Background: Clostridium difficile is the most common cause of healthcare-associated infections in the United States, and has been associated with adverse outcomes in the acute care setting. However, little is known regarding the burden or impact of C. difficile infection (CDI) in long-term acute care hospitals (LTACHs). Methods: A retrospective matched cohort study was performed among patients at an urban, university-affiliated LTACH between July 2008 and October 2015. The incidence rate of LTACH-onset CDI was assessed and patient characteristics associated with adverse outcomes examined. Patients with CDI were matched to concurrently hospitalized LTACH patients without a diagnosis of CDI. A multivariable model using logistic regression was developed to determine characteristics associated with a composite primary outcome of either 30-day readmission to an acute care hospital or mortality. Subgroup analyses were performed for patients with a diagnosis of severe CDI. Results: The overall incidence of CDI was 21.4 cases per 10,000 patient-days. Patients with CDI had a mean age (±SD) of 70 ±14 years and a mean admission Charlson Comorbidity Index (CCI) of 4 ±2. Median (IQR) time between admission and diagnosis of CDI was 16 days (range: 9-23 days). In the final multivariable model, CDI was not a significant risk factor for the primary outcome (OR, 1.06 [95% confidence interval {CI}, 0.53-2.10]). Congestive heart failure (OR, 2.27 [95% CI, 1.15-4.57]), albumin level (OR, 0.44 [95% CI, 0.22-0.79]), and immunosuppression (OR, 2.94 [95% CI, 1.06-8.39]) were independent risk factors for the primary outcome. On subgroup analysis, severe CDI and CCI were significant risk factors for the primary outcome in bivariable analysis (OR, 2.91 [95% CI 1.03-8.20] and OR, 1.36 [95% CI 1.06-1.80], respectively). Only CCI remained significant in the multivariable model (OR, 1.32 [95% CI 1.02-1.75]). Conclusions: LTACH-onset CDI was found to have a relatively high incidence in an urban, university affiliated LTACH. CDI was not a significant risk factor for the composite outcome of 30-day readmission or mortality. Future research should focus on infection prevention and antibiotic stewardship measures to decrease CDI specifically in the LTACH setting. / Epidemiology

Systemic lupus erythematosus (SLE) is complex autoimmune disease in which autoantibodies form against double stranded DNA (dsDNA) and nuclear antigens. Autoantigen immune complexes form, deposit in the vasculature, and cause multisystem organ damage. Both genetic and environmental factors contribute to the development of SLE. This thesis will explore three major themes found in the study of SLE: 1) Bacterial infection as an environmental trigger, 2) cytokine dysregulation in immune cells, and 3) the treatment of end organ damage in the form of lupus nephritis. Viral infections have long been associated with the development of systemic autoimmune disease, but the mechanisms by which chronic bacterial infections may promote autoimmunity remain unclear. In chapter three we show that a component of bacterial biofilms, the amyloid-like protein “curli”, irreversibly forms fibers with bacterial or eukaryotic DNA during biofilm formation. This interaction accelerates amyloid polymerization and creates potent immunogenic complexes that activate immune cells, including dendritic cells, to produce cytokines such as type I interferons, which are pathogenic in SLE. When given systemically, curli/DNA composites trigger immune activation and production of autoantibodies in lupus-prone and wild type mice. We also found that infection with curli-producing bacteria triggered higher autoantibody titers in lupus-prone mice compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity. Cytokine dysregulation is also common in SLE patients. Serum cytokines are often elevated during active disease, including type I IFNs and IL-10. In chapter four we demonstrate that Il10 is a type I IFN response gene and has increased basal expression in dendritic cells (DCs) derived from pre-disease lupus-prone Sle1,2,3 mice. We show that Sle1,2,3-derived DCs overproduce IL-10 in response to TLR ligands and that this is the result of autocrine signaling though the type I IFN receptor (IFNAR). These results suggest that dysregulation of cytokine signaling in the myeloid compartment may contribute to IL-10 dysregulation in SLE. Renal disease remains a major cause of morbidity and mortality in SLE. A number of mouse models of chronic kidney disease have implicated the EGFR-family receptors in the progression of renal fibrosis and dysfunction. In chapter five we show that renal expression of ErbB2 is increased in murine lupus. We therefore asked if EGFR-family inhibition could prevent murine lupus nephritis. To test this possibility we used lapatinib, an EGFR-ErbB2 dual kinase inhibitor, in an IFN-accelerated model of murine lupus. We found that lapatinib administration lowered autoantibody levels but worsened renal disease. Lapatinib failure to treat murine lupus nephritis despite lowered autoantibody levels suggests EGFR-family signaling is required for tissue repair in the acute phase of kidney injury. Together this thesis clearly demonstrates the complexity of systemic autoimmune disease – bringing us to the crossroads of immunity and tolerance. The combination of both environmental triggers (e.g. bacterial infection) and genetic susceptibility (e.g. intrinsic cytokine dysregulation) leads to end organ damage (e.g. lupus nephritis). Here we sought to explore each aspect of disease progression in the hopes to develop better interventions for systemic autoimmune disease. / Microbiology and Immunology

Toxoplasma gondii is a protozoan parasite that causes life-threatening disease in congenitally infected infants and immunocompromised patients, such as those inflicted with AIDS. Toxoplasmic encephalitis (TE) is a common presenting condition in an AIDS infection. People become infected with T. gondii by ingesting tissue cysts in undercooked meats or by ingesting oocysts excreted by cats. Methylmercury (MeHg) is a well-documented neurotoxicant that accumulates in the brain and causes severe mental and visual dysfunction, including chronic encephalopathy. Consumption of contaminated fish, grains, and seeds are common sources of human exposure to methylmercury. Studies from our laboratory suggest that oral exposure to a single high dose of 20 mg/kg MeHg does not increase the susceptibility to acute toxoplasmosis in CBA/J mice. Therefore, we further investigated endpoints associated with immunotoxicity and neurotoxicity in 6-week old, female CBA/J mice exposed to both MeHg and T. gondii during a chronic T. gondii infection. We examined both single and multiple doses of MeHg exposure in a chronic parasitic infection model. In the single high dose study, four groups of six-week-old, female CBA/J mice were either fed 25 T. gondii tissue cysts of the ME-49 strain or given vehicle. Six weeks later, two out of the four groups (T. gondii and vehicle control) were orally gavaged with a single dose of 20 mg/kg body weight of MeHg and sacrificed seven days post exposure. Experiments from the multiple MeHg dose study were performed under similar conditions with the same number of groups and dosed by oral gavage with 8 mg/kg body weight of MeHg on days 0, 2,4,7,10,13. These mice were sacrificed on day 17 or 18 after initiating MeHg exposure. Flow cytometry following exposure to a single dose of MeHg in mice with a chronic T. gondii infection revealed significant changes (P < 0.05) within the T cell subpopulation percentages caused by exposure to MeHg. For example, the thymic CD4+CD8+ T cell subpopulations were increased (P <0.05). However, MeHg had no significant effect on the CD4+CD8-, CD4-CD8+, or non-T cell subpopulations in the spleen. Furthermore, MeHg increased splenic cellularity and spleen-to-body-weight ratios with or without a concurrent T. gondii infection. MeHg also caused a significant decrease in mouse body weight. There was a significant (P <0.05) increase in brain tissue cyst counts within the group exposed to both MeHg and T. gondii (16 ± 4, mean ± SE, n=7) versus T. gondii alone (4 ± 1, n=8). Histopathological examination demonstrated that the brain was affected, as lesions, gliosis, and meningitis were notable in mice given T. gondii. Exposure of mice to multiple doses of MeHg also resulted in effects on the immune system of CBA/J mice with and without chronic toxoplasmosis. Total cellularity and numbers of CD4+CD8+, CD4+CD8-, CD4-CD8+, and CD4-CD8- T-cell subpopulations show a marked decrease in number in the thymus, while total cellularity was also decreased in the spleen following concurrent exposure to T. gondii and MeHg. Flow cytometric examination of lymphocyte populations (CD4+ and CD8+ lymphocytes) in the spleen and thymus demonstrated differences from control in the groups exposed to T. gondii and MeHg. Histopathological examination did not reveal any significant lesions. The data from experiments in which single or multiple doses of MeHg were given to mice with a chronic T. gondii infection indicate that concurrent exposure, to both MeHg and T. gondii, dependent on dose and time of exposure had notable effects, especially on the immune system (Supported by NIH Grant F36GM20301). / Ph. D.

Southwest Virginia has recently become an emerging focus of activity for La Crosse (LAC) virus, a mosquito-transmitted arbovirus in the California serogroup of Bunyaviruses. In 2005 and 2006, ovitrap surveys were conducted to access the spatiotemporal oviposition activity of LAC virus vectors Aedes triseriatus, Ae. albopictus and Ae. japonicus across a wide region of southwest Virginia. Egg abundance and oviposition patterns of these vectors were significantly different across the three study areas. The primary LAC virus vector, Ae. triseriatus, was collected in the greatest abundance from all three areas, and favored forested habitats. Aedes albopictus was the second most abundant species collected, and was found to favor urban environments. Aedes japonicus also has a preference for urban habitats, and is actively expanding its range throughout southwest Virginia. Dogs were used to determine their efficacy as sentinels for assessing the distribution of LAC virus in southwest Virginia. Canine serum samples were tested using plaque reduction neutralization (PRNT) assays. Of the 436 collected canine serum samples, 21 (4.8%) were positive for LAC virus antibodies. LAC virus seroprevalence was evident in dogs from each study region, including areas where LAC virus human cases and LAC virus positive mosquito isolates have not been reported. As a result, this study provided documentation of horizontal transmission of LAC virus throughout southwest Virginia, demonstrating that dogs make useful sentinels for assessing the distribution of LAC virus in an area. The final objective examined the effects of coinfection with D. immitis microfilariae and LAC virus in three species of Aedes mosquitoes. No significant differences were found between mosquitoes fed dually infected bloodmeals (i.e. D. immitis microfilariae and LAC virus) and those fed bloodmeals containing LAC virus only. A follow-up study found low mosquito midgut penetration rates by D. immitis, despite using biologically significant doses of microfilariae. Failure to demonstrate enhancement of LAC virus in vector mosquitoes suggests that D. immitis does not have a significant impact on LAC virus epidemiology in areas where these organisms co-exist. / Ph. D.

Microfilarial enhancement of viral transmission is well documented, however only one previously studied model used components that occur together in nature and therefore has realistic implications. La Crosse (LAC) virus encephalitis is the most common mosquito-borne illness affecting children in the United States. LAC virus is prevalent in the Great Lake and Mid-Atlantic states and coincidently this area overlaps the region of highest infection for Dirofilaria immitis, the nematode that cause canine heartworm disease. Ae. albopictus and Oc. triseriatus are important vectors of La Crosse virus and among the numerous species able to transmit D. immitis. In this study, Aedes albopictus and Ochlerotatus triseriatus were infected with La Crosse virus and Dirofilaria immitis to determine the effects of dual infection on the dissemination and transmission of the virus. The effects of dual infection varied between the species tested. Ae. albopictus had significantly higher tolerance to D. immitis infection than Oc. triseriatus. Dissemination for dually infected Ae. albopictus were higher than the control group for all days tested, except one. Transmission rates for D. immitis infected Ae. albopictus were significantly higher than the control group on day 14 post infection. No microfilarial enhancement of viral dissemination or transmission was observed for Oc. triseriatus. The infection, dissemination, and tranmission rates were low for both species compared to rates of previous studies. Low rates could be a result of low susceptibility for the strains tested. In a second study, mosquitoes were collected from two counties in Southwestern Virginia to determine the abundance of potential La Crosse virus and D. immitis vector species. The abundance and distribution of mosquito species were examined in 2003 and 2004 using gravid traps. An unexpected finding was the significant increase in the abundance of Ochlerotatus japonicus. In 2003, collections were made over 192 trap nights from June to August yielding 5,879 mosquitoes of which only 24 were Oc. japonicus. In 2004, 12,151 mosquitoes were trapped from June to September over 160 trap nights. Oc. japonicus was the second most abundant mosquito species and the dominant Ochlerotatus species collected in gravid traps. Oc. japonicus was collected in low numbers in June, but the abundance increased significantly in July and remained consistent throughout the rest of the season. Of the other major mosquito species collected in this study, only Aedes albopictus exhibited a similar seasonal pattern as Oc. japonicus. Other biological similarities of Oc. japonicus and Ae. albopictus are discussed. / Master of Science in Life Sciences

No / Not available

Yes / The behavior of a linear copolymer of N-isopropyl acrylamide with pendant vancomycin functionality was compared to an analogous highly branched copolymer with vancomycin functionality at the chain ends. Highly branched poly(N-isopropylacrylamide) modified with vancomycin (HB-PNIPAM-van) was synthesized by functionalization of the HB-PNIPAM, prepared using reversible addition-fragmentation chain transfer polymerization. Linear PNIPAM with pendant vancomycin functionality (L-PNIPAM-van) was synthesized by functionalization of poly(N-isopropyl acrylamide-co-vinyl benzoic acid). HB-PNIPAM-van aggregated S. aureus effectively whereas the L-PNIPAM-van polymer did not. It was found that when the HB-PNIPAM-van was incubated with S. aureus the resultant phase transition provided an increase in the intensity of fluorescence of a solvatochromic dye, nile red, added to the system. In contrast, a significantly lower increase in fluorescence intensity was obtained when L-PNIPAM-van was incubated with S. aureus. These data showed that the degree of desolvation of HB-PNIPAM-van was much greater than the desolvation of the linear version. Using microCalorimetry it was shown that there were no significant differences in the affinities of the polymer ligands for D-Ala-D-Ala and therefore differences in the interactions with bacteria were associated with changes in the probability of access of the polymer bound ligands to the D-Ala-D-Ala dipeptide. The data support the hypothesis that generation of polymer systems that respond to cellular targets, for applications such as cell targeting, detection of pathogens etc., requires the use of branched polymers with ligands situated at the chain ends. / MRC

Yes / Objective: this research seeks to investigate the impact the Industrial Revolution had on the population of England. Materials: Pre-existing skeletal data from 1154 pre-Industrial (1066–1700AD) and 4157 industrial (1700–1905) skeletons from 21 cemeteries (N = 5411). Methods: Context number, sex, age-at-death, stature and presence/absence of selected pathological conditions were collated. The data were compared using chi square, Kolmogorov-Smirnov, t-tests and logistic regression (α = 0.01). Results: There was a statistically significant increase in cribra orbitalia, periosteal reactions, rib lesions, fractures, rickets, osteoporosis, osteoarthritis, enamel hypoplasia, dental caries and periapical lesions in the industrial period. Osteomyelitis decreased from the pre-industrial to industrial period. Conclusion: Our results confirm the Industrial Revolution had a significant negative impact on human health, however the prevalence of TB, treponemal disease, maxillary sinusitis, osteomalacia, scurvy, gout and DISH did not change, suggesting these diseases were not impacted by the change in environmental conditions. Significance: This is the largest study of health in the Industrial Revolution that includes non-adults and adults and considers age-at-death alongside disease status to date. This data supports the hypothesis that the Rise of Industry was associated with a significant decline in general health, but not an increase in all pathologies. Limitations: This meta-analysis relies upon previously gathered data and diagnosis from a large number of researchers. Incomplete skeletons were often excluded from analyses. Few rural cemeteries were available for inclusion. Suggestions for further research: Data from unpublished and ongoing excavations should be investigated. Comparison with historical data is encouraged. / Funded by the Royal Society (IES\R1\180138)

No / Despite wide acknowledgment that hand hygiene is the pre-eminent measure in the control of health-care-associated infection, Didier Pittet and colleagues 1 have highlighted that there is still a need for a systematic programme of research that will allow the development of new¿as well as refinement of existing¿approaches to hand cleansing. One of the key priorities Pittet and colleagues identified is the need for investigations into the relative importance of between and within patient cross-transmission

Yes / African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. IMPORTANCE. Most African trypanosomes are lysed by the ApoL1 protein in human serum. Only the subspecies Trypanosoma b. gambiense and T. b. rhodesiense can resist lysis by ApoL1 because they express specific serum resistance proteins. We here report a complex interplay between trypanosomes and an ApoL1 variant characterized by a homozygous missense substitution (N264K) in the domain that we hypothesize interacts with the endolysosomal membranes of trypanosomes. The N264K substitution knocks down the lytic activity of ApoL1 against T. b. gambiense strains lacking the TgsGP defense mechanism and against T. b. rhodesiense if N264K is accompanied by additional substitutions in the SRA-interacting domain. Our data suggest that populations with high frequencies of the homozygous N264K ApoL1 variant may be at increased risk of contracting human African trypanosomiasis. / This work, including the efforts of Stijn Deborggraeve, was funded by Research Foundation Flanders (1501413N). This work, including the efforts of Bart Cuypers, was funded by Research Foundation Flanders (11O1614N). This work, including the efforts of Jean-Claude Dujardin and Etienne Pays, was funded by Interuniversity Attraction Poles Program of Belgian Science Policy (P7/41). This work, including the efforts of Jean-Claude Dujardin, was funded by Flemish Ministry of Sciences (SOFI-B SINGLE). This work, including the efforts of Etienne Pays, was funded by EC | European Research Council (ERC) (APOLs 669007).