds that are resistant or susceptible to Haemonchus contortus infection. Fifty-four Gulf Coast Native (resistant) and Suffolk (susceptible) lambs were allowed to acquire a natural GIN infection on pasture and were then randomly allocated into 4 groups. After being moved to parasite free housing for 2 months, lambs were given a challenge infection of 20,000 H. contortus L3. Fecal egg counts (FEC) were monitored throughout the study and animals were necropsied at 0, 1, 3, and 7 days post infection (DPI). FEC decreased beginning at 3 DPI in both breeds, with Native lambs having a higher percent reduction in FEC at 3 and 7 DPI compared to Suffolk lambs. Both Native and Suffolk lambs were able to expel their existing adult population. However, while Native lambs also successfully cleared the larval challenge, Suffolk lambs did not. The numbers of eosinophils within the abomasal mucosa reflect the magnitude and timing of fecal egg count reductions in both breeds. Additionally, the elevated levels of eosinophils within the abomasal mucosa of Native lambs at 3 DPI are likely to be involved with the clearance of the larval burden via eosinophil mediated larval killing. Suffolk lambs displayed a delayed cellular response that resulted in larvae to entering the mucosa before sufficient eosinophilic response could be established. Elevated mast cells within the abomasal mucosa coincide with the clearance of adult GIN and along with elevated levels of IL-13 seen in both breeds suggest their involvement. The results confirm that self-cure is an immune mediated response that can occur in both resistant and susceptible breeds of sheep while differences in the magnitude and time course of immune responses may prevent susceptible sheep from fully clearing the infection.

Hypoluteoidism in the bitch is described as a reproductive condition in which insufficient levels of endogenous progesterone are present resulting in failure to maintain a functional secretory endometrium. This condition can prevent normal embryo implantation, development, and ultimately end in pregnancy loss. Hypoluteoidism in the bitch is a rising concern in small animal theriogenology and current medical therapies available to veterinarians are limited. The aim of this study was to determine the pharmacokinetics (PK) of intravaginally (Crinone®, Serono Laboratories, Norwell, MA) and orally delivered micronized progesterone (Prometrium®, Solvay Pharmaceuticals, Inc., Marietta, GA) in the bitch. We hypothesized that both vaginal and oral treatments would result in a dose-dependent increase in concentrations of plasma progesterone. We further hypothesized that oral dosing of micronized progesterone would result in greater, sustained plasma progesterone than those recorded in bitches treated with intravaginal (IVa) micronized progesterone gel. Eight adult sexually intact bitches in anestrus were arranged in a 4x4 Latin square cross over experimental design. Each subject rotated through four different progesterone treatment groups with a minimum seven day-wash out period between treatments: 100 mg oral micronized progesterone, 200 mg oral micronized progesterone, 45 mg intravaginal micronized progesterone and 90 mg intravaginal micronized progesterone. Blood samples from each subject were obtained at time points 0, 0.5, 2, 1.5, 2, 4, 6, 8, 12, 24, 36, 48 and 72 hours following one initial dosing of each treatment. Concentrations of plasma progesterone were determined by RIA (ImmuChem Double Antibody, 125I RIA Kit, MP Biomedicals, Costa Mesa, CA). Pharmacokinetic analysis was carried out using commercially available software (Phoenix WinNonlin 6.4, Certara Inc., Princeton, NJ). One-compartmental (intravaginal) and non-compartmental (oral administration) modeling were performed to analyze data using the mean concentrations for each dosing to calculate the area under the curve (AUC), maximum plasma concentration (Cmax), time elapsed to reach Cmax (Tmax), and elimination half-life ( t1/2). Results for the 100 mg and 200 mg oral doses and 45 mg and 90 mg IVA doses were as follows: AUC, 30.86, 187.96, 90.64, and 226.68 ng.h.mL-1, respectively; Cmax, 13.47, 169, 8.68, and 13.24 ng.mL-1, respectively; Tmax, 0.5, 0.5, 0.84, and 1.67 hr, respectively, and half-life, 5.87, 6.76, 6.6, and 10.65 hr, respectively. Micronized progesterone was readily absorbed in bitches when administered either orally or intravaginally. Contrary to our initial hypothesis, micronized progesterone exposure over time, as indicated by the area under the curve, was greater when intravaginal micronized progesterone was used. The ability of intravaginal preparations of micronized progesterone to induce sustained progesterone exposure may provide an alternative strategy for treating pregnant dogs whenever hypoluteoidism is being suspected.

Pythium insidiosum is an aquatic oomycete that causes invasive, progressive granulomatous lesions of the skin in dogs, horses, and cats, and of the gastrointestinal tract in dogs. Quantitation of anti-P. insidiosum IgG antibodies can be used in dogs to both confirm a suspected diagnosis and to monitor response to therapy. Recently, an immunotherapeutic product (IP) has been marketed for the treatment of pythiosis in dogs, horses, and people. The aim of this study was to evaluate the effect of administration of this product on anti-P. insidiosum IgG concentrations in dogs. The IP was administered to seven, healthy hound mixes on days zero, seven and 21. Serum was collected on days zero, seven, 14, 21, 28, 35, 42, 49, and 56. Anti-P. insidiosum antibody concentrations were measured using a previously-described ELISA that utilizes a soluble mycelial-based antigen, with results reported as percent positivity (PP) in comparison to a strong positive control serum. Prior to immunotherapy administration, average PP was 7.45% +/- 3.02%. Following immunotherapy administration, there was no significant change in anti-P. insidiosum antibody concentrations, with PP values in all dogs remaining within the range expected for healthy dogs (3% - 15%) for the entire study period. In conclusion, the IP did not produce a significant change in anti-P. insidiosum IgG concentrations when administered to healthy dogs using the protocol suggested by the manufacturers. Further investigation will be required to determine whether a similar effect is observed in naturally infected dogs.

Research suggests that the cyclic AMP (cAMP) signaling pathway including CREB-CRE regulated expression of various genes is implicated in the predisposition to and development of alcoholism in humans. Alcohol also induces changes in inflammatory and immune responses; these changes increase the incidence of pneumonias and other infections, which can negatively affect recovery from infections. Cyclic AMP (cAMP) is known for its immunosuppressive effects and is also required for proper development of the immune system. Previous work in our laboratory has demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform-specific manner; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesize that the AC isoform expressed in the cells will play a role in ethanols effects on cAMP regulated gene expression. We further hypothesize that alcohol modulates cAMP signaling in immune cells by enhancing the activity of AC7; thus, AC7 may play a role in ethanols effects on immune function. Our objectives include: 1) evaluate the AC isoform specific effects of ethanol on cAMP regulated gene in NIH 3T3 cells by overexpressing two AC isoforms: AC3 and AC7; 2) employ immune cell lines endogenously expressing AC7, RAW 264.7 and BV-2, to further elucidate the role of AC7 in the effect of ethanol on cAMP regulated gene expression. To examine these objectives, time-lapse fluorescent resonance energy transfer (FRET) and cAMP accumulation assays were used to monitor cAMP levels within the cells. A reporter gene (luciferase) driven by an artificial promoter inducible with cAMP was utilized to evaluate the effect of ethanol on cAMP regulated gene expression. CREB phosphorylation and nuclear translocation of transducers of regulated CREB (TORCs) were examined by western blotting. Stimulation of AC activity by the addition of dopamine caused an increase in the reporter gene activity. Ethanol potentiated the increase of reporter gene activity in NIH 3T3 cells expressing AC7, while cells expressing AC3 did not respond to ethanol. Cyclic AMP pathway activation via stimulation with prostaglandin E1 (PGE1) showed an increase in cAMP and reporter gene expression in RAW 264.7 and BV-2 cells. The effect observed was potentiated in the presence of ethanol. Cyclic AMP analog, 8-Bromo-cAMP, induced luciferase activity was not significantly affected by ethanol. The level of CREB phosphorylation did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 may play a more critical role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity. Furthermore, the ethanol effect on cAMP regulated reporter gene expression is due to a change in the amount of cAMP, which most likely results from the enhancement of AC7 activity by ethanol.

Semen cryopreservation has allowed the establishment of genome banks and the large scale propagation of species. The development of simple techniques to cryopreserve semen or alternatives to efficiently use cryopreserved semen from males of valuable genetics that have become infertile will permit continuous propagation of the genetics from these males and may serve as a model for preservation and propagation of endangered species. Sperm cryopreservation without cryoprotectants is a simple process, and offspring have been produced following intracytoplasmic sperm injection (ICSI); however the ability of frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI was unknown. In the series of experiments performed, bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants was used to activate intra- and interspecies oocyte following ICSI. Additionally, equine cumulus-oocyte complexes (COCs) glucose metabolism during in vitro maturation was evaluated. The first experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had their plasma membrane damaged; however the DNA was unaffected. The second experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had the ability to activate bovine oocytes following intracytoplasmic sperm injection; although at a lower rate compared to frozen-thawed sperm. The third experiment demonstrated that frozen-thawed stallion sperm refrozen without the addition of cryoprotectants was unable to activate equine oocytes. The exact reason for this failure could not be explained from the experiment; however COC metabolism during in vitro maturation impacts embryo activation/development and required further investigation. The fourth experiment demonstrated that equine COCs consume and metabolize glucose through glycolysis during in vitro maturation; however, results from this experiment were unable to explain the failure of refrozen stallion sperm to activate equine oocytes. To our knowledge, this is the first report of the use of bull or stallion frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI. Furthermore, this is also the first report of equine COCs glucose metabolism during in vitro maturation.

Histone H3K79 methylation has been shown to play roles in different DNA repair pathways. Histone H4 residues serine 64 to threonine 80 surround histone H3K79 residue. We have analyzed the effect of the mutations of the residues on UV sensitivity, H3K79 methylation, nucleotide excision repair, chromatin state and homologous recombination. We found that mutation of the residues 64 to 72 cause resistance to killing by UV whereas mutation of the residues 73 to 80 cause sensitivity to killing by UV compared to wild type. In general, we found that the mutations make nucleotide excision repair more proficient at the constitutively active RPB2 loci. We found global genomic repair is faster in most of the mutants except H75E. Transcription coupled repair is normal in most of the mutants except mutant Y72T. In mutant H75E, Rad26 independent transcription coupled repair is also defective. The mutations T73D, T73F and T73Y affect mono, di and tri methylation of H3K79 but they have faster or normal nucleotide excision repair. We have also found that these histone mutations make chromatin more accessible to micrococcal nuclease. The UV sensitive histone mutants have normal or faster nucleotide excision repair. Methyl methane sulfonate (MMS) sensitivity test, Rad14 and Rad52 epistasis analysis suggests that the UV sensitive histone H4 mutants could play role in homologous recombination repair pathway. Taken together, the results imply that the histone mutations remodel the chromatin that helps to recruit nucleotide excision repair factors for efficient repair.