Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant pathogen responsible for ~1/3 of antimicrobial resistance-associated mortality in the USA. The glycopeptide, vancomycin, remains the primary treatment for invasive MRSA infections while lipopeptides (e.g. daptomycin) and lipoglycopeptides (e.g. dalbavancin) are common alternatives. All MRSA are resistant to beta-lactams (e.g. nafcillin) but synergistic antimicrobial activity between vancomycin or daptomycin and beta-lactams is commonly observed. Some investigators attribute this synergy to the “seesaw effect”, a phenomenon where the susceptibility to beta-lactams increases with declining vancomycin or daptomycin susceptibility. However, the association between synergy and the seesaw effect hasn’t been rigorously tested. The objective of this study was to determine whether the seesaw effect was necessary for synergy. We used standard time-kill methods to test for synergy between nafcillin and vancomycin, daptomycin or dalbavancin in a series of isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin. Two strains exhibited the seesaw effect (N315-VAN8, N315-D1) with nafcillin while 1 didn’t (N315-DAL0.5). All time-kills were performed using half the minimum inhibitory concentration(MIC) for each drug. Nafcillin concentrations were capped at peak physiological concentrations if the MIC was above this value and all experiments were performed in duplicate. Bacterial survival was counted at 0, 4, 8, and 24-hours. Synergy was defined as ≥2 log10 colony forming units per milliliter increase in bacterial killing of the combination compared to the most active single agent. All strains tested exhibited synergy between nafcillin and vancomycin, daptomycin, and dalbavancin, independent of the seesaw effect, suggesting that these two phenomena are distinct. This is important because the emergence of the seesaw effect cannot be detected clinically. This suggests that the seesaw effect is not a therapeutically relevant phenomenon. Further work is warranted to characterize strains that don’t exhibit beta-lactam synergy to identify which strains we should target with combination therapy.

Human Immunodeficiency Virus (HIV) remains on the forefront of research due to the ongoing global epidemic. HIV is comprised of two genetically different types, HIV-1 and HIV-2. HIV-2 is inherently resistant to some classes of antiretroviral drugs, and many HIV-2 patients develop drug resistance to first-line and subsequent regimens. HIV-2 can further be divided into two distinct genetic groups: A and B. While both are endemic to West Africa, group A accounts for the majority of infections and remains the most studied of the two groups. In-depth knowledge of drug resistance in HIV-2 group B is lacking, as only a few patients with drug-resistant virus are described in the literature and there have been no systematic efforts to characterize the drug resistance patterns of HIV-2 group B isolates in cell culture. My project's goal is to build drug resistance mutations, documented in literature, for HIV-2 group A into a full-length HIV-2 group B infectious molecular clone. Those results are used to compare the relative drug resistance conferred by those mutations to the phenotypes observed for equivalent mutants of HIV-2 group A. More specifically, common drug resistance mutations are introduced into the pol gene of a group B clone, individual mutant clones are isolated, and these are used to transfect replication-competent cells for virus production and drug susceptibility testing. Inhibitors targeting the reverse transcriptase, protease and integrase targets of HIV-2 are evaluated. The resultant drug resistance profiles are then compared to those found in published datasets for HIV-2 group A to determine how HIV-2 group A and group B mutants differ in terms of the magnitude and/or scope of drug resistance. These data are essential for developing evidence-based treatment guidelines for HIV-2–infected patients that harbor drug-resistant group B strains.

Polycystic ovarian syndrome (PCOS) is a prevalent gynecological concern affecting women of reproductive age. However, the exact cause of PCOS has yet to be identified. As described by the Western medical field, PCOS is characterized by polycystic ovaries, hyperandrogenism, and irregular menstruation. In contrast to this, PCOS is described as a “kidney- and spleen-deficiency syndrome” by the Traditional Chinese Medicine (TCM) community. By comparing and contrasting the treatment for PCOS in the West and in the East, we can propose a more comprehensive mechanism for phenotypically different PCOS patients, and hence improve medical support in the future. This literature review addresses the gaps in PCOS research for both Western and Eastern medical fields and compares their account of and treatment for the syndrome. In recent studies, PCOS has been shown to have association with vitamin D deficiency. Vitamin D concentration and anti-Müllerian hormone (AMH) are reportedly inversely associated. In addition, elevated AMH reflects abnormal maturation of the ovarian follicle in PCOS women. Given that kidneys are significant in Vitamin D activation, the kidney imbalance condition described by TCM is consistent with the vitamin D deficiency condition. Since both the Western metabolic school and TCM advocate lifestyle and diet change in PCOS treatment, TCM’s account of PCOS aligns with that of the metabolic studies in the West. Such findings have innovative interesting implications for the future of possible integrations between Western and Eastern medicine in PCOS diagnosis and treatment options for phenotypically-varied PCOS patients.

Gene therapy may be an effective way to treat vascular disease. However, current approaches to achieve efficient gene delivery to the blood vessel wall require invasive surgery. Others have reported that high-concentration lentiviral vectors injected into the jugular vein preferentially transduce cells in the vascular wall of mice. If reproducible, this approach would be a major advance in vascular gene therapy. To test reproducibility, we injected high-concentration lentivirus into mouse jugular veins and measured transduction 4 days later. Eight mice received 10e8 particles of a green-fluorescent protein-expressing lentivirus; six mice received vehicle only. Mice were euthanized and their aortas, hearts, lungs, livers, kidneys, spleens, quadriceps and gastrocnemius muscles, carotid and femoral arteries were excised. These tissues were prepared for DNA analysis (to measure transduction) and histology (to detect GFP expression). Tissues used for DNA analysis were frozen in liquid nitrogen and DNA was extracted. We used quantitative PCR targeting the GFP gene to measure integrated proviral DNA, which we quantified as proviral copies per cell. We found a mean of 3 proviral copies (range 1-6) per cell in the liver samples and 13 proviral copies (range 4-28) per cell in the spleen samples. Essentially no proviral DNA was detected in the liver or spleen of vehicle-injected mice (p<0.003 for both; limit of detection 0.3 copies per cell). Proviral DNA was not reproducibly detected in the other organs/tissues listed above. Tissue for histology was fixed and embedded in frozen blocks for immunostaining. We anticipate that immunostaining will enable detection or exclusion of lower levels of transduction. Intravenously injected lentiviral vectors seem to efficiently transduce the spleen and liver and do not efficiently transduce cells in the blood vessel wall. Our data suggest that better vector targeting is needed to deliver gene therapy to the vessel wall by IV injection.

In recent years, numerous studies have investigated the transfer of rare cells between a mother and her fetus during pregnancy, a phenomenon known as microchimerism. Maternal microchimerism (MMc) refers to maternal cells transferred to the fetus, whereas fetal microchimerism (FMc) refers to fetal cells transferred to the mother. FMc is found in both normal and abnormal pregnancies, and in particular is increased in settings of placental dysfunction such as pre-eclampsia. Plasmodium falciparum infection during pregnancy may lead to sequestration of infected erythrocytes in the placenta, known as placental malaria, that results in a phenotype similar to pre-eclampsia. The Harrington lab previously found that the fetus acquires more MMc in the setting of placental malaria. My research project investigates the reciprocal transfer of FMc to the mother in this setting. I hypothesize that malaria infection during pregnancy is associated with an increased prevalence of FMc. To complete this project, high-quality genomic DNA was extracted from samples gathered from a cohort of women in Mali. These samples came from women both with and without malaria infection during their pregnancy. After collecting the DNA, HLA-typing between a mother and her offspring were compared to determine a unique marker of fetal DNA. I am currently using quantitative PCR to amplify fetal alleles in the background of the mother in order to quantify FMc. Lastly, I will compare the level of assessed FMc in malaria exposed and non-exposed women. The findings from this research project have the potential to contribute important information about the complex role of microchimerism in immune function.

Maternal microchimerism (MMc) is the transfer of maternal cells to the fetus across the placenta during pregnancy. MMc is found in both normal and abnormal pregnancies. The Harrington lab has previously shown that the amount of MMc increases with Plasmodium falciparum infections during pregnancies. In the same cohort, children with detectable levels of MMc were more likely to become infected with malaria but interestingly less likely to experience symptoms, suggesting that these maternal cells may regulate or educate the infant immune response against malaria. I aim to assess whether maternal cells selectively proliferate in the infant during the first infection with Plasmodium falciparum in order to mount an immune response by comparing the levels of MMc throughout three time points. To investigate the role of maternal cells in the infant immune response, I will test blood samples of infants exposed to placental malaria from three time points (a cord blood sample, a blood sample immediately preceding the infant’s first parasitemia, and a blood sample immediately following the infant’s first parasitemia) for the presence and amount of MMc. I hypothesize that MMc will be found in higher quantities in blood samples following an infant’s first infection with parasitemia, indicating that maternal cells help mount an immune response in the infant. Blood samples stored as dried blood spots (DBS) have been received from Ugandan field sites. First, the DNA was extracted from DBS samples. After comparison of class II HLA markers between a mom and her offspring, quantitative PCR (qPCR) will be used to target a unique, maternal HLA-class II marker in order to quantify MMc levels. The levels of MMc will be compared across the three time points. The results of my research have important implications for the current understanding of how MMc regulates the infant immune system.

Human immunodeficiency virus (HIV) infection is a significant global health issue, with approximately 75 million infections, and over 35 million deaths, since the beginning of the AIDS pandemic. The majority of these are attributable to HIV type 1 (HIV-1). A second form of HIV – HIV type 2 (HIV-2) – is endemic in West Africa and has spread to other areas with socioeconomic ties to the region. Historically, regimens for first-line treatment of HIV-2 have differed from those used in HIV-1-infected patients due to the intrinsic resistance of HIV-2 to nonnucleoside reverse transcriptase inhibitors. This distinction is coming to an end, as countries throughout West Africa are implementing a new WHO-recommended treatment regimen for first-line treatment of all HIV-infected patients, including those with HIV-2. The regimen, known as TLD, is comprised of the nucleoside reverse transcriptase inhibitors tenofovir and lamivudine and the integrase inhibitor, dolutegravir that has potent activity against both HIV-1 and HIV-2. Although treatment-emergent drug resistance has been well characterized for HIV-2 patients receiving tenofovir and lamivudine, there are few data regarding resistance mechanisms in patients receiving the third component of TLD, dolutegravir. The two objectives of my project are: (1) to construct a system for generating recombinant HIV-2 clones that encode and express integrase sequences from TLD-treated HIV-2 patients, and (2) to determine the in vitro susceptibility of viruses produced from the patient-derived clones to the integrase inhibitor dolutegravir. Specifically, I am engineering a plasmid vector into which patient-derived integrase sequences can be ligated for virus production and drug resistance testing in culture. The plasmid vector produced in this study will be used to characterize novel genetic pathways to dolutegravir resistance in HIV-2 and will help identify patients who are failing TLD treatment due to drug resistance. This information is crucial for improving treatment outcomes in HIV-2-infected individuals worldwide.

Syphilis is caused by infection with Treponema pallidum subsp. pallidum (T. pallidum). The Enhanced CDC (ECDC) and Multi-locus Sequence Typing (MLST) are two different T. pallidum strain typing systems. Our lab previously showed that neurosyphilis is more common in patients infected with ECDC type 14d/f. In this project, we aimed to compare the two typing systems, and to determine if there is an association between neurosyphilis and MLST strain type. We performed MLST analysis on 75 T. pallidum isolates by analysis of regions of the tp0136, tp0548, and tp0705 genes. Samples were chosen because they were completely typeable by ECDC. We determined association between MLST type and neurosyphilis by using Chi-Square or Fisher’s exact test to compare each MLST type to all other types grouped together. We excluded MLST types with N<5 from the analysis. We determined a complete MLST type for 74 (99%) of 75 isolates and found that MLST subdivided two of the most common ECDC types, 14d/f and 14d/g, into 7 and 3 types. However, MLST failed to distinguish between some ECDC types, such as 14d/f and 15d/f. Compared to all other types, a reactive cerebrospinal fluid Venereal Disease Research Laboratory (CSF-VDRL) test, the diagnostic test for neurosyphilis, was more common in individuals infected with MLST type 1.1.2 (P=0.02), which corresponds to ECDC type 14d/f. A reactive CSF-VDRL was also more common in patients with tp0705 type 2 (P=0.01). A reactive CSF-VDRL was less common in patients infected with MLST type 1.3.1 (P=0.002), which corresponds to ECDC type 14d/g. In the future, we will examine whether one MLST typing target, tp0705, could be used alone on blood to identify individuals with syphilis who are at greatest risk for neurosyphilis.